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Impact of obesity and overweight on DNA stability: Few facts and many hypotheses
Accepted Manuscript Title: Impact of obesity and overweight on DNA stability: Few facts and many hypotheses Authors: Tahereh Setayesh, Armen Nersesyan, Miroslav Miˇs´ık, Franziska Ferk, Sabine Langie, Vanessa M. Andrade, Alexander Haslberger, Siegfried Knasmuller ¨ PII: DOI: Reference:
S1383-5742(17)30096-0 https://doi.org/10.1016/j.mrrev.2018.07.001 MUTREV 8246
To appear in:
Mutation Research
Received date: Revised date: Accepted date:
27-10-2017 3-5-2018 6-7-2018
Please cite this article as: Setayesh T, Nersesyan A, Miˇs´ık M, Ferk F, Langie S, Andrade VM, Haslberger A, Knasmuller ¨ S, Impact of obesity and overweight on DNA stability: Few facts and many hypotheses, Mutation Research-Reviews in Mutation Research (2018), https://doi.org/10.1016/j.mrrev.2018.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Impact of obesity and overweight on DNA stability:
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few facts and many hypotheses
Tahereh Setayesh1, Armen Nersesyan1, Miroslav Mišík1, Franziska Ferk1, Sabine Langie2,
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Vanessa M. Andrade3, Alexander Haslberger4, Siegfried Knasmüller1*
Institute of Cancer Research, Department of Medicine I, Medical University of Vienna,
Environmental Risk and Health Unit, Flemish Institute for Technological Research
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Vienna, Austria
Laboratório de Biologia Celulare Molecular, Programa de Pós-Graduação em Ciências da
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3.
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(VITO), Mol, Belgium
Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul
Department of Nutritional Sciences, University Vienna, Vienna, Austria
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4.
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Catarinense (UNESC), Brazil
* Corresponding author: Siegfried Knasmüller, Institute of Cancer Research, Department
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of Medicine I, Medical University of Vienna, Vienna, Austria, Tel: +43 1 4016057562; Fax: +43 1 40160957500; E-mail: [email protected]
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Abstract: Health authorities are alarmed worldwide about the increase of obesity and overweight in the last decades which lead to adverse health effects including inflammation, cancer, accelerated aging and infertility. We evaluated the state of knowledge concerning the impact of elevated body mass on genomic instability. Results of investigations with humans (39 studies) in which
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DNA damage was monitored in lymphocytes and sperm cells, are conflicting and probably as
a consequence of heterogeneous study designs and confounding factors (e.g. uncontrolled
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intake of vitamins and minerals and consumption of different food types). Results of animal studies with defined diets (23 studies) are more consistent and show that excess body fat
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causes DNA damage in multiple organs including brain, liver, colon and testes. Different
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molecular mechanisms may cause genetic instability in overweight/obese individuals. ROS
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formation and lipid peroxidation were found in several investigations and may be caused by
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increased insulin, fatty acid and glucose levels or indirectly via inflammation. Also reduced DNA repair and formation of advanced glycation end products may play a role but more data
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are required to draw firm conclusions. Reduction of telomere lengths and hormonal imbalances are characteristic for overweight/obesity but the former effects are delayed and
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moderate and hormonal effects were not investigated in regard to genomic instability in obese
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individuals. Increased BMI values affect also the activities of drug metabolizing enzymes which activate/detoxify genotoxic carcinogens, but no studies concerning the impact of these alterations of DNA damage in obese individuals are available. Overall, the knowledge
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concerning the impact of increased body weight and DNA damage is poor and further research is warranted to shed light on this important issue. Abbreviations: AGEs, advanced glycation end products; AP-1, activating protein -1; APE1, apurinic/apyrimidinic endonuclease 1; BER, base excision repair; BMI, body mass index; CAF, cafeteria diet; CAT, Catalase; CBMN-Cyt, cytokinesis block micronucleus cytome assay; CEL, carboxyl ester lipase; CML, Nε-carboxymethyllysine; CRP-1, C-reactive protein; DDR, DNA damage 2
response; DFI, DNA fragmentation index; DMBA, 7,12-dimethylbenz(α)anthracene; DNA, deoxyribonucleic acid; DNMTs, DNA methyltransferases; DSBs, double strand breaks; EGCG, Epigallocatechin gallate; ELISA, enzyme linked immunosorbent assay; ENDO III, endonuclease III; ER, estrogen receptor; FA, fatty acid; FACS, fluorescence-activated cell sorting; FPG, formamidopyrimidine DNA glycosylase; GPx, glutathione peroxidase; GSH/GSSG, glutathione/ oxidised glutathione disulphide; GSK-3β, glycogen synthase kinase-3β; GST, glutathione-Stransferase; HFD, high fat diet; Hras, harvey rat sarcoma virus oncogene 1; HPLC, high-performance
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liquid chromatography; 5hmU, 5-hydroxymethyluracil;4-HNE, 4-hydroxy-2-nonenal; HO-1, haem oxygenase-1; HUVEC, human umbilical vein endothelial cells; IGF, insulin-like growth factor; IL-6,
interleukin-6; IR, insulin receptor; IRAC, international agency for research cancer; JNK, Jun N-
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terminal kinase; LC-MC, liquid chromatography-mass spectrometry; LP, lipid peroxidation; MAPK,
mitogen activated protein kinase; MDA, malondialdehyde; MetS, metabolic syndrome; MLH-1, MutL homolog 1; MMR, mismatch repair; MN, micronuclei; NAT, N-acetyltransferase; NBs, nuclear bridges; Nbuds, nuclear buds; NER, nucleotide excision repair; NF-kB, nuclear factor kappa-light-
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chain-enhancer of activated B cells; NOX1, NADPH oxidase 1; 8-OHdG, 8-hydroxy-2'– deoxyguanosine; 8-oxodG, 8-oxo-7,8-dihydro-2' –deoxyguanosine; 8-OH-Gua, 8-OH-guanosine; 8-
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oxoGuo, 8-oxo-7,8-dihydroguanosine; 16α-OHE1, 16α-hydroxyestrone; PAHs, polycyclic aromatic
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hydrocarbons; PCNA, proliferating cell nuclear antigen; PDK1, phosphatidylinositol-4,5-
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bisphosphate 3-kinase; PI3K, phosphatidylinositol-3-kinase; PKC, proteinkinase C; PTEN, phosphatase and tensin homolog; RAGE, receptor advanced glycation end products; ROS, reactive oxygen species; SAM, S-adenosyl-L-methionine; SCGE, single cell gel electrophoresis assay; SDH,
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sorbitol dehydrogenase; SOD, superoxide dismutases; SSBs, single strand breaks; SCSA, sperm chromatin structure assay; TBARS, thiobarbituric reactive substances; T2D, type 2 diabetes; TdT,
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terminal deoxynucleotidyl transferase; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor; TP53, tumor suppressor gene p53; TUNEL, terminal deoxynucleotidyl transferase dUTP
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nick end labelling UGT, uridine diphosphate glucuronosyltransferase; YAP, yes-associated protein
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Keywords: overweight; obesity; DNA damage; genomic instability; somatic cells; germ cells
Contents 1.
Introduction ................................................................................................................... 4
2.
Data collection .............................................................................................................. 7 3
3.
Methods used in investigations concerning associations between genetic instability and overweight/obesity ................................................................................................ 8
4.
Human studies ............................................................................................................... 9 4.1. Studies with overweight/obese individuals ............................................................... 9 4.2. Studies with MetS patients ...................................................................................... 11 4.3. Impact of body weight on DNA damage in human sperm cells .............................. 12 4.4. Comprehensive evaluation of human studies .......................................................... 13 Results of animal studies ............................................................................................ 15
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5.
5.1. DNA damage in somatic cells ................................................................................. 15
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5.2. DNA damage in germ cells...................................................................................... 17 5.3. Comprehensive evaluation of animal studies .......................................................... 18 Impact of obesity/overweight on mutations in oncogenes .......................................... 18
7.
Results of in vitro studies............................................................................................ 19
8.
Molecular mechanisms ............................................................................................... 21
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6.
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8.1. Direct formation of ROS ......................................................................................... 22 8.2. Obesity induced lipid peroxidation and DNA damage ............................................ 25
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8.3. Impact of overweight on sex hormones ................................................................... 26
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8.4. Glycation end products ............................................................................................ 29 8.5. Impact of obesity on DNA repair ............................................................................ 30
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8.6. Obesity and telomere lengths................................................................................... 33 8.7. Impact of obesity/overweight on the activities of enzymes that activate and detoxify mutagens .................................................................................................... 35
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8.8. Inflammation and DNA damage .............................................................................. 37 Consequences of obesity induced DNA damage ........................................................ 38
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10. Conclusions and knowledge gaps ............................................................................... 40 Conflict of interest statement ............................................................................................. 43
Acknowledgments.................................................................................................................. 43
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References .............................................................................................................................. 44
1.
Introduction Obesity and overweight are characterized by abnormal accumulation of body mass and
are associated with increased health risks, in particular with diseases such as type 2 diabetes 4
(T2D), cardiovascular diseases, polycystic ovary syndrome, infertility and cancer [1]. Scientists and health authorities are alarmed by the fact that the increase of overweight/obesity is a worldwide phenomenon. A global estimate indicates that about 640 million adults were obese in 2014, this number corresponds to a 6-fold increase since 1975 [2]. Obesity has a prevalence of 10.8 % among men and 14.9 % among women [3]. The worldwide incidence
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of childhood overweight and obesity increased from 4.2% in 1990 to 6.7% in 2010. This trend is expected to reach 9.1% or 60 million, in 2020 [4].
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A typical feature of excess body weight is the formation of ROS and of cytokines which are characteristic for inflammation. These processes lead to damage of the genetic material
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which in turn plays a key role in the induction of cancer and infertility; these disorders are
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increased in overweight/obese humans [5, 6]. Aim of this article is to describe the current
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state of knowledge of the relation between excess body weight and genomic instability.
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Recently, several studies have been published which concern the association between MetS and DNA damage [7-9], however they are based on mechanistic considerations but not on a
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systematic evaluation of experimental data. The first part of the present paper focuses on the description of results of animal and human studies in which different parameters of DNA
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instability and chromosomal damage were measured in overweight/obese individuals in somatic and germ cells. Furthermore we describe also measurements which were performed
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in these studies concerning biological processes that lead to DNA stability such as inhibition of DNA repair processes, decrease of the telomere lengths, changes of the redox status and of
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the activities of antioxidant enzymes, lipid peroxidation and of the immune status. The association between overweight/obesity and cancer is well documented; it is, apart
from cardiovascular diseases, the most important health consequence of excess body weight. In 2016, the IARC published a report which is based on the evaluation of more than 1000 individual epidemiological studies. The Agency comes to the conclusion that the absence of 5
excess body fatness lower the risks of most cancer [2]. A comprehensive study from the US states that 14% of all cancer cases in man and 20% in women are due to obesity [10]. Numerous investigations showed that gene mutations as well as chromosomal aberrations play a key role in malignant transformation [11, 12]. Nevertheless, it is apparent
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that most studies which concerned the mechanisms by which excess body weight leads to cancer focused on other modes of action such as alterations of signaling pathways, increased
production of growth factors and hormones and activation of transcription factors (for reviews
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see [13-15]).
In the last decades, a variety of methods was developed which enable the investigation
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of DNA stability in human and animal models [16]. These approaches were frequently used
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to study occupational, lifestyle and dietary factors [16-20]. Furthermore, in vitro assays [21]
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and rodent mutagenicity tests [22, 23] are conducted worldwide for routine monitoring of
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chemicals. These models can be also used to identify drugs and dietary components which protect humans against DNA damage and its consequences [16, 24, 25] .
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This review describes human and animal studies which concern the impact of overweight/obesity on DNA integrity. The results of these investigations may contribute to a
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better understanding of the mechanisms by which excess body weight causes adverse health
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consequences such as cancer and infertility and can also contribute to the development of strategies to prevent these effects. We included also investigations concerning the MetS, which is the most important consequence of abdominal obesity. Studies concerning T2D and
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the polycystic ovary syndrome were excluded; these disorders are related to obesity but are characterized by severe additional pathological changes, which may have an impact on DNA stability but are not attributable to excess body weight [26, 27]. The following chapter defines the search strategy (chapter 2) and the methods which were used (chapter 3). The subsequent paragraphs describe investigations with humans 6
(chapter 4) and animals (chapter 5), the impact of obesity on mutations in oncogenes (chapter 6) and in vitro studies (chapter 7). Furthermore we describe the molecular mechanisms by which overweight/obesity may cause DNA damage (chapter 8), the consequence of obesity induced DNA damage and the links between DNA damage and cancer (chapter 9) and define
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knowledge gaps and future research strategies (chapter 10).
2. Data collection
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Medicine,
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The literature search was performed by using the MedLine/PubMed data-base (National http://www.ncbi.nlm.nih.gov/PubMed),
SCOPUS
(Elsevier,
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http://www.scopus.com), Thomson ISI’s Web of Science (Thomson Reuters Corporation,
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http://apps.webofknowl-edge.com), Google Scholar (http://www.google.scholar.-com) and
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covered the period between December 1961 and August 2017.
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Additionally, a manual search of the reference list of studies and of reviews was conducted. References of retrieved articles were analysed to identify further publications
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which may have been missed. Only articles in English were considered. The keywords and combinations of search terms were: body mass index, chromosomal aberrations,
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chromosomal damage, comet assay, DNA damage, DNA adducts, DNA repair, DNA
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instability, γ-H2AX, genomic instability, metabolic syndrome, micronuclei, mutation, obesity, 8-OHdG, 8-oxodG; overweight, single cell gel electrophoresis (SCGE), sister chromatid exchange, sperm DNA integrity, telomere length and TUNEL assay.
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The quality of the individual studies was assessed by a quality score (QS) system, which
is similar to a model which was used for MN- studies with occupationally exposed humans [28]. A detailed description of this system is described in the section “supplementary information”. According to the criteria, the studies were classified and grouped in different
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categories (Low-, Medium- and High- quality). The score and the categories are indicated in the supplementary tables S1 and S2. Exclusion criteria for human studies were as follows (i) an insufficient number of participants (less than 20 individuals), (ii) lack of information concerning the number of
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individuals per group, (iii) lack of information concerning the number of scored cell, (iv) an insufficient number of evaluated cells (<1000 per experimental point in MN studies, in comet
assays <100 cells per experimental point, in TUNEL tests <200 and in SCSA experiments
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<5000 cells per experimental point respectively and (v) small differences in the BMI-values of different study group (≤0.5%). Exclusion criteria for animal studies were (i) an insufficient
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number of animals per group (less than 3) and (ii) an inadequate number of scored cells
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(<1000 per experimental point in MN studies with bone marrow and lymphocytes, in comet
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assays <100 cells per experimental point, in TUNEL tests <200 and in SCSA experiments
Methods used in investigations concerning associations between genetic instability
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3.
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addition of the individual scores).
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<5000 cells per experimental point respectively The overall QS values were determined by
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and overweight/obesity
Figure 1 gives an overview of the methods which are used in studies with overweight/
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obese humans and laboratory animals.
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Table. 1 describes different methods which were used to study impact of overweight/obesity on DNA stability. Most of these approaches are also used to investigate the effect of 8
chemicals, lifestyle factors and chemical exposure in somatic cells, for studies with sperm cells additional test systems such as TUNEL assay and SCS assays were employed.
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The most frequently method in human studies are comet assay experiments with and without restriction enzymes (in total 12 studies were published) followed by measurement of
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8-OHdG (7 studies), MN (8 studies), only two publications concern γ-H2X measurements.
Also in animal studies, comet assays are dominating (8 investigation), followed by 8-
Human studies
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4.
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OHdG or 8-oxodG (4 studies) and by MN and γ-H2X (3 studies per endpoints).
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The following chapters describe results which were obtained with somatic cells of healthy individuals (chapter 4.1) and MetS patients (chapter 4.2), chapter 4.3 summarizes the
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human studies.
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results with sperm cells and the last paragraph contains a comprehensive evaluation of the
4.1. Studies with overweight/obese individuals
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Only relatively few studies (in total 22) were realized which focused on the impact of
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body weight on genomic instability in somatic cells (Table. 2 to 5).
In five out of six comet assay trials with parallel design, increased DNA migration was
seen in lymphocytes of overweight/obese individuals [41-45]. In MN experiments lymphocytes, positive effects were found only in two of five studies with parallel design [47, 50]. Other markers of genomic instability (Nbuds and NBs) which were additionally scored
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in a MN study by Donmez-Altuntas et al. [47] were also increased while no association was found between MN induction and formation of oxidised guanine (8-OHdG) levels in plasma [47]. Another endpoint which was elevated in the study by Scarpato et al. was γ-H2AX [50] Two studies were conducted with buccal cells. Unexpectedly the authors found in Malaysian farmers, which were exposed to pesticides and fertilizers, higher MN-rates in
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individuals with normal BMI compared to participants with higher body weights [51]. A second trial was realized with school children in Mexico and no differences were seen
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between normal, obese and overweight participants [52]. In the latter study a Giemsa based
stain was used which may leads to false positive results [60] and in both trials [51, 52] only
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1000 instead of 2000 cells were scored per participants which is not in agreement with the
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standard protocol [61].
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Table 2. describes also the results of several intervention trials. Clear reduction of DNA
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damage was seen in a study with calorie restriction [38]. No clear effect was seen in an intervention trial in which comparisons were made between consumption of red meat and a
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high carbohydrate diet [46]. A clear difference of the rate of Nbuds was observed at the end of this study but no differences between meat and carbohydrate consumption were detected.
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It is mentioned that the weight loss at the end of intervention was significant but the extent is not specified [46]. The most recent study was conducted with bariatric surgery patients
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(n=56); the authors found no alteration of the comet formation in lymphocytes after 6 months after the surgery, but reduced DNA damage was observed after 1year [39].
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Table 5. concerns findings from experiments in which phosphorylation of H2X (which
is indicative for DSBs) was monitored. Seven studies concerned formation of oxidised guanosin in plasma, whole blood or urine. In three trials a positive association between base oxidation and overweight was found [54, 56, 57], three studies reported an inverse relation [47, 53, 55]. No effect was seen in an Italian study; but clear evidence for formation of FPG
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sensitive sites was detected in the same trial [41]. Two γ-H2AX studies were performed with lymphocytes and significant correlations were found in both trials [50, 58]. The last line of Table 5. summarizes the results of a study concerning associations between telomere lengths and damage of DNA bases before and after calorie restriction. The authors found increased telomere lengths and in parallel reduced formation of abasic sites
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which were monitored with a commercial test kit in rectal biopsy samples. This assay was rarely used and not standardized [59].
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It is notable, that changes which were seen in intervention trials are not necessarily due to reduction of the BMI but may be caused by other factors, i.e. by alterations of the intake of
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vitamins and macronutrients which have an impact on genomic instability [62].
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4.2. Studies with MetS patients
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Table 6. summarizes the results of currently available studies. Five investigations concerned comparisons between overweight MetS patients and healthy individuals. Karaman
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et al. [63] and Demiberg et al. [64] found evidence for increased comet formation in lymphocytes with higher BMI values. Furthermore, also evidence for increased MN
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frequencies and higher levels of malondialdehyde (MDA) and decreased activities of
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antioxidant enzymes was found in one of the studies [63]. On the contrary, no elevated comet formation was seen in a trial which was realized in Croatia [65]; the authors hypothesized that the lack of an effect may be due to the fact that the study was performed with young
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participants who had a high repair capacity. The most recent study concerned DNA damage in morbidly obese women [45]. The
authors found no evidence for increased DNA damage in lymphocytes, also oxidised purines (FPG-sensitive lesions) were not increased. Interestingly, the number of individuals with gene
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polymorphism in the fat mass and obesity associated gene (AA-FTO) was higher in the obese group, while another polymorphism in the same gene (TT-FTO) was found less frequently. Results of 8-OHdG measurements were performed in two MetS studies. Abdilla and co-workers [66] monitored several endpoints in different MetS groups and in healthy controls and detected no differences while Milić et al. [65] found even lower levels of 8-OHdG in the
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urine of patients compared to the controls; they hypothesized that this unexpected result may
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be due an adaptive response.
4.3. Impact of body weight on DNA damage in human sperm cells
The findings of different studies are listed in Table 7. The results of comet experiments
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are controversial. No effects were seen in a trial in which the neutral version (which detects
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mainly DSBs and cross-links) was used [67], while clear differences were found in a study
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from Brazil with the standard protocol i.e. under alkaline conditions which detect DSBs, SSBs
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and apurinic sites [68].
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The results of TUNEL assays indicate that differences exist between obese and normal weight individuals while no effects were found between overweight and lean participants [69,
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70]. Vignera et al. [69] analysed additional parameters and found a higher rate of sperm cells
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with increased chromatin compactness in the former group. The authors postulated that this is a sign of early uncontrolled apoptosis. This assumption was supported by the observation of increased externalization of phosphatidylserine and reduced mitochondrial membrane
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potential as a consequence of elevated body weight. These processes lead to programmed cell death and DNA fragmentation [78]. Interestingly, an increase of the DFI by 50% was observed in obese but not in overweight individuals in a large study (n=330) by Dupont et al. [70].
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The results of 7 studies in which the effects of body weight were analysed in SCSA assays are controversial. The findings of Bandel et al. [79] are unexpected as the authors found an inverse association between BMI values and sperm DNA integrity, also the motility of the cells increased with the body weight. No such effects were found in several other trials [71, 73-76].
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Two further studies are notable; one is an intervention trial in which the DFI of the
participants was compared after a weight loss period of 14 weeks in three groups. Several
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sperm parameters were improved after weight reduction, also the DFI values were lower at
the end of the trial. The strongest reduction of this parameter (from 18 to 10) was found in the
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group with the highest BMI values (46.1-60.9), however the effect did not reach significance
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[71].
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An interesting study from Denmark concerned the impact of the BMI of mothers on the
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DFI in sperm of the sons. Higher values were found in the offspring of overweight mothers compared to mothers with normal weight [72].
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4.4. Comprehensive evaluation of human studies
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The main methods which were used in human studies are MN assays and the comet experiments. It is notable that MN reflect persisting chromosomal aberration, while “comets”
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are caused by SSBs and DSBs and apurinic sites and may disappear as a consequence of repair processes. For MN studies a standard protocol is available and MN can be easily identified [80]. As described above, MN experiments yielded conflicting findings, only 3 out of 8
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investigations found positive correlations with the BMI. In comet experiments different parameters (tail intensity (synonymous with % DNA in tail), tail moment and olive tail moment) can be measured by automated scoring systems while other authors use “manual scoring” and determine DNA migration by definition of arbitrary units. All these parameters were found acceptable, however it was emphasised by various expert groups that tail intensity 13
(%DNA in tail) is the most reliable one [23, 81, 82]. While studies which used this marker yielded strongly controversial results (6 negative, 9 positive) consistently positive findings were obtained in all manual scoring experiments which may be indicative for a biased evaluation.
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Also the results of studies which concerned the induction of DNA damage in sperm are conflicting. It is difficult to assess which endpoints are the most reliable ones (SCSA, TUNEL assay and comet assay). It was emphasized that the different techniques reflect different
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aspects i.e. the TUNEL assay and the comet assay are indicative for “real” damage while the
SCSA reflects the susceptibility of the DNA to denaturation [83]. Only for the later assay a
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standardized protocol is available [84].
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In the case of experiments which concern the quantification of the oxidation of DNA
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bases not only differences of antioxidants in the diet but also methodological aspects may
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play a role. It was emphasize that HPLC measurements are more reliable as compared to
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ELISA based measurements [85]. Furthermore, 8-oxodG and oxoGuo were monitored either in urine, plasma or in different target organs which may add to heterogeneity of the results.
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The calculation of the QS shows that the quality of the most studies is in a relatively
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narrow range, therefore it is not possible to explain the conflicting results by shortcomings of the study design of the individual studies. As described in more detail in chapter 10 none of the investigations which were published so far was controlled in regard to the mineral and
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vitamin intake of the participants which may have a strong impact on the results. It is conceivable that the composition of the foods in lean and overweight human has a strong impact on the outcome of the studies, also weight loss programs are based on substantial changes of the dietary patents.
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5. Results of animal studies 5.1. DNA damage in somatic cells Different models were used in experiments with laboratory rodents. In most trials, the
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animals were fed with a high fat diet (HFD) or a cafeteria diet (CAF) for several weeks which
lead to an increase of the body fat and also to excess body weight. These studies were mainly
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conducted with C57BL/6j mice; these animals develop symptoms which are characteristic for
obese humans namely hyperinsulinemia, hyperglycemia, and hypertension [86]. In some
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studies Zucker rats were used; obesity is in these animals an autosomal recessive trait and
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resembles the early onset of human obesity [87] i.e. the animals have hyperlipidaemia,
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hyperinsulinemia and peripheral insulin resistance. In one study, WNIN/Ob rats were used
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which are hyperglycaemic and insulin–resistant and reflect the metabolic syndrome in humans [88].
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The first paper which described chromosomal aberrations as a consequence of obesity was published by the Swedish scientist Karl Fredga [89] more than 50 years ago. He observed
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in a pilot study (2 animals per group) that a specific strain of hyperglycaemic mice had
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aberrant numbers of chromosomes in epithelial cells from the cornea. Our literature search identified 18 further studies which concerned the effects of
overweight on DNA stability in somatic cells (Table 8. to 11.). Results of all comet trials
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(n=8) indicate that excess body fat causes genomic instability in a variety of inner organs [88, 90-96]. Tenorio and co-workers [94] analysed DNA migration in different tissues of rats and found larger comets in liver and lymphocytes but not in kidneys, brain and heart. Also in a Brazilian study [96] clear evidence for increased DNA damage was seen in obese mice which were fed with a carbohydrate and fat rich cafeteria diet (consisting of various supermarket
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products). The consumption of this chow induced glucose intolerance and DNA damage in lymphocytes, kidneys, liver and in the brain [96]. A positive result was also obtained in MN experiments with bone marrow cells in the same study [96]. Also in HFD fed mice induction of DNA–migration was detected in liver and colon in a number of studies [90-93]. Remely et al. [90, 91] found an effect in both organs; Setayesh et al. [92] detected additionally increased
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damage in brain, liver and colons while no significant induction of DNA migration was
observed in blood cells and adipose tissue. Gutzkow et al. [93], analysed in a more recent
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study the extent of damage in different cell types (liver, blood and testes) of HFD fed mice.
They found in standard comet assays only in the liver an effect; furthermore an increase of
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formation of oxidised purines was detected in hepatocytes and also in white blood cells. Table
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8. describes the results of a study with genetically modified rats, which are a model for the
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MetS. Clear evidence for increased damage was observed in different regions of the brain.
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The authors state that the effects which they observed in 3-months old obese rats are similar to those seen in 15-months old parental control animals and conclude that obesity leads to
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premature ageing [88].
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Also in γ-H2AX measurements which are indicative for DSBs, clear effects were seen in livers and colons of obese animals [101]. Furthermore, alterations of the urinary excretion
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of 8-oxodG were monitored in the same trial. All these markers of DNA damage were decreased as a consequence of weight loss after gastric bypass surgery [101]. Another γ-
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H2AX study was published with obese Zucker rats; the authors found clear induction of DSBs in the lungs of the animals when they conducted the measurements after intraperitoneal injection of glucose, which leads to hyperglycemia, but not in fasting animals [105]. A further γ-H2AX study concerned the relation between DSB induction in ovaries and alterations of DNA repair proteins [106]. Also in a study with obese rats in which 5-hydroxymethyluracil
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was measured in the livers and in the mammary glands, a protective effect was detected after calorie restriction [107]. Most studies, which are available so far were carried out with nuclear DNA; only few investigations concerned on mitochondrial DNA [103, 104, 108]. In one of them, the authors measured damage in liver and skeletal muscles of mice and detected pronounced differences
in liver, heart, kidney and testes of HFD mice were detected [103].
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between obese and normal weight animals [108]. In another study, higher levels of 8-oxodG
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Table 10. describes also findings of a study in which obese and lean rats were treated
with the breast carcinogen 7,12-dimethylbenz(α)anthracene (DMBA). The authors found in
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obese animals increased oxidative damage (8-OH-guanosine) in the liver and in parallel an
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altered ratio of reduced to glutathione/oxidised glutathione disulphide (GSH/GSSG), which
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is indicative for oxidative stress. This effect was was paralleled by changes of the overall
DNA damage in germ cells
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5.2.
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DNA methylation status [104].
We found in total five animal studies which concerned the effects of obesity on sperm
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integrity. In four of them clear associations were observed, regardless if the damage was
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quantified in comet assay, TUNEL assays or SCSA experiments [93, 109-112]. Only in one study, the authors did not find an increase of DNA damage in testes of HFD mice; it is notable that the differences of the body weights of the experimental groups were only marginal in this
A
study [93].
17
5.3.
Comprehensive evaluation of animal studies In contrast to the conflicting findings which were obtained in the human studies in
lymphocytes and sperm, consistent results were found in animal studies with rodents, i.e. most of the comet experiments which have been published so far with somatic and sperm cells yielded positive results. The reasons for the unequivocal results of experiments with rodents
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are probably due the use of indicator organisms with a similar genetic background and feeding
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of standardized diets.
In contrast to the findings of comet assay studies, several MN experiments with cells from the haematopoietic system failed to detect an association between DNA damage and
U
increased body weight. The reason for the discrepancy to the comet results may be differences
N
in the sensitivity of the tissues which were analyzed in comet assay and MN experiments and
A
not differences in the sensitivity of the endpoints. Sasaki et al. [114] stressed that 54 out of
M
165 proven rodent carcinogens were not detected in MN experiments with haematopoietic
ED
cells and that 49 of these chemicals (which gave false negative results) were positive in comet
Impact of obesity/overweight on mutations in oncogenes
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6.
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assays in at least one or more organs in mice in comet experiments.
Only few studies concerned mutations in cancer associated genes of laboratory rodents
and humans. These investigations are important as they enable to establish direct causal links
A
between overweight induced genomic instability and malignant transformation. Shen et al. [115] found in whole genome sequencing experiments with tissue from
hepatocellular carcinomas of obese mice increased rates of mutations in the carboxyl ester lipase (Cel) gene and in the Harvey rat sarcoma virus oncogene 1 (Hras). In Cel, multiple mutations were detected which lead to functional loss, while in Hras single mutations were 18
found in a specific codon (61). Mutant Cel causes accumulation of cholesteryl ester in liver cells which leads to induction of endoplasmic reticulum stress and consequently activates IRE1 α/c-Jun N-terminal kinase (JNK)/c-Jun/activating protein -1 (AP-1) signalling cascade to promote liver cell growth. In the case of Hras, gain of function mutations promote liver cell growth through activation of mitogen-activated protein kinase (MAPK) and
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phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt pathways [116].
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A study on Kras mutations in colon cancer found in colon tissue of obese and lean
patients differences in the mutation rates in several genes [117]. The most striking observation was a lower number of mutations in patients with high BMI. The authors hypothesised that
N
U
the reduced number found in “driver genes” of overweight individuals is due to the fact that mutations in genes which lead to malignant cells are not required in obese patients as in this
M
A
case “other alterations of signalling pathways” may substitute them [117]. The impact of overweight on tumor suppressor gene p53 mutations was analysed by
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Ecke et al. [118] in bladder cancer patients and no impact of the BMI on the mutation
Results of in vitro studies
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7.
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frequencies was detected.
It is not possible to investigate biochemical changes which are caused by excessive
calorie intake and elevated body weight under in vitro conditions. Nevertheless, results of
A
studies were published with mammalian and human cells which reflect physiological conditions that are characteristic for obese individuals such as increased concentrations of glucose, fatty acids and insulin. These findings contribute to a better understanding of the mechanisms by which overweight/obesity lead to the damage of the genetic material.
19
Cavazos et al. [119] exposed human prostate cells to serum from obese and non-obese mice. They monitored induction of DSBs in γ-H2AX experiments and found increased damage in malignant and non-malignant cells with sera from overweight animals. This effect correlated with ROS formation; furthermore, several markers of aerobic glycolysis were induced. The authors found that a potent antioxidant (N-tert-butylhydroxylamine) prevents
involved in aerobic glycolysis) and formation of γ-H2AX foci [119].
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induction of hypoxia-inducible factor 1-α (a transcriptional regulator of genes which is
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The effects of insulin were investigated in several studies by Othman et al. [120-122]. They observed induction of DNA damage in colon derived cells (HT29) and also in
U
lymphocytes in MN and comet experiments and explained these effects by intracellular ROS
N
formation [120, 122]. Also with primary kidney cells, positive results were obtained.
A
Subsequent in vivo studies with rats confirmed the assumption that insulin causes ROS
M
formation [120, 121]. Results of a further experiment in which the authors investigated the mechanisms of insulin induced DNA damage are described in chapter 7.
ED
Several investigations concerned the impact of glucose, which is increased in the blood of diabetic patients, but also as a consequence of consumption of carbohydrate and sugar rich
PT
diets. Lorenzi et al. [123] studied the effects of glucose on induction of DNA damage with a fluorometric technique which detects the unwinding of DNA in endothelial cells from
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umbilical veins and human fibroblasts already 30 years ago. They found positive results in the former cell type; furthermore they detected also increased hydroxyurea incorporation of
A
radio-labelled thymidine which is indicative for DNA repair. Quagliaro et al. [124] used human umbilical vein endothelial cells (HUVEC) and found evidence for formation of 8OHdG as well as increased concentrations of nitrotyrosine (an indicator of NO· formation) and apoptosis as a result of high glucose levels [124]. The authors postulated that the oxidative stress is caused by protein kinase C (PKC) dependent activation of NAD(P)H oxidase. This
20
mechanism was also proposed by a Japanese group which worked with aortic smooth muscle and endothelial cells [125]. The findings of the latter studies are in contrast to results of Shimoi et al. [126], they found in HUVEC induction of oxidised purines but failed to explain these effects by formation of ROS formation and postulated that this phenomenon is caused by glyoxal.
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Increased levels of fatty acids (FA) are typical for obese and diabetic individuals.It was reported by Beeharry et al. [127] that palmitic acid (a major saturated FA in plasma) causes
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comet formation in an insulin producing cell line and also in fibroblasts. This effect was
inhibited by linoleic acid and the authors postulated that the protection is due to prevention of
U
oxidative stress. The assumption of ROS formation from palmitate was confirmed by
N
Inoguchi et al. [125] who found in electron spin resonance spectroscopy experiments evidence
8.
Molecular mechanisms
M
A
for the involvement of PKC.
ED
The following paragraphs concern mechanisms which may play a role in the association between excess body weight and genomic instability (Figure 2). Apart from enzymatic
PT
processes which lead to formation of ROS also other modes of action could be involved. Lipid
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peroxidation (LP) processes are induced by ROS and lead to formation of DNA reactive products. Furthermore, inhibition of DNA repair and induction of inflammation have been found in a number of studies. Also formation of advanced glycation products, hormonal
A
effects and telomere shortening are phenomena which may lead to genomic instability.
Figure 2
21
8.1.
Direct formation of ROS The evaluation of human and animal studies (Tables 2-5 and 8-11) shows that in some
of them evidence for induction of oxidative DNA damage and/or decreased total antioxidant capacity and induction of antioxidant enzymes was observed [41, 53, 54, 56, 57, 63, 101, 103, 108]; also increased LP by ROS was found in a number of investigations (see chapter 8.2).
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The results of Othman et al. [120] concerning the effects of insulin are of particular
interest as the authors measured in their experiments also genetic damage in comet assays. By
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use of specific inhibitors they developed a model for the induction of DNA migration in colon and kidney cells (Figure. 4). Insulin binds either to its receptor (IR) or to the insulin-like growth
factor-1 (IGF-1) receptor. After internalization of the receptor-insulin-complex into the cells,
U
the hormone is digested by proteases. Binding to both types of receptors leads to activation
N
of PI3K. Results with a specific PI3K inhibitor showed that this enzyme plays a key role in
A
signalling and also in the induction of genomic damage by insulin. The subsequent step is the
M
activation of Akt by PI3K which translocates from the membrane to the nucleus where it
ED
phosphorylates a wide array of downstream proteins.
PT
Figure 3
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The reduction of PI3K/Akt levels which was seen few hours after insulin exposure was
explained as a consequence of activation of p53 which controls the phosphatase and tensin
A
homolog (PTEN), a deactivator of Akt and PI3K. Two pathways lead subsequently to ROS formation: (i) insulin enhances the translocation of Akt into mitochondria where it causes ROS formation; (ii) another pathway is the activation of NADPH-oxidase. Different isoforms of this enzyme are involved in specific cell types. While NADPH oxidase 1 (NOX1) was found to be activated in colon cells, NOX4 is active in the kidney.
22
In this context it is notable that is known that cancer cells produce their energy by increasing the rate of anaerobic glycolysis (Warburg-Effect), therefore it is possible that elevated glucose levels may accelerate the proliferation of transformed cells [128]. This phenomenon has been demonstrated under in vitro conditions, and it is notable in this context that lower blood glucose levels in cancer patients correlated with better outcomes [129].
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Increased glucose levels in blood lead to ROS formation via different pathways. (i) In
cultured vasoendothelial cells, radical formation was observed via PKC-dependent activation
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of NADPH-oxidase (Figure 4) [124]. (ii) Glycoxidation products are formed as a consequence
of sequential glycation and oxidation reactions between proteins and reducing sugars [130]. (iii) Furthermore, an alteration of the ratio of NADH/NAD+ was found in hyperglycaemic
U
tissues. This condition is also termed “pseudohypoxia” as the tissue levels of oxygen are
N
normal and is characterized by increased production of O2· and NO·[131]. (iv) Finally,
M
A
intracellular oxidation processes may cause O2· formation in mitochondria. This process starts with glycolysis in the cytoplasm which generates NADH and pyruvate. Cytoplasmic NADH
ED
can donate reducing equivalents to the transport chain in the mitochondria. Pyruvate is reduced in the cytoplasm to lactate or migrates into the mitochondria. Two main sites in these
PT
organelles generate O2· namely the NADH-dehydrogenase complex I and the interface
A
CC E
between ubiquinone and complex III [132].
Figure 4
The assumption of a relation between insulin levels and DNA damage is supported by
the results of a calorie restriction study; the authors found lower hormone levels and in parallel less DNA damage at the end of the intervention phase. Notably, also the blood glucose levels were reduced at the end of the trial [38]. Also in a Korean study associations were detected
23
between insulin and glucose levels in blood and comet formation [42]. Furthermore, it is notable that correlations between DNA damage in sperm and insulin and glucose levels were found in the blood of mice [111]. It is well known that lipid levels are elevated as a consequence of excess consumption of fat and in metabolic diseases [133]. In this context, it is notable that palmitate, which is the
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most abundant fatty acid in plasma was shown to increase the activity of PKC and to cause ROS production [125].
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Only few studies measured ROS formation in obese and lean individuals and monitored its relation to DNA damage. Demirbag et al. [64] found reduced total antioxidant capacity in
U
a study with MetS patients and in parallel increased comet formation in lymphocytes. Higher
N
mitochondrial ROS levels in sperm cells were found in two studies (i.e. in a human
A
intervention trial and in an experiment with mice); in both, elevated DNA damage was
M
detected [110, 111].
Also investigations which concerned the reduction of the activities of antioxidant
ED
enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-Stransferase (GST)) and of oxidised glutathione as a consequence of excess body weight
PT
provide evidence for oxidative stress. Such effects were seen in a number of studies with humans [55, 63, 88, 108]. Also in the brain activities of SOD and catalase (CAT) were
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decreased in a rat model which reflects the MetS in humans; in parallel higher MDA levels were found. These effects correlated with induction of DNA damage in comet experiments
A
[88].
In a study with 7,12-dimethylbenzo anthracene (DMBA; a polycyclic aromatic
hydrocarbon which causes breast cancer) reduction of the ratio of oxidised/reduced glutathione (GSH/GSSG) which is indicative for oxidative stress was observed in obese rats. This effect was paralleled by increased 8-OHdG levels [104].
24
Another marker of oxidative stress are protein carbonyl concentrations in serum [134], no alterations in this marker were seen in a 6-months calorie restriction study with overweight individuals [38].
8.2. Obesity induced lipid peroxidation and DNA damage
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Oxidative stress leads to oxidation of fatty acids in biological membranes; as a consequence of this chain reaction, lipid peroxides and alkoxyl radicals as well as aldehydes
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and ketones are formed [135]. Some of these molecules attack DNA and were postulated to
play a key role in the aetiology of cancer and other diseases [136]. The formation of DNA adducts by LP products for example by MDA, acrolein, 4-hydroxy-2-nonenal (4-HNE) and
U
other compounds are described in recent reviews [137, 138]. Elevated levels of different LP
N
markers were repeatedly seen in blood and muscles and also in adipose tissue of obese
M
A
individuals [42, 56, 63, 88, 95, 101, 103, 139-143].
The most commonly monitored biomarkers which are used in LP studies are MDA,
ED
thiobarbituric reactive substances (TBARs), hydroperoxides, 4-HNE, isoprostanes and
PT
conjugated dienes [134]. We found only few publications in which LP markers and DNA stability were monitored in parallel in obese/overweight and in normal weight subjects.
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Karaman et al. [63] conducted such a study in Turkey; they found increased MDA levels in individuals with MetS and also higher MN rates in peripheral lymphocytes. Also in experiments with obese rats evidence for an association between MDA formation and DNA
A
damage was observed [95]. In a study from Poland, the serum levels of MDA and 4hydroxyalkenals were measured as an index for LP, in parallel 8-oxodG concentrations were determined in blood [56]. The authors found that both parameters are associated with the BMI and with hip circumference. Furthermore, correlations between LP and 8-oxodG were observed. 25
Notably, also a clear relation between isoprostane (8-epi-prostaglandin F2α) concentrations in urine and MDA in plasma (which are both markers for LP) and comet formation in lymphocytes was seen in a study which focused on the effect of visceral body fat in non-obese Korean men [42]. Yu et al. [103] compared the association between MDA in different organs of rats which
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received a HFD and formation of oxidised DNA (8-OHdG) in mitochondria. The LP product was increased in heart, kidney, testes and liver tissue after 10 weeks but in the hepatic tissue
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even a decrease was observed after 8 weeks.
A large number of studies focused exclusively on the impact of obesity on LP markers
U
but not on genomic stability. In total, about twenty papers have been published and in most
N
of them evidence for increased LP as a consequence of excess body weight was found (for
A
review see [139]).
M
Also mitochondrial membranes are attacked by ROS in overweight individuals and in MetS patients. This process leads to alterations of proteins which are involved in the electron
ED
transport chain. As a consequence, increased release of ROS formation was postulated [144].
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8.3. Impact of overweight on sex hormones
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It is well documented that excess body weight causes hormonal imbalance. One of the enzymes which plays a key role is aromatase which catalyses the conversion of androgens to estrogens [145]. Ovaries are the main site of estrogen production in pre-menopausal women,
A
but peripheral tissues including body fat become the primary source in overweight postmenopausal individuals [15, 146, 147]. Cauley et al. [148] showed that estriol and estradiol levels are increased by more than 40% in obese post-menopausal women. Also in obese prepubertal girls, elevated levels of these hormones were found [149]. This effect was
26
explained by enhanced production of aromatase as a consequence of secretion of TNF-alpha and IL-6 adipocytes. Apart from enhanced production of humans also decreased levels of serum hormonebinding globulin were found in obese individuals [150]. This glycoprotein binds estrogens
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and testosterones and inhibits their activities. Alterations of the hormonal status were proposed to play a key role in the induction of breast and endometrial cancer which have an increased prevalence in post-menopausal obese
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women [151-154]. Furthermore, it was also found that excess body weight in children increases the overall cancer death rates as well as breast cancer mortality [155].
U
Receptor-dependent as well as independent mechanisms are involved in estrogen
N
induced breast carcinogenesis [156]. The hormones enhance the proliferation of breast cells
A
by binding to specific receptors. As a consequence of increased mitotic activity, errors in
M
DNA-replication are induced, the repair of mutations is less efficient and DNA adducts,
ED
breaks and other lesions are more frequently converted to persisting mutations [157]; furthermore, cell divisions cause mitotic recombinations which lead to errors that are passed
PT
to the daughter cells [157].
It is well documented that certain estrogen metabolites cause direct DNA damage.
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Estradiol (17-βE2) is converted to catechol metabolites which are hydroxylated at the 2- or 4positions to form 2-hydroxyestrogen (2-OHE) and 4-hydroxyestrogen (4-OHE). The latter
A
compound is mutagenic in rat and human cells; i.e. it is converted to E(2)-3,4-quinone which forms covalent estrogen-DNA-adducts [158]. Additionally, E(2)3-4 quinoline can undergo redox cycling with E(2)3-4 hydroquinone leading to release of ROS. Quinones are backconverted via quinone reductase to create a cycle which leads to continuous release of radicals and also to formation of unstable depurination products with adenine and guanine [159].
27
Estrone undergoes a similar metabolism as estradiol but can be also converted to 16αhydroxyestrone (16α-OHE1) which binds covalently to DNA and enhances the proliferation of cells via binding to the ER [155, 160]. The genotoxic effects of estrogens in human derived breast cancer cells are well studied.
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For example, Mobley and co-workers [161-163] found evidence for induction of oxidative damage under conditions which are relevant for humans. Comparisons of effects in cells with
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and without ERs indicate that their presence increases the extent of oxidative damage.
The associations between formation of genotoxic estrogen metabolites and breast cancer have been investigated in a number of studies. It was shown that E(2)3-4 quinoline
U
causes cancer in animals [160, 164-168]. However, it is not clear, if the concentrations of
N
DNA reactive estrogen metabolites are indeed increased in obese individuals. Mauras et al.
A
[149] found elevated concentrations of 16α-OHE1 in a small study in sera of obese
M
prepubertal girls (12 lean, 24 obese individuals) while no relation between the 4- and the 16α-
ED
OHE1 pathway and BMI values was found in a larger study (n=603) with post-menopausal women [169].
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Hormonal effects are also involved in the development of endometrial tumors [170]. Apart from enhanced cell proliferation, alterations of signalling pathways may play a role.
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However, at present experimental evidence is lacking that genomic instability is enhanced in the endometrium of obese women and further studies are required to assess the impact of
A
overweight induced genomic instability on this form of cancer.
28
8.4. Glycation end products Advanced glycation end products (AGEs) are formed as a consequence of reactions between sugar moieties and proteins; their biological effects are described in a number of reviews [171-173].
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The endogenous formation of these products in the human body takes place nonenzymatically via the Maillard reaction but also via the so called “polyol pathway”, a reaction
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which is catalysed by sorbitol dehydrogenase (SDH) and phosphokinase. Reactive αoxoaldehydes which are formed during these reactions (glyoxal, methylglyoxal and 3deoxyglucosone) are converted irreversibly by further reactions to products such as Nε-
U
carboxyethyllysine, arginine pyrimidine, pentosidine, pyrralin, methylglyoxal, glyoxal lysine
N
and Nε-carboxymethyllysine (CML) [171].
A
AGEs bind in the human body to specific receptors (receptor advanced glycation end
M
products (RAGE)) which are involved in different signalling pathways; the most relevant ones
ED
are shown in Figure 5. These interactions cause formation of ROS via activation of NADH-
Figure 5
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PT
oxidase which leads to activation of various transcription factors including NF-κB [174].
A
Consumption of carbohydrate and sugar rich Western diets causes increased formation
of AGEs; also excess body fat may lead to elevated concentrations in specific tissues. Some findings indicate that the serum levels of CML are inversely related to the BMI and also to body fat, but enhanced levels were found in adipose tissue [175-178]. A different observation was made with sera from the MetS patients, i.e. the concentrations of AGEs were elevated in
29
these individuals and the concentrations correlated with the levels of markers which are indicative for insulin resistance and inflammation [179]. Also in experiments with HFD fed mice it was found that the concentrations of AGEs are increased in adipose tissue and in the liver [180].
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AGEs cause DNA damage directly and via interactions with signalling pathways. It is well documented that methylglyoxal causes gene mutations in bacterial test [181, 182] and also in yeast [182]. Furthermore, it was shown that CML accumulates in nuclear proteins and
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causes DNA strand breaks in human skin cells [183].
Stopper and co-workers [184] studied the effects of methylglyoxal and CML in comet
U
assays with different cell types. They found induction of DNA damage and postulated on the
N
basis of results obtained with inhibitors that formation of ROS as a consequence of the
A
activation of the transcription factor NF-κB plays a key role (Figure 5). One of the proteins
M
which is regulated by NF-κB is angiotensin which is vasoconstrictive and causes down
ED
regulation of peroxisomes. The authors observed pronounced reduction of DNA damage when they added a specific receptor antagonist (candesartan) to the incubation mix [184, 185].
PT
Apart from formation of AGEs, sugars undergo also direct reactions with DNA-bases [186]. The products cause depurination [187] as well as SSBs and mutations in vitro [188]. In
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recent years, improved techniques (for example LC-MS and HPLC measurements) as well as antibodies have been developed which enable to quantify these products in humans [189-191]
A
but it is not known if increased concentrations are present in obese individuals.
8.5. Impact of obesity on DNA repair Overweight has an impact on different DNA repair pathways (Figure 6), but the tissue specificity and the underlying molecular mechanisms are only partly understood.
30
Recently, results of a study with young adults were published in which the authors found an association between the BMI and NER in a modified comet experiments with lymphocytes [192]. Langie and co-workers [102] performed interesting experiments with mice in which the mothers received a low folate diet and the offspring a HFD. They found reduction of BER in several brain regions of the progeny. Furthermore, they analysed the impact of this feeding
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is involved in repair of oxidised DNA bases in the hippocampus [102].
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scheme on the expression of BER related repair genes and observed alterations of Ogg1 which
U
Figure 6
N
It is well documented that defects of BER and NER are associated with increased cancer
A
rates. Single nucleotide polymorphisms in genes that encode for proteins involved in these
M
pathways were found to cause reduced activities of repair enzymes and to increase the cancer
ED
risks in humans [193]. Furthermore, it is known that defective NER in Xeroderma pigmentosum patients leads to skin cancer and glioblastomas [194, 195].
PT
It is notable that also other repair pathways are affected by oxidation of the DNA [196198] and by LP products [199, 200]. A study with transgenic overweight mice concerned the
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induction of DSBs, the synthesis of DNA repair proteins and transcription of repair associated genes after administration of phosphoramide mustard. The authors found clear evidence for
A
induction of DNA damage and activation of certain proteins (PRKDC and XRCC6) as well as increased levels of the ATM protein in ovaries of obese animals [106]. A number of investigation concerned the impact of obesity on the methylation status of genes which are directly or indirectly involved in DNA repair. The MutL homolog 1 (MLH1) is one of seven proteins which play a role in DNA mismatch repair (MMR) [201]. Remely 31
et al. [91, 98] found an inverse association between DNA damage in liver tissue of obese mice (which was monitored in comet experiments) and alterations of the methylation status of MLH1 in CpG1 in the same organ [91, 98]; on the contrary a positive association of the methylation of the same gene was seen in the liver in CpG4 [98]. In colon mucosa cells an inverse relation was seen with this gene in CpG2. These effects were reversed by
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supplementation of the drinking water with the green tea constituent, EGCG and also with vitamin E. [91] On the contrary, no relation between BMI and MLH-1 methylation was
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detected in a human study (n=128) with biopsies from endometrial tumors [202].
DNA methyltransferases (DNMTs) are responsible for the transfer of methyl groups
U
from the universal methyl donor S-adenosyl-L-methionine (SAM) to the 5-position of
N
cytosine residues in DNA. One of the members of this group, DNMT1 encodes for the
A
maintenance methyltransferase and plays a key role in MMR [201]. Remely et al. [91, 98],
M
found in the aforementioned studies no significant differences in the CpG methylation status in the promoter region of DNMT1 in colon and liver tissue of obese HFD mice.
ED
Hypomethylation of this gene leads to increased transcription of genes which are involved in
PT
MMR.
A recent study with lymphocytes of obese and normal weight adolescents indicated that
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the capacity mitomycin C induced DNA repair is lower in the latter groups [58]. The authors measured in this investigation the time kinetics of disappearance of γ-H2AX foci in
A
lymphocytes as a parameter of the repair capacity. A further protein which may play an important role in obesity associated repair is p53.
The activation of this tumor suppressor protein as a consequence of excess body weight is well documented [7]. One of its functions is the inhibition of cell divisions which prolongs the repair phase [7]. It is known that p53 is activated by different types of DNA damage,
32
including oxidation of bases [7, 203]. Apart from beneficial effects of p53, newer findings indicate that its activation in adipocytes contributes to the development of insulin resistance [203, 204]. The impact of obesity on sex hormones is described in chapter 8.3. It was emphasized
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that ER signalling leads to suppression of DNA repair and apoptosis in favour of cell proliferation [205]. The most important processes which are influenced by steroid hormones concern the regulation of DSB repair, non-homologous end-joining and homologous
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recombination [206]. Most data come from in vitro experiments and it is not known at present
to which extent obesity induced alterations of the hormonal status have an impact on repair
U
function in humans.
N
Also telomeres have an impact on DNA repair; the impact of increased BMI on their
M
A
length is described in the following chapter.
ED
8.6. Obesity and telomere lengths
Telomeres are DNA sequences (tandem repeats of the sequence TTAGGG) which are
PT
localized at the end of chromosomes [207]. It was shown in vitro that shortening leads to
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cellular senescence and that overexpression of the enzyme telomerase (which maintains the levels of telomeres) leads to immortalization [208]. Also results of animal experiments
A
support the assumption of the involvement of these structures in senescence and ageing [209]. The impact of obesity on these structures may be an important factor which affects DNA
stability. The mechanisms which cause this phenomenon are partly known [210]. One of the modes of action which may be involved is the dependency of DSB repair on telomeric regions. Chromosomal fusion in mammalian cells involves homologues end joining which is one of the major forms of DSB repair. Chromosome fusion initiates chromosome instability 33
involving breakage fusion cycles in which dicentric chromosomes form bridges and breaks when the cells attempt to divide [211]. One of the mechanisms which could lead to telomere shortening in overweight/obese individuals is oxidative stress as it was shown in in vitro that telomeres are highly sensitive to damage by ROS [212, 213].
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Only one intervention study has been conducted which concerned the correlation between alterations of telomere lengths and DNA base damage in overweight humans [59]. The authors monitored alterations of these parameters after weight loss (for details see Table
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5) and found an increase of telomere lengths and in parallel reduction of apurinic/apyrimidinic
sites. In another study with a parallel design, no connections were detected between obesity
U
and telomere length and DNA damage [40].
N
A large number of studies was published which concerned the effects of body weight
A
on telomeres. The results are reviewed in two papers, which contain meta-analyses [214, 215].
M
In the first, the authors evaluated 16 studies. Only in cross-sectional investigations (n=14), a
ED
inverse relation was observed [214]. A second analysis [215] comprised 63 studies (119.439 subjects); in 39 an inverse correlation between obesity and telomere lengths was observed.
PT
The authors found in a pooled analysis a trend for an inverse association. However, they emphasize the need for further longitudinal studies and stress that the assumption of a
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relationship between obesity and telomere lengths remains open due to the heterogeneity of the results.
A
Several studies indicate that the relation between telomere lengths and adiposity is more
pronounced in younger subjects; this observation was also confirmed in a meta-analysis of Müezzinler [214]. Another aspect which was addressed in a study by Martens et al. [216] concerns the impact of the maternal BMI on telomere integrity in new-borns. The authors found evidence for shorter telomeres in babies of overweight/obese mothers.
34
The impact of weight loss on telomere lengths was investigated in several trials; overall the findings are controversial. Moreno-Navarrete et al. [217] concluded that shorter telomere length in obese subjects is an established irreversible feature which cannot be improved by weight loss while O’Callaghan et al. [59] state that weight loss as a consequence of consumption of a calorie restricted diet prevents telomere shortening. Also results of studies
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with bariatric surgery patients are inconsistent. No effect was seen in a trial with 97 subjects
[218] one year after the operation, while an increase was found in a 10 year prospective study
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[219].
N
U
8.7. Impact of obesity/overweight on the activities of enzymes that activate and detoxify mutagens
A
Many dietary and environmental carcinogens such as polycyclic aromatic
M
hydrocarbons, heterocyclic aromatic amines, aflatoxins and nitrosamines are converted in the body to DNA reactive metabolites by specific phase I enzymes and partly also by phase II
ED
enzymes [220]. The latter ones catalyse also the detoxification of DNA reactive intermediates and of directly active compounds [221]. A number of human and animal studies addressed
PT
the question if obesity affects the activities of these enzymes. In experiments with humans,
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the clearance rates of enzyme specific drugs were studied with chemical analytical methods while in animal models mainly hepatic enzyme activities were monitored. A comprehensive review of human studies was published by Brill et al. [222]; the
A
authors state that there is evidence that the clearance of CYP3A4 substrates it reduced in individuals with high BMI while increased excretion was observed with substrates for CYP2E1 and CYP1A2. Also in children, increased activities of the later isozymes was found [223]. These observations are partly in agreement with animal data; increased i.e. CYP2E1 was observed for example in genetically modified obese female mice but not in males [224],
35
in a trial with monosodium glutamate treated overweight male mice an increase of this enzyme and also of CYP2A5 and 3A were detected in hepatic tissue and in colon mucosa [225]; furthermore, there is also evidence for CYP2E1 induction in obese rats [226]. This isozyme plays a key role in the activation of genotoxic nitrosamines. In regard to phase II enzymes, increased clearance of N-acetyltransferase (NAT) and
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uridine diphosphate glucuronosyltransferase (UGT) substrates was found in humans [222]; the later effects were also seen in overweight children [223]. UGT activity was also increased
SC R
in livers of obese mice with steatosis [227].
Data concerning the impact of obesity on GST are controversial. In diabetic mouse
U
model, decreased activity was found in the liver [224]; no such effects were seen in genetically
N
modified (ob/ob) mice in females but a higher activity was detected in animals in the same
A
study [228].
M
It is conceivable that alterations of the activities of the different drug metabolizing enzymes have a strong impact on the genotoxic properties of chemicals; however we are not
ED
aware of studies in which the consequences of overweight on the genotoxic effects of dietary, occupationally and environmentally relevant groups of DNA reactive carcinogens were
PT
investigated and related to alterations of drug metabolizing enzymes. An interesting study was published by Johnson et al. [229], who quantified cyclophosphamide DNA crosslinks in blood
CC E
cells of overweight and lean patients who underwent hematologic cell transplantation and found no evidence for BMI associated differences despite the fact that the overweight patients
A
received substantially higher doses of the drug. This cytostatic is activated by CYP2B6 and the authors hypothesized that their finding may be due to altered metabolism of the drug in obese individuals but no enzyme measurements were conducted to confirm this assumption [229]. Data concerning the impact of polycyclic aromatic hydrocarbon (PAH) exposure on oxidative DNA damage are controversial [55]. In a large study with PAH exposed coke oven
36
workers, an inverse relation with the BMI was observed in lymphocytes, while elevated levels of 8-OHdG were detected in livers of obese mice which were treated with PAH in comparison to lean controls [104].
Inflammation and DNA damage
IP T
8.8.
Inflammations are defence mechanisms which protect the host against infections and
SC R
other insults (for reviews see [230, 231]). It was found in many investigations that overweight/obesity are associated low grade inflammation [232, 233].
Adipose tissue plays a central role in this reaction. Adipocytes release so-called
U
“adipokines” into the blood stream which exert systemic effects [234]. These inflammatory
N
mediators belong to several groups including hormones (e.g. leptin which is pro-inflammatory
A
and adiponectin which is anti-inflammatory), chemokines and pro-inflammatory cytokines
M
(e.g. interleukins 1β, 6, 10 and TNF-α), acute phase proteins and growth factors such as IGF-
ED
1 [235]. Apart from adipocytes also macrophages and other cell types accumulate in adipose tissue and are important contributors to the production of substances which trigger
PT
inflammation [236].
Pro-inflammatory proteins which are produced in adipose tissue including the enzyme
CC E
haem oxygenase-1 (HO-1) cause substantial alterations of several pathways which activate transcription factors [237]. Probably the most relevant one is NF-kB which is activated by ROS [174] and adipokines [238] and causes increased transcription of genes encoding for
A
inflammatory molecules including COX-2. This enzyme catalyzes the formation of proinflammatory eicosanoids from arachidonic acid [239]. Inflammations cause DNA damage which in turn induces further inflammatory reactions [240] and it is likely that genomic instability plays a key role in the aetiology of
37
diseases which are associated with inflammations, in particular with cancer [241, 242]. However, there is only limited evidence for formation of oxidised DNA bases in obese humans by ROS as a consequence of inflammation (see Tables 2-6). In a study by Scarpato et al. the levels of inflammation markers (IL-6 and C-reactive protein) were found to be
9.
IP T
significantly higher in individuals with increased body weight [50].
Consequences of obesity induced DNA damage
SC R
Overweight/obesity are associated with increased prevalence of different forms of cancer in humans [243, 244]. The assumption of an association between excess body weight and cancer is also supported by results of rodent experiments [2, 90-92]. We are not aware of
U
any targeted studies in which correlations between DNA damage and cancer rates were
N
investigated in obese humans and animals; however, direct links can be postulated on the
A
basis of mechanistic studies.
M
Another relevant issue is the impact of obesity on infertility which was seen in a number
ED
of studies. As described in chapter 4.3 there is some evidence for increased DNA damage in sperm cells of obese infertile men, but further investigations concerning this relation are
PT
warranted. It is also conceivable that obesity causes mutations in germ cells which lead to heritable diseases in the offspring but this issue has never been investigated according to our
CC E
knowledge.
The term “DNA damage response” (DDR) concerns the consequences of genomic
A
instability [7, 205, 245, 246]. It describes pathways by which cells react to damaged DNA to prevent adverse health effects such as initiation of mitotic arrest, senescence, repair and cell death [205]. It was postulated that overweight and the MetS cause processes which prevent malignant transformation by activation of processes which lead to senescence and programmed cell death. The induction of inflammation via DNA damage and the activation
38
of transcription factors as well as telomere dysfunctions play a key role in these response mechanisms. A detailed description of this concept can be found in the papers of Shimizu et al. [246] and Erol [7]. The results of comet experiments with rodents indicate that excess body weight causes
IP T
genomic instability in multiple organs. The extent of damage which we measured in experiments with overweight mice in different tissues was similar (e.g. in colon, brain and liver) and no pronounced gender specific differences was observed [92]. It is conceivable that
SC R
the formation of tumors involve different organs specific tumors promoting mechanisms
which cause clonal expansion of damaged cells. These mechanisms are described in several
U
reviews [14, 247]. In regard to breast and endometrial cancer hormonal effects play an
N
important role, whereas in the liver the key mechanisms include lipogenesis and insulin
A
resistance which cause inflammation. In regard to formation of pancreatic tumors,
M
dysfunctional autophagy and toxic effects are discussed while obesity associated colorectal cancer is probably a consequence of dysbiosis and thinning of the intestinal mucosa layer
ED
resulting in increased permeability of the intestinal epithelial to microbial products.
PT
Apart from these organ specific effects, several mechanisms have been identified in obese individuals which may favour the development of tumors at multiple sites from initiated
CC E
cells including sustained angiogenesis, tissue invasion and metastasis and specific metabolic disturbances. These effects are described in detail in a recent working report of the IRAC [2,
A
248].
39
10. Conclusions and knowledge gaps Obesity and metabolic diseases are among the most relevant health risk factors for humans worldwide. The evaluation of the literature show that the impact of overweight/obesity on DNA stability which plays a key role in the etiology of cancer, infertility and ageing is not adequately investigated. We found in total 27 human studies
IP T
concerning the effects of obesity/overweight individuals and MetS patients on genomic instability in somatic cells, 12 human and 5 animal investigations concerning on DNA damage
SC R
in sperm and 18 investigations with rodents in which DNA damage was studied in blood cells
and in inner organs. It is interesting that a substantially higher number of human studies has been published which concern the genotoxic effects of chemicals. On the basis of a computer
U
aided search we estimate that about 220 articles deal with the effects of selected heavy metals
N
(Cr, Ni, Pb, As and Hg), the impact of radioactivity was monitored in more than 700 trials.
A
The quality of studies concerning the impact of overweight/obesity on DNA stability is
M
in general low since potential confounding factors such as intake of vitamins, minerals and
ED
the effects of plant derived DNA protective components were not taken into consideration in the most trials. Only in two studies the intake of micronutrients were recorded [53, 63]. It is
PT
known from a number of human studies that vitamins such as folate, B6, B12 and minerals such as Se, Zn and Fe have a strong impact on genomic instability [249, 250]. Furthermore it
CC E
is notable that most animal trials were conducted with diets which differ in regard to their macronutrient levels, this leads to differences of the body weight and to accumulation of fat
A
but the differences of the composition of the chows may cause results which do not necessarily reflect the effects of excess body weight. The literature provides only limited evidence for an association between overweight/obesity and damage of the genetic material in men. The evaluation of human studies on the basis of the QS values shows that in most comet trials with medium quality (no 40
study reached a high score) evidence for an association was detected. Four of six investigations with medium quality found that increased BMI values are associated with comet formation while results of MN studies with medium quality are conflicting (50% positive, 50% no relation). Also 8-OHdG measurements in humans do not allow to draw firm conclusions (positive, as well as negative correlations were detected); notably the study with
IP T
the highest QS found even a significant inverse association [53]. Notable, significant positive
correlations between induction of DSBs and excess body weight were detected in all γ-H2AX
SC R
experiments. Overall, 24 human studies with somatic and sperm cells found evidence for an
increase of DNA damage with the BMI while 22 trials reported no or even inverse
U
associations. It is probable that the highly conflicting results are due to the differences in the
N
diets, i.e. it is likely that normal and slim individuals consume higher amounts of vegetables,
A
fruits and full grain products which contain a broad variety of DNA protective compounds
M
while obesity is associated with increased intake of fats and foods which contain sugars and purified flour.
ED
The results of animal studies in which standardized diets were fed under controlled conditions provide clearer answers; i.e. positive associations between excess body weight and
PT
comet formation were seen in all studies with a medium quality (no high quality trials were
CC E
published so far) in somatic cells. Also the findings of sperm experiments with medium quality are in good agreement (two TUNEL and two SCSA trials, found consistently positive associations). Results of MN studies with animals are conflicting (one positive and two
A
negative were reported) but all of them had only low quality scores. The assumption of a causal relation between excess body weight and DNA damage is
strongly supported by results of mechanistic studies which indicate that several modes of action may cause genetic instability in overweight/obese individuals. Many investigations indicate that oxidative stress may play an important role; the formation of ROS may be a 41
consequence of increased insulin and glucose levels. ROS may cause either direct oxidative DNA damage (which was seen not in all studies), or via formation of DNA reactive LP products. As mentioned above, pronounced LP was seen in many trials with animals and humans. Also inflammation may be casually related as it leads to formation of oxygen and nitrogen radicals. Increased levels of pro-inflammatory cytokines and activation of NF-kB are
IP T
characteristic features of increased excess body fat; however, further investigations are warranted to allow firm conclusion. As described in chapters 8.3 to 8.6 also hormonal effects,
SC R
formation of AGEs, inhibition of DNA repair process and reduction of telomere lengths may play a role in the association between increased BMI values and genomic instability.
U
However, further experimental data are required which address these issues. In the case of
N
hormonal effects it is conceivable that they are organ specific, but similar DNA damage was
A
seen in multiple organs in a number of studies and no gender specific effects were observed.
M
The impact of excess body weight on telomere lengths was quite moderate and was only observed after longer time periods while DNA protective effects were seen in obese humans
ED
and laboratory rodents after short intervention periods with dietary components indicating that the impact of the BMI on telomeres may be of minor relevance. The effects of AGEs are
PT
difficult to assess at present as no studies are available in which their formation was correlated
CC E
with DNA stability in humans and animals. Many important knowledge gaps exist at present. It is not known exactly which BMI
values lead to a significant increase of genomic instability and if the effects increase linear or
A
exponentially. Further research is also warranted to elucidate the consequences of overweight/obesity induced DNA damage in regards to malignant transformation of cells. So far, research concerning the induction of cancer by excess body weight focused mainly on inflammation, signaling pathways and hormonal effects [251-253]. Other relevant topics for further studies concern the consequences of DNA damage on cognitive dysfunctions and 42
investigations concerning induction of mutations in germ cells. As mentioned above, some animal studies indicate that increased BMI leads to DNA damage in different regions of the brain [88, 92, 96], furthermore some animal studies found elevated levels of DNA damage in sperm cells as a consequence of excess body weight.
IP T
Genotoxicity studies will also enable to assess if and to which extent weight loss strategies as well as therapeutic interventions with drugs and bioactive natural dietary components lead to an improvement of the stability of the genetic material. Weight loss diets
SC R
which are advocated worldwide are generally not designed on the basis of scientific data and
it is conceivable that drastic reduction of the caloric intake and imbalanced consumption of
U
vitamins and minerals (which are required to maintain the stability of genetic material) will
N
lead to adverse health effects including destabilization of the DNA. Also this important issue
ED
Conflict of interest statement
M
A
should be addressed in future investigations.
PT
There are no conflict of interest.
CC E
Acknowledgments This works was supported by COST Action CA15132 (hCOMET); the authors are
A
also thankful to participants of this research initiative for discussions.
43
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PT
66.
84.
A
85.
86. 87.
47
95.
96.
97.
98.
99. 100. 101.
IP T
CC E
102.
SC R
94.
U
93.
N
92.
A
91.
M
90.
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Lawrence, T., The nuclear factor NF-κB pathway in inflammation. Cold Spring Harb Perspect Biol, 2009. 1(6): p. a001651. Baker, R.G., M.S. Hayden, and S. Ghosh, NF-κB, inflammation, and metabolic disease. Cell Metab, 2011. 13(1): p. 11-22. Pálmai-Pallag, T. and C.Z. Bachrati, Inflammation-induced DNA damage and damageinduced inflammation: a vicious cycle. Microbes Infect, 2014. 16(10): p. 822-832. Jackson, A.L. and L.A. Loeb, The contribution of endogenous sources of DNA damage to the multiple mutations in cancer. Mutat Res, 2001. 477(1): p. 7-21. Reuter, S., et al., Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med, 2010. 49(11): p. 1603-1616. Renehan, A.G., et al., Body-mass index and incidence of cancer: a systematic review and meta-analysis of prospective observational studies. The Lancet, 2008. 371(9612): p. 569578. Bracci, P.M., Obesity and pancreatic cancer: overview of epidemiologic evidence and biologic mechanisms. Mol Carcinog, 2012. 51(1): p. 53-63. Erol, A., Metabolic syndrome is a real disease and premalignant state induced by oncogenic stresses to block malignant transformation. Med Hypotheses, 2010. 74(6): p. 1038-1043. Shimizu, I., et al., DNA damage response and metabolic disease. Cell Metab, 2014. 20(6): p. 967-977. Simpson, E.R. and K.A. Brown, Obesity and breast cancer: role of inflammation and aromatase. J Mol Endocrinol, 2013. 51(3): p. T51-9. O'Flanagan C.H, B.L.W., Allott E.H,Hursting S. D., Molecular and metabolic mechanisms underlying the obesity-cancer link, I.w.g. No.10, Editor. 2017, IARC. p. 95-104. Fenech, M., The Genome Health Clinic and Genome Health Nutrigenomics concepts: diagnosis and nutritional treatment of genome and epigenome damage on an individual basis. Mutagenesis, 2005. 20(4): p. 255-69. Fenech, M.F., Dietary reference values of individual micronutrients and nutriomes for genome damage prevention: current status and a road map to the future. Am J Clin Nutr, 2010. 91(5): p. 1438s-1454s. Sun, B. and M. Karin, Obesity, inflammation, and liver cancer. J Hepatol, 2012. 56(3): p. 704-713. Ramos-Nino, M.E., The role of chronic inflammation in obesity-associated cancers. ISRN Oncol, 2013. 2013. Jung, U.J. and M.-S. Choi, Obesity and its metabolic complications: the role of adipokines and the relationship between obesity, inflammation, insulin resistance, dyslipidemia and nonalcoholic fatty liver disease. Int J Mol Sci, 2014. 15(4): p. 6184-6223. Fenech, M., Nutritional treatment of genome instability: a paradigm shift in disease prevention and in the setting of recommended dietary allowances. Nutr Res Rev, 2003. 16(01): p. 109-122. Azqueta, A. and A.R. Collins, The essential comet assay: a comprehensive guide to measuring DNA damage and repair. Arch Toxicol, 2013. 87(6): p. 949-68. Bakos, H.W., et al., Sperm DNA damage is associated with assisted reproductive technology pregnancy. Int J Androl, 2008. 31(5): p. 518-26. Evenson, D.P., K.L. Larson, and L.K. Jost, Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl, 2002. 23(1): p. 25-43.
PT
238.
255.
A
256.
257.
Legends for figures
55
Figure 1. Use of different methods for the detection of DNA damage in obesity studies. DNA damage was analysed in animal experiments mainly in comet assays (in different organs) which were also used in human studies with lymphocytes. Micronucleus (MN) experiments are conducted in bone marrow cells of rodents and in human studies with lymphocytes and with exfoliated epithelial cells. For the analysis of sperm cells, terminal deoxynucleotidyl-
IP T
transferase dUTP nick end labelling (TUNEL) and sperm chromatin structure assays (SCSA) are conducted. Oxidative damage of the DNA and alterations of DNA repair processes (BER,
SC R
NER) can be monitored with a modified protocol of the comet assay (not shown) and by
U
determination of 8-OHdG and/or 8-oxodG in urine, plasma and bloods cells.
N
Figure 2. Induction of DNA damage as a consequence of obesity/overweight. DNA damage
A
can be induced directly via estrogen metabolites, formation of advanced glycation end
and
alterations
of
the
activation
of
enzymes
which
catalyse
the
ED
shortening
M
products, LP products and radicals or indirectly via increased mitotic activity, telomere
activation/detoxification of mutagens and by inhibition of repair pathways. Phenomenona
PT
which were observed in overweight/obese humans (H) or animals (A) are indicated in circles. Solid lines indicate that experimental evidence shows that they lead to DNA stability or ROS
CC E
formation in rodents or men. Dotted lines are indicative for effects which were not demonstrated so far in overweight/obese subjects were observed in in vitro experiments.
A
Numbers indicate chapters in which the different effects are described.
Figure 3. Induction of ROS by insulin. The hormone binds to receptors (insulin receptor and insulin growth factor receptor) which leads to activation of phosphatidylinositol-3-kinase (PI3K) and Akt, the subsequent steps are activation of NADPH-oxidase and translocation of 56
Akt into mitochondria. Both processes lead to formation of ROS. Most findings come from in vitro experiments with mammalian cells but it was also shown that increased insulin levels cause DNA damage in rodents.
IP T
Figure 4. Induction of ROS by increased glucose levels. ROS can be formed by different molecular mechanisms, namely via activation of PKC or as a consequence of glycolysis in
SC R
the cytosol. AGEs are formed as a consequence of reactions of sugars with proteins, they activate transcription factors and cause also direct DNA damage. Another pathway of ROS
formation is caused by pseudohypoxia. All findings come from in vitro experiments with
A
N
U
mammalian cells but it was shown that increased insulin levels cause DNA damage in rodents.
M
Figure 5. Formation of AGEs and activation of NF-kB via RAGEs. Dotted line: intermediate processes which are not described in detail. AGEs are formed via Maillard reactions or via
ED
the polyol pathway and cause activation of transcription factors via interactions with signalling pathways. All findings come from in vitro experiments with mammalian cells but
CC E
PT
it was shown that increased insulin levels cause DNA damage in rodents.
Figure 6. Impact of obesity on DNA repair processes. Inflammation and estrogens increase
A
the mitotic activity of cells which results in errors of DNA replication and reduces the duration of the repair phase. Hormones have also a direct impact on different forms of repair. Telomere shortening was found in a number of investigations with overweight humans and leads to chromosomal aberrations as a consequence of inhibition of repair pathways. It is documented that ROS have an impact on repair functions (e.g. on NER); they also activate p53 which results in inhibition of cell divisions. Few studies are available indicating that 57
overweight/obesity have an impact on the methylation of DNA repair enzymes. Phenomenon which were observed in overweight/obese humans (H) or animals (A) are indicated in circles. Solid lines indicate that experimental evidence shows that they lead to DNA stability or ROS formation in rodents or men. Dotted lines are indicative for effects which were not
A
CC E
PT
ED
M
A
N
U
SC R
IP T
demonstrated so far in overweight/obese subjects but were observed in in vitro experiments.
58
59
A ED
PT
CC E
IP T
SC R
U
N
A
M
60
A ED
PT
CC E
IP T
SC R
U
N
A
M
A ED
PT
CC E
IP T
SC R
U
N
A
M Table. 1
61
I N U SC R
Table. 1. Overview of genotoxicity methods which are used in studies concerning the impact of overweight/ obesity on DNA stability Endpoints
Principle
BER/NER
cells are exposed to ROS generating chemicals e.g. (hydrogen peroxide or radiation) [30] detection of alterations of DNA repair activities by induction of BER and NER specific DNA damage [31] MN are formed as a consequence of structural and numerical chromosomal aberrations, additional markers in MN cytome studies are nuclear buds and nuclear bridges which reflect genomic instability, furthermore acute toxicity markers are evaluated i.e. karyolysis, karyorrhexis, pyknotic nuclei, binucleated cells and condensed chromatin [32] determination of oxidised deoxyguanosine by use of HPLC, ELISA or calorimetric assays [30]
ED
FPG/Endo sensitive sites ROS sensivity
A
detection of DNA migration in an electric field. in humans: lymphocytes and whole reflect formation of single and double strand breaks blood in rodents: different inner organs (SSBs and DSBs) and apurinic sites [29] *the methods can be also used to detect DNA damage in sperm cells III detection of oxidised purines and pyrimidines
M
Comet formation Standard conditions
CC E
PT
Micronuclei
A
8-OXOdG/ 8-OHdG/
γ-H2X
Target cells/ tissues and body fluids
in humans: lymphocytes and exfoliated cells from the mouth in rodents: bone marrow and erythrocytes
Remarks the predictive value of comet formation in regard to human cancer risks is not known.
MN in lymphocytes of humans are a reliable marker for cancer [33]
in humans: lymphocytes, plasma and relation to urine human cancer in rodents: plasma and urine risks not known. Induced by inflammatory diseases detection of double strand breaks by measurement of in humans: lymphocytes relation to human phosphorylation of a specific histone as a consequence of in rodents: different inner organs cancer risks not double strand DNA repair [34] known.
62
I N U SC R
Table. 1. continued Principle
Terminal deoxynucleotidyltransferase dUTP nick end labelling (TUNEL) assay
based on the ability of the enzyme terminal sperm cells in humans and animals deoxynucleotidyl- transferase (TdT) to incorporate labelled dUTP into free 3hydroxyl termini generated by fragmentation of genomic DNA into low molecular weight double stranded DNA and high molecular weight single stranded DNA [35] based on heat treatment or acidic denaturation sperm cells in humans and animals of sperm DNA and subsequent staining with acridine orange which enables to discriminate between DSBs and SSBs (double strands stain green, single strands red) [36]
M
ED
A
CC E
PT
Sperm chromatin structure assay (SCS)
Target cells/ tissues
A
Endpoints
63
Remarks in some animal and human studies results correlated with reduced fertility.
in some animal and human studies results correlated with reduced fertility.
I N U SC R
Table 2. Results of comet assay in overweight/obese subjects1 Study design
Parameter (indicator cells)
Participants and treatments
Intervention
tail intensity (lymphocytes)
48♂/23♀, BMI=18.0–23.9, n=21 BMI=24.0–26.9, n=26 BMI=27.0–39.9, n=24 age (all groups) 35-64
Intervention
arbitrary units (leukocytes)
Results
QS
Remarks
Ref
↔ DNA damage ↔ FPG in groups with different BMI
22 (M)
↑ oxidative DNA damage after consumption of sugar (1.8-fold) ↔ gender ↔ age
[37]
calorie restriction (CR): n=6♂/6♀, CR + exercise: n=6♂/6♀, very low- calorie diet: n=6♂/6♀, C: n=6♂/6♀, baseline BMI (all groups)=27.8, age (all groups) 26-49 9♂/47♀, BMI=51.1 n=56 before surgery 6 and 12 months after surgery age (all groups) 44.70 ± 1.4
↓ DNA damage after CR and after CR+exercise and very low-calorie diet groups at month 6 vs. baseline (1.03-fold)
18 (M)
weight loss after very low-caloric diet (12 kg) weight loss after CR + exercise and CR (8 kg) ↓ fasting insulin level after 3 and 6 months in CR + exercise and CR ↔ fasting glucose ↔ serum protein carbonyl
[38]
↓ DNA damage after 12 months (38%) ↔ DNA damage after 6 months
20 (M)
[39]
OB: n=20, BMI ≥30 OB+ type 2 diabetes (T2D): n=21, BMI ≥30 C: n=20, BMI ≤30 age (all groups) 44-55
↔ DNA damage OB vs. C
19 (M)
↓ ferric reducing antioxidant power assay after 6 and 12 months ↓ weight (22% after 6 months) and (29% after 12 months) ↑ total glutathione after 6 months (1.1-fold) ↑ total oxidised glutathione after 6 months (1.6-fold) ↔ total glutathione and oxidised glutathione after 12 months ↔MDA ↓ MDA in T2D vs. C (75%) ↓ telomere length in OB+T2D vs. OB
CC E
PT
ED
M
A
Comet assay2
tail intensity (whole blood)
A
Intervention
Parallel
tail intensity (visceral adipose tissue)
64
[40]
I N U SC R
Study design
Parameter (indicator cells)
Participants and treatments
Parallel
tail intensity (lymphocytes)
OB: n=20♀, BMI ≥25 C: n=20♀, BMI ≤25 normal weight obese syndrome3 (NWO): n=20♀, BMI ≤25 age (all groups) 25-35
Parallel
tail intensity (lymphocytes)
ED
M
A
Comet assay
QS
Remarks
Ref
↑ DNA damage OB vs. C (1.5-fold) ↑ DNA damage NWO vs. C (1.5-fold) ↑ FPG NWO vs. C (1.7fold) ↑ FPG OB vs. C (1.6-fold) ↑ DNA damage in high visceral fat vs. normal visceral fat (1.6-fold)
18 (L)
↔ 8-OHdG in whole blood
[41]
↑ MDA in high visceral fat vs. normal visceral fat (1.419 (M) fold)
[42]
↑ DNA damage in OB vs. C (5.0-fold)
↔ WC 21 (M) ↔ waist to hip ratio ↔ gender ↔ age
[43]
CC E
PT
healthy, non-obese Koreans, BMI=23.8, high visceral fat ≥100 cm2, n=27♂ normal visceral fat,<100 cm2, n=27♂ age (all groups) 36.5±0.8 OB: n=18♂/17♀, BMI= 31.9 (age <20: n=12, age 25-53: n=23) C: n= 8♂/7♀, BMI= 23.3 (age<20: n=9 and age 2553: n=6) OB: n=80♂/80♀, BMI=29 C: n=80♂/80♀, BMI =16.022.0 age (all groups) young and old (n.s)
Results
A
Parallel
Parallel
arbitrary units (leukocytes)
length of DNA migration (μm) (peripheral blood)
↑ DNA damage in ♀ young 17 OB vs. C (2.5-fold) (L) ↑ DNA damage in ♂ young OB vs. C (2.2-fold) ↑ DNA damage in ♀ old OB vs. C (2.6-fold) ↑ DNA damage in ♂ old OB vs. C (2.3-fold)
65
alterations of DNA damage and plasma homocysteine correlated with triglycerides, LDL-cholesterol, systolic blood pressure, cholesterol, MDA and total oxygen stress in OB. (-) association DNA damage and plasma homocysteine with total oxidant status and globulin.
[44]
I Study design
Parameter (indicator cells)
Participants and treatments
tail intensity (lymphocytes)
OB-MetS pts.: n= 30♀, BMI=45.7, age 32.4±6.8 OB-without MetS: n= 30♀, BMI=21.9 age 32.4±6.5
ED
M
A
Comet assay Parallel
N U SC R
Table 2. Continued
1
Results
QS
Remarks
Ref
↑ DNA damage in OB vs. C (1.7-fold) ↑ FPG in OB vs. C (1.4fold) ↑ endonuclease (ENDO) in OB vs. C (1.5-fold)
19 (M)
↑ AA-FTO genotype and allele A OB♀ (1.2-fold) ↓ TT-FTO genotype and allele T in morbidly OB♀ (1.2-fold)
[45]
3
Unless otherwise indicated comet assays were conducted under standard condition (SC), alkaline version which detects single/ double and apurinic sites.
CC E
2
PT
Abbreviations: BMI, body mass index; C, control; FPG, formamido pyrimidine glycosylase; LPO, lipid peroxidation; MN, micronuclei; MDA, malondialdehyde; NMO, normal weight obese; n, number of participants; n.s., not specified; OB, obese; OW, overweight; 8-OHdG, 8-hydroxy-2’-deoxyguanosine; SC, standard conditions; T2D, type 2 diabetes; wk, week; WC, waist circumference; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table)
A
Normal weight obese (NWO): according to the article by Tomasello et al [41], NWO known as a metabolic condition which observed in women with normal index but fat mass (FM) >30%.
66
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Table 3. Results of cytokinesis – block micronucleus cytome (CBMN-Cyt) assay in overweight/obese subjects1 Parameter Participants and treatments (indicator cells) Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay
MN (lymphocytes)
CC E
M
PT
Parallel
OB: n=33♂, 2 dietary interventions (both diets: 7000 kJ): either high protein-high red meat group (n=16), BMI=32.0 or high carbohydrate-low red meat diet group (n=17), BMI=31.0 age (all groups) 20-65 OB: n=36♂/47♀, BMI= 38.0, age 37.9±10.5 OW: n=10♂/11♀, BMI= 26.0, age 40.5±10.6 C: n=7♂/14♀, BMI= 22.0, age 34.8±11.5
ED
Intervention MN (randomized (lymphocytes) trial)
A
Study design
A
Parallel
Parallel
MN (lymphocytes)
OW/OB: n=14♀/♂, BMI>25 C: n=23, 20
MN (lymphocytes)
OB: n=33♂, BMI= 33.6 OW: n=55♂, BMI= 27.9, age elderly population (all groups) 68.0±6.8
Results
QS
Remarks
Ref
↔ MN (after weight loss) ↔ MN (by diet and time) ↑ nuclear buds during intervention (specific diet)
17 (M)
only 1000 lymphocytes scored for MN ↓ weight loss by both interventions after 12 wks (12%)
[46]
↑ MN in OB vs. C (1.7-fold) ↑ MN in OB vs. OW (1.5-fold) ↑ nucleoplasmic bridges in OB vs. C (1.7-fold) ↑ nucleoplasmic bridges in OB vs. OW (1.5-fold) ↑ nuclear buds in OB vs. C (2.2-fold) ↑ nuclear buds in OW vs. C (1.6fold) ↔ MN in OW/OB vs. C
16 (M)
only 1000 lymphocytes scored for MN ↓ 8OHdG in OB vs. C (7%) ↔ correlation waist to hip ratio and CBMN assay parameters and 8-OHdG (+) correlation between MN and BMI in OW and OB (+) correlation between BMI and nuclear buds in OW and OB
[47]
15 (M)
↔ gender ↔ age ↔ smoking ↔ effect of mobile phone
[48]
↔ MN in OB vs. OW
14 (M)
↔ smoking ↔ age ↔ BMI lower DNA damage in former uranium workers who received X-ray examination
[49]
67
I N U SC R
Table 3. Continued
Study Parameter Participants and treatments design (indicator cells) Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay
Parallel
MN (buccal cells)
Crosssectional
MN (buccal cells)
OB: n= 31♂/30♀, BMI= 29, age: 10.8±3.4 OW: n= 9♂/11♀, BMI= 24.4 age: 12.2±2.6 C: n=16♂/22♀, BMI= 17.5 age:10.8±2.6 Malaysian farmers ♀/♂ OB: n=3, 35≤BMI OB: n=5, 30≤BMI<34.9 OW: n=35, 25≤BMI<29.9 normal: n=21, 18.5≤BMI<24.9 UW: n=4, BMI <18.5 age (all groups) 26-65 OB: n=20, BMI=22.4 OW: n=20, BMI=18.3 C: n=20, BMI=16.2 age (all groups) 7-11
A
MN (lymphocytes)
QS
↑ MN in OW vs. C (2.5fold) ↑ MN in OB vs. C (2.7fold)
17 (M)
↑ MN in normal vs. OW (1.25-fold) ↔ MN in normal vs. OB
17 (M)
↔ MN in different groups
18 (M)
A
CC E
PT
ED
M
Parallel
Results (fold)
1
Remarks
Ref
↑ γ-H2AX foci in OW vs. C (5.6-fold) ↑ γ-H2AX foci in OB vs. C (8-fold) ↔ tumor necrosis factor-alpha in all groups ↑ interleukin 6 in OW and OB vs. C (9- fold) ↑ C-reactive protein in OW and OB vs. C (17.7-fold) only 1000 cells scored for MN ↔ binucleated between different groups ↑ MN in pesticide exposed farmers vs. C ↔ age
[50]
only 1000 cells scored for MN ↔ binucleated cells ↔ pycnosis cells ↔ broken-eggs cells ↔ karyorrhexis cells ↔ condensed chromatins ↔ total nuclear abnormalities
[52]
[51]
Abbreviations: BMI, body mass index; C, control; CBMN-Cyt, cytokinesis block micronucleus cytome assay; MN, micronuclei; n, number of participants; n.s., not specified; OB, obese; OW, overweight; SC, standard conditions; UW, under-weight; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table)
68
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Table 4. Results of 8-hydroxy-2' -deoxyguanosine (8-OHdG) in overweight/obese subjects1 Parameter (indicator cells)
Participants and treatments
8-hydroxy-2' -deoxyguanosine (8-OHdG)
A
Study design
HPLC (urine)
n=31♂/52♀, BMI= 26.0, age (all groups) 40-64
Parallel
colorimetric assay (whole blood)
OB: n=20♀, BMI ≥25 C: n=20♀, BMI <25 NWO2: n=20♀, BMI ≤25 age (all groups) 25-35 OB: n=12♂/8♀, BMI= 46.8, age 44.2±12.0 and laparoscopic gastric banding C: n=10♂/10♀, BMI= 22.5, age 40.7±12.3
ED
ELISA (plasma & urine)
CC E
PT
Parallel
M
Intervention (randomizedtrial)
HPLC (plasma)
A
Parallel
Parallel
HPLC (plasma)
coke oven workers ♂ BMI ≤ 24: n=18 exposed, 12 non exposed BMI > 24: n=29 exposed, 19 non exposed, age according to BMI (n.s) OW and OB: n=6♂/52♀, BMI=31.9 C: n=3♂/17♀, BMI=22.3 age (all groups) 46-54
Results
QS
Remarks
↓ 8-OHdG in high BMI (4%) ↑ 8-OHdG in ♂ vs. ♀(1.3fold) ↔ 8-OHdG
18
↔ intake of vitamin C, E, A on DNA damage [53] ↑ 8-OHdG in smokers (2.0-fold)
(H) 14
(M) ↑ urinary 8-OHdG in OB vs. C (2.6-fold) ↔ plasma 8-OHdG ↓ urinary 8-OHdG after surgery (59%) ↓ plasma 8-OHdG after surgery (50%) ↓8-OHdG with BMI (not significant)
14
↑ 8-oxodG in OW and OB vs. C (2.0-fold)
17 (M)
69
(M)
15
(M)
Ref
↑ FPG NWO vs. C (1.7-fold) ↑ FPG OB vs. C (1.6-fold) ↑ DNA damage NWO vs. C (1.5-fold) ↑ DNA damage OB vs. C (1.5-fold) ↓ BMI after surgery (20%) ↓ body weight after surgery (19%)
[41]
↑ 8-OHdG and glutathione S-transferase in the exposed group (not significant)
[55]
↑ LPO in OW and OB vs. C (3.0-fold) (+) correlation 8-oxodG and BMI, WC, hip circumference, triglycerides (+) correlation LPO and BMI
[56]
[54]
I Study design
Parameter (indicator cells)
Participants and treatments
ELISA (plasma)
Parallel
ELISA (urine)
OB: n=36♂/47♀, BMI= 38.0, age 37.9±10.5 OW: n=10♂/11♀, BMI= 26.0, age 40.5±10.6 C: n=7♂/14♀, BMI= 22.0, age 34.8±11.5
↓ 8-OHdG in OB vs. C (7%)
QS
Remarks
Ref
17 (M)
↔ association waist to hip ratio and CBMN assay parameters and 8-OHdG ↑ MN in OB vs. C (1.7-fold) ↑ MN in OB vs. OW (1.5-fold) ↑ nucleoplasmic bridges in OB vs. C (1.7-fold) ↑ nucleoplasmic bridges in OB vs. OW (1.5-fold) ↑ nuclear buds in OB vs. C (2.2-fold) ↑ nuclear buds in OB vs. C (1.6-fold) (+) correlation between MN and BMI in OW and OB (+) correlation between BMI and nuclear buds in OW and OB association between 8-oxoGuo and waist-hip ratio association between 8-oxodG and adrenaline (↔) no association between 8-oxoGuo and 8-oxodG with leptin, adiponectin and high sensitive-Creactive protein
[47]
CC E
1
PT
ED
M
Parallel
Results
A
8-hydroxy-2' -deoxyguanosine (8-OHdG)
N U SC R
Table 4. Continued
OB hypertensive (OB-H): n=63♂, BMI≤30 OB normotensive (OB-N): n=40♂, BMI≤30 lean normotensive (L-N): n=27♂, 20< BMI≤25 age (all groups) 39-58
↔ 8-oxodG ↑ 8-oxoGuo in OB-H vs OB-N (1.1-fold) ↑ 8-oxoGuo in OB-N vs LN (1.2-fold) ↑ 8-oxoGuo in OB-H vs LN (1.3-fold)
14 (M)
[57]
A
Abbreviations: BMI, body mass index; C, control; ELISA, enzyme linked immunosorbent assay, FACS, fluorescence-activated cell sorting; FPG, formamido pyrimidine glycosylase; HPLC, high-performance liquid chromatography, LPO, lipid peroxidation; MN, micronuclei; MDA, malondialdehyde; NWO, normal weight obese; n, number of participants; n.s., not specified; OB, obese; OW, overweight; 8-OHdG, 8-hydroxy-2’-deoxyguanosine; SC, standard conditions; wk, week; WC, waist circumference; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table) 2
Normal weight obese (NWO): according to the article by Tomasello et al [41], NWO known as a metabolic condition which observed in women with normal index but fat mass (FM) >30%.
70
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Table 5. Results of Gamma-H2AX (γ-H2AX) in overweight/obese subjects1 Participants and treatments
Parallel
FACS (lymphocytes)
OB: n= 11♂/12♀, BMIZscore2=2.8,age:11.0±0.8 C: n=7♂/8♀, BMIZ-score =0.7 age: 12.4±0.4
Parallel
FACS (lymphocytes)
M
A
Parameter (indicator cells) Gamma-H2AX (γ-H2AX)
ED
Study design
QS
Remarks
Ref
↑ γ-H2AX foci in OB vs. C after 2 hours inducing DNA damage by mitomycin C (2.0-fold) ↑ γ-H2AX foci in OB vs. C after 4 hours inducing DNA damage by mitomycin C (1.7-fold) ↑ γ-H2AX foci in OW vs. C (5.6-fold) ↑ γ-H2AX foci in OB vs. C (8.0-fold)
14 (M)
↑ expression of the high mobility group box-1 protein (HMGB1) involved in doublestrand breaks repair in OB
[58]
15 (M)
↑ MN in OW vs. C (2.5-fold) ↑ MN in OB vs. C (2.7-fold) ↔ TNF-α in all groups ↑ IL-6 in OW and OB vs. C (9.0- fold) ↑ CRP in OW and OB vs. C (17.7-fold)
[50]
↓abasic sites in DNA at 12 wks (30%) ↓abasic sites in DNA at 52 wks (65%)
-
↑ telomere lengths after weight loss (not significant)
CC E
PT
OB: n= 31♂/30♀, BMI= 29, age: 10.8±3.4 OW: n= 9♂/11♀, BMI= 24.4 age: 12.2±2.6 C: n=16♂/22♀, BMI= 17.5 age:10.8±2.6
Results
Miscellaneous
A
Intervention
test kit for abasic sites (apyrimidinic/ apurinic) (midrectal)
OB: after weight loss for 12wks (7000 kJ/day): n=45♂, BMI=31.9, age 51±1.0 and after 52 wks weight loss: n=12, BMI=31.6, age 56±1.1
1
[59]
Abbreviations:; BMI, body mass index; C, control; CRP, c-reactive protein; FACS, fluorescence-activated cell sorting; IL-6, interleukin 6; MN, micronuclei; n, number of participants; OB, obese; OW, overweight; TNF-α, tumor necrosis factor-alpha; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table) 2
BMI Z-score: Body mass index z-scores, also called BMI standard deviation (s.d.) scores, are measures of relative weight adjusted for child age and sex.
Table 6. Genomic instability in metabolic syndrome (MetS) patients1
71
I Parameter (indicator cells) Comet assay2
tail intensity early stage of MetS pts: (lymphocytes) n=65 ♂, BMI=33.0,age 47.4±5.5 C: n=56♂, BMI=24.5 age (all groups) 46.8±4.8 arbitrary units MetS pts: n= 38♂/27♀, (lymphocytes) BMI= 27.8 C: n=42♂/23♀, BMI= 26, age (all groups) 54-57 arbitrary units MetS pts: n=20♂/32♀, (whole blood) BMI=30.3, age 32-80 C: n=♂17/♀20, BMI=25, age (all groups) 27-70
Results
QS
Remarks
Ref
↔ DNA damage ↔ hOGG1 modified comet assay ↔ neutral comet assay
19 (M)
↓ 8-OHdG in urine MetS pts. (38%)
[65]
↑ DNA damage in MetS pts (1.7fold)
18 (M)
↓ total antioxidant capacity in MetS pts (50%)
[64]
↑ DNA damage in MetS pts (3.3fold)
20 (M)
↔ DNA damage in OB-MetS pts vs. OB-without MetS ↔ FPG in OB-MetS pts vs. OBwithout MetS ↔ endonuclease (ENDO) in OBMetS pts vs. OB-without MetS
19 (M)
↑ MN in MetS pts (2.2-fold) [63] ↑ malondialdehyde (MDA) in MetS pts (2.0-fold) ↓ superoxide dismutase in MetS pts (44%) ↓ glutathione peroxidase in MetS pts (27%) ↑ AA-FTO genotype and allele A OB♀ (1.2- [45] fold) ↓ TT-FTO genotype and allele T in morbidly OB♀ (1.2-fold)
tail intensity OB-MetS pts.: n= 30♀, (lymphocytes) BMI=45.7, age 32.4±6.8 OB-without MetS: n= 30♀, BMI=21.9 age 32.4±6.5
A
CC E
Parallel
ED
Parallel
PT
Parallel
M
A
Parallel
Participants and treatment
N U SC R
Study design
72
I N U SC R
Table 6. Continued
Study Parameter Participants and treatment design (indicator cells) Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay MetS pts: n=20♂/32♀, BMI=30.3, age 32-80 C: n=17♂/20♀, BMI=25, age 27-70
A
MN (lymphocytes)
QS
Remarks
Ref
↑ MN in MetS pts (2.2-fold)
17 (M)
↑ DNA damage in MetS pts (3.3-fold) ↑ MDA in MetS pts (2.0-fold) ↓ superoxide dismutase in MetS pts (44%) ↓glutathione peroxidase in MetS pts (27%)
[63]
↔ 8-OHdG levels in different MetS groups
13 (M)
↔ oxidative stress in the different groups ↑ 8-OHdG in hypertensive pts vs. C (1.5-fold)
[66]
↓ 8-OHdG in MetS pts.(38%)
15 (M)
↔ DNA damage in lymphocytes ↔ hOGG1 modified comet assay ↔ neutral comet assay
[65]
M
Parallel
Results
HPLC (genomic and mitochondrial – mononuclear cells)
CC E
PT
Parallel
ED
8-hydroxy-2' –deoxyguanosine (8-OHdG)
A
Parallel
ELISA (urine)
MetS pts: n=108♂/77♀ number of groups of MetS: 1- n=10♂/11♀, BMI=23 2- n=28♂/12♀, BMI=28 3- n=40♂/26♀, BMI=29 4- n=24♂/20♀, BMI=32 5- n=6♂/8♀, BMI=32 C (no MetS): n=16♂/14♀, BMI= 24 age (all groups) 25-50 early stage of MetS pts: n=65 ♂, BMI=33.0,age 47.4±5.5 C: n=56♂, BMI=24.5 age (all groups) 46.8±4.8
1
Abbreviations: BMI, body mass index; C, controls; CBMN-Cyt, Cytokinesis block micronucleus cytome assay; ENDO, endonuclease; FPG, Formamidopyrimidine DNA glycosylase; HPLC, high-performance liquid chromatography; MN, micronuclei; MetS, metabolic syndrome; 8-OHdG, 8-hydroxy-2' –deoxyguanosine; n, number of participants; OB, obese; OW, overweight; 8-oxo-dG, 8-hydroxy-2' –deoxyguanosine; pts, patients; SC, standard conditions; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table) 2
Unless otherwise indicated comet assays were conducted under standard condition (SC), alkaline version which detects single/ double and apurinic sites
73
I Method/ Participants and treatments Results parameter Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL assay)
A
Study design
N U SC R
Table 7. Obesity related DNA damage in sperm
♂ in subfertile couples, n=330 OB: n=43, BMI≥30 OW: n=137, BMI=25-29.9 C: n=151, BMI=18.5-24.9 age (all groups) 37.6±6.2 Sperm chromatin structure assay (SCSA) TUNEL assay
Intervention
SCSA, DFI
Intervention
SCSA, DFI
ED
M
Parallel
PT
CC E
A
♂n=43, BMI=33-61, 14 wks residential weight loss program n=15, BMI=46.0-61.0 n=14, BMI=41.7-46.0 n=14, BMI=33.3-41.6 age (in all group) 20-59 weight of women before pregnancy: under weight (UW): n=33 BMI<18.5 normal weight (C): n=263 BMI=18.5-24.9 OW: n=22, BMI≥25.0 off spring: UW: n=11 BMI<18.5 C: n=243 BMI=18.5-24.9 OB: n=83, BMI≥25.0
QS
Remarks
Ref
↑ DFI in OB vs. C (1.5-fold) ↔ DFI in OW vs. C (1.2-fold)
16 (M)
↑ viable spermatozoa OB vs. C (30%)
[70]
↔ DFI after weight loss
16 (M)
(+) correlation DFI and BMI weight loss associated with increased testosterone levels
[71]
↑ DFI in sons of OW mothers vs. sons of C mothers (1.2-fold)
15 (M)
↑ DFI in OW vs. C (but not significant), after 20years of follow-up cohort study
[72]
Table 7. Continued
74
I N U SC R
Table 7. Continued Study design
Method/ parameter
Participants and treatments
Parallel
SCSA (flow cytometry) DFI
Parallel
SCSA DFI
Crosssectional
SCSA (flow cytometry), DFI
PT
CC E A
♂ in subfertile couples, n=153 OB: n=16, BMI≥30, age 32.5±4.0 OW: n=63, BMI=25.0-29.9, age 32.8±6.7 C: n=74, BMI<24.9, age 30.2±5.9 ♂n=287, C: n=91 OW: n=136 OB: n=59 age (all groups) 23-45 ♂n=468, n=72, BMI≥35 n=122, BMI=30-35 n=191, BMI=25-29 n=83, BMI<25 age (all groups) 31.8 ± 4.8 n=175♂, BMI=25.5 age (all groups) 25-60
QS
Remarks
Ref
↔ DFI
13 (M)
↔ levels of chromatin integrity impairment ↑ cells with defective chromatin condensationin OB vs. C ↔ BMI and basic semen parameters, chromatin integrity and chromatin condensation
[73]
↔ DFI in different BMI ↑ high DNA sustainability in OB vs OW (4-fold) ↔DFI in different BMI
15 (M)
↑ cell phone use (11-25 years) vs (6-10 years) ↑ occupational stress (work)
[74]
14 (M)
(-) association WC and sperm count no significant association body size and semen concentration , motility, vitality or morphology
[75]
↔ BMI and DFI
12 (L)
(-) significant between association DFI and age
[76]
M
SCSA (flow cytometry) DFI
ED
Parallel
A
Sperm chromatin structure assay (SCSA)
Results
75
I Study Method/ Participants and treatment design parameter Sperm chromatin structure assay (SCSA)
♂;cohort A: n=275, age 18 cohort B: n=304 Swedish military recruits, BMI=22.6, age 18 cohort C: n=724 fertile men and men without fertility problems, BMI=25.3, age 18-69 cohort D: n=200 of men without known fertility problems, BMI=24.6, age 19-40
A
SCSA DFI
ED
M
Crosssectional
N U SC R
Table 7. Continued
Results
QS
Remarks
Ref
↑ DFI in C vs. OW (when excluding cohort C, the significance was lost)
14 (M)
↑ progressive motile spermatozoa in OW vs. C (when excluding cohort C, the statistical significance was lost)
[77]
A
CC E
PT
Abbreviations: AU, arbitrary unit; BMI, body mass index; DFI, DNA fragmentation index; N, number; n.s. , not specified; OB, obese; OW, over weight; SCSA, sperm chromatin structure assay; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; UW, underweight; WC, waist circumference; Y, year; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS) (For details see supplementary Table)
76
Participants and treatments
I
Method/ parameter
Results
N U SC R
Study design Comet assay
♂ in subfertile couples, n=413 ↔ DNA damage between high obese (OB): n=40, BMI=39.4, groups age 35.7±5.6 low OB: n=87, BMI=32.0. age 37.2±5.9 overweight (OW): n=233, BMI=27.0, age 36.3±5.2 control (C): n=123, BMI=23.0, age 35.7 ±5.2 Parallel standard ♂ in subfertile couples ↑ DNA damage in OB vs. C comet assay OB: n=36, BMI≥30, age 34.3±4.9 (1.8-fold) (arbitrary OW: n=187, BMI=25.0-29.9, age ↑ DNA damage in OW vs. C units) 34.7±7.9 (1.1-fold) C: n=82, BMI<24.9, age 33.5±6.1 Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL assay) neutral comet assay (tail intensity)
Remarks
Ref
19 (M)
↑ estradiol in both OB groups vs. C (1.2-fold) ↓ total testosterone in both OB groups vs. C (36%) ↑ sex hormone-binding globulin in both OB groups vs. C (40%)
[67]
18 (M)
↓ mitochondrial activity in OB vs. C (27%) ↓ sperm motility in OB vs. C (17%)
[68]
14 (M)
↓ viable spermatozoa in OB vs. C ↑ spermatozoa with decondensed chromatin in OB and OW vs. C (2.0-fold) ↑ spermatozoa with low mitochondrial membrane potential OB vs. C (14.0-fold) ↑ spermatozoa with low mitochondrial membrane potential OW vs. C (10.0-fold)
[69]
PT
TUNEL assay, flow cytometry
♂ healthy non-smokers, n=150 OB: n=50, BMI≥30, 31.6 ± 1.7 OW: n=50, BMI=25.0-29.9, age 31.2 ± 1.2 C: n=50, BMI=19.0-24.9, age 31.5 ± 1.1
↑ DFI in OB vs. C (3.0-fold) ↔ DFI in OW vs. C (1.5fold)
A
CC E
Parallel
ED
M
A
Parallel
QS
Table 8. Results of comet assay in animal studies concerning the impact of obesity on DNA stability1
77
I Zucker rats
Results
QS
Remarks
Ref
obese (OB) and control (C), n=5 p.g.♀ 3, 6, 12 months old weight after 12 months: OB: 747g; C: 259g OB: n=40♂, C: n=10♂ fed either with a hypercaloric (weight 620g) or a standard (STD) diet (weight 450g) for 30 wks after 20 wks , half of the OB rat were fed a 25%-caloric restriction diet (CRD) of STD for 10 wks n=6 p.g.♂ fed either STD or cafeteria diet (CAF) weight after 13 wks: STD: 44g, CAF:46g
↑ DNA damage in peripheral blood cell and liver OB vs. C (2.0-fold) ↔ DNA damage in kidney, brain and heart OB vs. C
13 (M)
↑ DNA damage in old rats vs. younger rats (3.0-fold in blood cells)
[94]
↑ DNA damage in OB vs. CRD (2.1-fold) ↑ DNA damage in OB vs. STD (10.0-fold)
13 (M)
↑ malondialdehyde (MDA) in OB vs. STD (2.2-fold) ↑ MDA in OB vs. CRD (1.8-fold) ↑ body weight in OB vs. STD animal lost weight after CRD vs. OB
[95]
↑ DNA damage in kidney CAF vs. STD (3.1-fold) ↑ DNA damage in peripheral blood CAF vs. STD (5.0-fold) ↑ DNA damage in liver CAF vs. STD (3.8-fold) ↑ DNA damage in brain CAF vs. STD (5.2-fold)
14 (M)
[96]
n=5p.g. ♂ and ♀ fed with either HFD (60% fat) or STD weight after 12 wks: ♂. OB: 43g; STD: 32g ♀. OB: 30g; STD:23g
↑ DNA damage in liver OB vs. STD in ♂ (1.6fold) and ♀ (2-fold) ↑ DNA damage in colon OB vs. STD in ♂ (1.5fold) and ♀ (1.5-fold) ↑ DNA damage in brain OB vs. STD in ♂ (1.9fold) and ♀ (1.7-fold) ↔ DNA damage in blood OB vs. STD in ♂ and ♀
15 (M)
↔ ratio polychromatic: normochromatic erythrocytes from bone marrow ↑ MN in CAF vs. STD (4.0-fold) ↑ oral glucose tolerance OB vs. STD (2.0-fold) ↑ TNF-α in plasma OB vs. STD in ♂ (1.9-fold) and ♀ (2.1-fold) ↑ MCP-1 in plasma OB vs. STD in ♂ (1.9-fold) and ♀ (3.3-fold) ↑ insulin in plasma OB vs. STD in ♂ (9.0-fold) and ♀ (9.9-fold) only abstract available
M
ED
tail moment (kidney, liver, heart, brain, blood cells) Wistar rats
N U SC R
Experimental design
A
Animal/parameter (indicator cells) comet assay2
CC E
PT
tail intensity (lymphocytes)
Swiss albino mice
A
arbitrary units (peripheral blood, kidney, liver, brain) C57BL/6J mice tail intensity (liver, colon, brain, blood)
78
[92]
I Experimental design
C57BL/6J mice
A
tail intensity (liver, colon)
Remarks
Ref
n=14 per group.♂ fed with HFD (45% fat) or STD weight after 4 months: OB: 46g STD: 27g
↑ DNA damage in liver OB vs. STD (2.1-fold) ↑ DNA damage in colon OB vs. STD (2.4-fold)
14 (M)
[97]
n=6 per group ♂ 5-6 wks fed either STD (10% fat) or HFD (45 % fat) weight after 6 wks.: HFD: 32g, STD: 28g
↑ DNA damage in in liver STD vs. HFD ↔ DNA damage in testes and blood ↑ FPG in white blood cells and liver in STD vs. HFD (1.1-fold) ↔ FPG in testes
14 (M)
↓ gene expression of MLH1 in liver OB vs. STD (50%) ↔ gene expression of MLH1 in colon ↓ gene expression of IL-6 in colon OB vs. STD (70%) ↓ gene expression of Dnmt1 in colon OB vs. STD (60%) ↓ gene expression of Dnmt1 in liver OB vs. STD (50%) ↓ bacterial abundance in OB vs. STD ↑ haemoglobin glycidamide adducts in HFD vs. STD ↑ weight gain HFD vs. STD (1.5-fold) glycidamide-induced sperm DNA fragmentation higher in HFD vs. STD ↔DFI in sperm HFD vs. STD
n=14 per group ♂ fed with STD or HFD (45% fat) weight after 4 months: OB: 46g, STD : 27g
↑ DNA damage in liver OB vs. STD (2.3-fold) ↑ DNA damage in colon OB vs. STD (2.6-fold)
14 (M)
↓ gene expression of MLH1 in liver OB vs. STD (49%) ↔ gene expression of MLH1 in colon OB vs. STD ↓ gene expression of Dnmt1 in colon OB vs. STD (61%) ↓ gene expression of Dnmt1 in liver OB vs. STD (61%) ↓ bacterial abundance in OB vs. STD ↔ gene expression Nrf2 and MGMT
[98]
ED PT
CC E
tail intensity (testes, blood, liver) and SCSA (sperm)
C57BL/6J mice
QS
M
tail intensity (liver, colon)
C57BL/6J mice
Results
A
Animal/method/parameter (indicator cells) Comet assay
N U SC R
Table 8. Continued
79
[93]
I N U SC R
Table 8. Continued
Experimental design
Results
QS
Remarks
Ref
WNIN/Ob rat (Met S- model)
n=6 per group ♂, OB: WNIN/Ob-obese (3-months) C: WNIN/Ob-lean (3months)
↑ DNA damage (SSB) in hippocampus in OB vs. C (1.2-fold) ↑ DNA damage (SSB) in neocortex in OB vs. C (1.4-fold) ↑ DNA damage (double strand breaks (DSBs) in hippocampus in OB vs. C (2.0-fold) ↑ DNA damage (DSBs) in neocortex in OB vs. C (1.9-fold)
13 (M)
↑ MDA in OB vs. C (1.5-fold) ↓ superoxide dismutase in OB vs. C (50%) ↓ activity of catalyse in OB vs. C (44%) weight (n.s)
[88]
M
ED
tail moment (neocortex & hippocampus)
A
Animal/method/parameter (indicator cells) Comet assay
1
Unless otherwise indicated comet assays were conducted under standard conditions (SC), alkaline version which detects single/ double and apurinic sites
A
2
CC E
PT
Abbreviations: C, controls; CAF, cafeteria diet; CBMN-Cyt, cytokinesis block micronucleus cytome assay; CRD, calorie restricted diet; DIO, diet induced obesity FPG, formamido pyrimidine glycosylase; HFD: high fat diet; IL-6, interlukin 6; MN, micronuclei; MDA, malondialdehyde; n, number of participants; OB, obese; OGG1, 8oxoguanine DNA glycosylase; OW, overweight; 8-oxodG, 8-oxo-7,8-dihydro-2' –deoxyguanosine; p.g.: per group; RSD, restricted diet; SC, standard conditions; STD, standard diet, TNF-α, tumor necrosis factor-alpha; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table)
80
I N U SC R
Table 9. Results of Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay in animal studies concerning the impact of obesity on DNA stability Animal (indicator cells)
Experimental design
Results
QS
Remarks
Ref
↑ DNA damage in kidney CAF vs. STD (3.1[96] fold) ↑ DNA damage in peripheral blood CAF vs. STD (5.0-fold) ↑ DNA damage in liver CAF vs. STD (3.8-fold) ↑ DNA damage in brain CAF vs. STD (5.2fold) ↑ oral glucose tolerance test OB vs. STD the promoters of p16 and Dapk genes were [99] methylated in the livers of C57BL/6J mice fed a HFD (irradiated and non-irradiated) Mgmt promoter was methylated in irradiated and HFD mice.
11 (M)
n=5, C57BL/6J DIO: fed for 3 wks, age=14 wks C3H/HeJ: fed for 12 wks, age=5 wks both strains fed either HFD (60% fat) or STD (10 % fat) gender (n.s) n=36♂ fed either with HFD (60% fat) or with LFD (10% fat) for 18 wks, weight 28 g treated either with N-ethylN-nitrosourea (ENU) or with the vehicle control for 3 days
↔ MN in HFD in vs. STD (in both strains) ↔ P/N ratio in HFD vs. STD (in both strains)
10 (M)
↔ MN-NCEs HFD vs. LFD ↔ MN RETs HFD vs. LFD ↔ MN after ENU treatment in HFD vs. LFD ↑ MN-NCEs after ENU treatment in HFD and LFD vs. control (1.1-fold) ↑ MN RETs after ENU treatment in HFD and LFD vs. control (4-fold)
9 (M)
CC E
PT
C57BL/6J DIO mice (prediabetic T2D) and C3H/HeJ mice
C57BL/6J mice
flow cytometry (blood)
A
↑ MN in CAF vs. STD (4.0 -fold) ↔ polychromatic: normochromatic (P/N) ratio in bone marrow
ED
(bone marrow)
(bone marrow)
n=6 per group ♂ fed either STD or cafeteria diet (CAF) weight after 13 wks: STD: 44g, cafeteria diet (CAF): 46g
M
Swiss albino mice
A
Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay
81
↑ Pig-α mutant RBCs in HFD vs. LFD (2.5fold)
[100]
I N U SC R
Table 10. Results of 8-oxo-7,8-dihydro-2' -deoxyguanosine (8-oxodG) in animal studies concerning the impact of obesity on DNA stability1
C57BL/6J mice
n=10 per group ♀ mother: either folate-depleted (2 mg folic acid/kg diet) or folate-adequate (0.4 mg folic acid/kg diet) 4 wks before mating off-spring: at weaning (age: 22-25 days), either HFD (20% anhydrous milk fat) or C (5% anhydrous milk fat) for 5 months n=40 per group ♂ fed either with HFD (45 %) or C (12%) for 10 wks.
PT
OB Zucker fa/fa rat and lean Zucker fa/+ rats
M
HPLC (urine)
♂, OB: weight=440 g, lean (C): weight=350 g, weight loss via roux-en-Y gastric bypass surgery (n=15), weight 410 g or sham surgery (n=17), weight 580 g
ED
A
Method/parameter Experimental design Results QS Remarks (indicator cells) 8-oxo-7,8-dihydro-2' -deoxyguanosine (8-oxodG) and 8-OH-Guanosine (8-OH-Gua) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo)
CC E
HPLC (subcortical regions of brain)
A
Diet induced obesity (DIO) rat HPLC (in mitochondria of heart, liver, kidney and testes)
weight: HFD: 628 g, C: 570 g
↑ 8-oxodG after sham surgery vs. C (3.5-fold) ↔ 8-oxodG in OB vs. C ↑ 8-oxoGua in OB vs. C (2.5-fold) ↑ 8-oxoGuo in OB vs. C (3-fold)
11 (M)
↓ 8-oxodG in subcortical region of HFD vs. C (not significant)
11 (M)
↑ 8-oxodG in heart of HFD vs. C (1.1-fold) ↑ 8-oxodG in kidney of HFD vs. C (1.2-fold) ↑ 8-oxodG in testes of HFD vs. C (1.2-fold) ↑ 8-oxodG in liver of HFD vs. C in 10 wks.(1.1-fold) ↓ 8-oxodG in liver of HFD vs. C in 2 and 8 wks. (25%)
11 (M)
82
Ref
↑ MDA in OB vs. C (3.0-fold) ( + ) correlation between DNA damage and basal plasma insulin values ↑ DSBs in sham surgery vs. C (3.0fold) ↑ γ-H2AX in OB rats (7.0-fold) ↓ after intervention by bypass surgery and caloric restriction (3.2-fold) ↑8-OxodG levels correlated with lower BER (not significant) ↓ BER activity in off-spring HFD vs. C in cortex (40%), hippocampus (40%) and subcortical regions (45%)
[101]
↑ MDA in liver of HFD vs. C (1.3fold) ↑ MDA in heart of HFD vs. C (1.2fold) ↔ MDA in kidneys and testes of HFD vs. C
[103]
[102]
I N U SC R
Table 10. Continued
Method/parameter Experimental design Results QS Remarks (indicator cells) 8-oxo-7,8-dihydro-2' -deoxyguanosine (8-oxodG) and 8-OH-Guanosine (8-OH-Gua) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo)
A
↓ 8-OH-Gua in OB vs. C (24%)
ED
HPLC with ultraviolet (liver)
n=46♀ obese fa/fa: n=20 lean (C): n=26 mice received 7,12 dimethylbenz(α)anthracene (DMBA) and after 155 days, liver removed.
M
Zucker rat
1
11 (M)
↑ ratio of glutathione/ oxidised glutathione disulphide in OB vs. C (1.3-fold) ↓ S-adenosylmethionine/Sadenosylhomocysteine levels (45%) ↑methionine in OB vs. C (1.3-fold) ↑5-methylcytosine in OB vs. C (1.1-fold)
Ref
[104]
A
CC E
PT
Abbreviations: C, controls; CAF, cafeteria diet; CBMN-Cyt, cytokinesis block micronucleus cytome assay; CRD, calorie restricted diet; DIO, diet induced obesity; DMBA, 7,12 dimethylbenz(α)anthracene, ELISA, enzyme linked immunosorbent assay; ENU, N-ethyl-N-nitrosourea; GSH/GSSG, glutathione/ oxidised glutathione disulphide; HFD: high fat diet; HPLC, high-performance liquid chromatography; MDA, malondialdehyde; n, number of participants; OB, obese; OGG1, 8-oxoguanine DNA glycosylase; OW, overweight; 8-oxodG, 8-oxo-7,8-dihydro-2' –deoxyguanosine; 8-OH-Gua, 8-OH-Guanosine; 8-oxoGuo, 8-oxo-7,8-dihydroguanosine; RSD, restricted diet; SC, standard conditions; STD, standard diet; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS) (For details see supplementary Table).
83
I N U SC R
Table 11. Results of Gamma-H2AX (γ-H2AX) in animal studies concerning the impact of obesity on DNA stability
Method (indicator cells)
Experimental design
Results
QS
Remarks
Ref
OB: n=22♂, weight 360g C: n=18♂, weight 300g either fasting or acute hyperglycemia ♂, weight 440 g, lean Zucker fa/+ rats (12 wks), weight 350g weight loss either via Roux-en-Y gastric bypass (RYGB) surgery (n=15), weight 410g or sham surgery (n=17), weight 580g
↑ γ-H2AX in OB vs. C (4.5-fold) ↑ γ-H2AX in OB/acute hyperglycemia vs. C/acute hyperglycemia (4.5-fold)
9 (L)
↑ insulin in OB vs. C (2.6-fold)
[105]
↑ DSBs in sham surgery vs. C (3.0-fold) ↑ γ-H2AX in OB rats (7.0-fold) and decrease after intervention by RYGB surgery and caloric restriction (3.2-fold)
[101]
♀, OB: n=10, lean (c): n=10, 15 wks in both the lean and obese groups were ip dosed once with SO and phosphoramide mustard (PM)
↑ γ-H2AX in OB (PM) vs. lean (PM) (1.5-fold) ↑ γ-H2AX in OB(PM) vs. OB (1.5-fold)
↑ 8-OxodG after RYGB vs. lean 11 (M) (2.5-fold) ↔ 8-OxodG in OB vs. lean ↑ 8-oxoGua in OB vs. lean (2.5-fold) ↑ 8-oxoGuo in OB vs. lean (3.0-fold) ↑ malondialdehyde in OB vs. lean (3.0-fold) (+) correlation DNA damage and basal plasma insulin ↔ DNA repair genes Prkdc, Parp1 9 (M) and Rad51 mRNA by obesity ↓relative Xrcc6 mRNA in OB vs. C (45%) ↑ relative Atm mRNA in OB vs. C (1.8-fold) ↑relative Brca1 mRNA in OB vs. C (1.3-fold)
A
Gamma-H2AX (γ-H2AX)
M
Zucker rat
OB Zucker fa/fa rat
CC E
PT
(colon and kidney)
ED
(lung)
A
Normal nonagouti (a/a; designated lean), agouti lethal yellow (KK.Cg-Ay/J; designated obese) Western blot (ovaries)
84
[106]
I Method/parameter (indicator cells) Miscellaneous
Experimental design
n=344♀ groups: LFD (3% fat) hydroxymethyluracil, weight=136 g, gas spectrometry HFD (20 % fat) weight 146 (liver and mammary g, dietary restriction (CRD, gland) 40 %) weight 113 g, C (5% fat) weight 137 g, for 2 wks. C57BL/6J mice n=36♂ fed either with HFD (60% Pig-α mutation, flow fat) or LFD (10% fat) for 18 cytometry wks, weight 28 g (in normochromatic treated either with N-ethylerythrocytes (NCEs) and N-nitrosourea (ENU) or reticulocytes(RET)) vehicle control for 3 days C57BL/6J mice n=7-9♂ fed either with HFD (60% quantitative alkaline fat) or LFD (10% fat) for 16 Southern blot wks. (liver and skeletal weight: muscles) HFD: 38g LFD: 28g
Results (fold)
QS
Remarks
Ref
↓ 5-hydroxymethyluracil in liver in CRD vs. C (43.3%) ↓ 5-hydroxymethyluracil in mammary gland in HFD vs. LFD (31.8%)
-
↓ body weight in CRD vs. HFD (28%) ↑ body weight in HFD vs. STD (1.1-fold) ↑ body weight in HFD vs. LFD (1.1-fold) ↑ body weight in HFD vs. CRD (1.3-fold)
[107]
↑ Pig-α mutation in RBCs in HFD vs. LFD (2.5-fold)
-
[100]
↑ DNA damage (mitochondrial) in HFD in liver and muscle
-
↔ MN-NCEs HFD vs. LFD ↔ MN RETs HFD vs. LFD ↔ MN after ENU treatment in HFD vs. LFD ↑ MN-NCEs after ENU treatment in HFD and LFD vs. control (1.1-fold) ↑ MN RETs after ENU treatment in HFD and LFD vs. control (4-fold) ↓ ratio glutathione/glutathione disulfide ↔APE1 in skeletal muscles in mitochondria base excision DNA repair: ↑ apurinic/apyrymidinic Endonuclease 1 in liver (2.0-fold) and skeletal muscles (1,8-fold) ,in nucleus in HFD ↑ OGG1 (8-oxoguanine DNA glycosylase/AP lyase), in skeletal muscles in nucleus (1.6-fold) and mitochondrial (1.5fold) in HFD vs. LFD ↔ OGG1 in liver (nucleus and mitochondrial) ↓ GSH/GSSG HFD vs. LFD in liver (2.0-fold) ↓ GSH/GSSG HFD vs. LFD in muscle (2.8-fold) ↑ free fatty acid HFD vs. LFD (2.0-fold) ↑ insulin HFD vs. LFD (2.0-fold) ↑ triglyceride HFD vs. LFD (2.5-fold)
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A
Fischer rats
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Table 11. Continued
85
[108]
I N U SC R
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Abbreviations: C, controls; CAF, cafeteria diet; CRD, calorie restricted diet; DIO, diet induced obesity; DMBA, 7,12 dimethylbenz(α)anthracene; ENU, N-ethyl-N-nitrosourea; GSH/GSSG, glutathione/ oxidised glutathione disulphide; HFD: high fat diet; MN, micronuclei; MDA, malondialdehyde; n, number of participants; OB, obese; OGG1, 8oxoguanine DNA glycosylase; OW, overweight; 8-oxodG, 8-oxo-7,8-dihydro-2' –deoxyguanosine; 8-OH-Gua, 8-oH-Guanosine; 8-oxoGuo, 8-oxo-7,8-dihydroguanosine; PM, Phosphoramide mustard; RET, reticulocytes; RSD, restricted diet; SC, standard conditions; STD, standard diet, TNF-α, tumor necrosis factor-alpha; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS) (For details see supplementary Table).
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Table 12. Results of animal studies concerning obesity related DNA damage in sperm
86
I Zucker rat
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Experimental design
Results
QS
Remarks
Ref
n=6 per group ♂ after 64 and 100 days OB: weight in day 64=344g, day100=490g C: weight in day 64=233g, day 100=344g
↑ DNA damage (tail length) in OB vs. C (1.3-fold) ↑ DNA damage (tail extent moment) in OB vs. C (1.8-fold) ↔ DNA damage (tail DNA and olive tail moment)
12 (M)
↑ daily sperm production in C vs. OB (1.3-fold) ↑ testes weight in C vs. OB (1.5-fold)
[113]
n=40♂, 6 wks old, HFD: n=7, STD: n=8 fed either STD (6% fat) or HFD (21% fat) weight after 18wks: HFD=34.2g, STD=28.3g
↑ sperm DNA damage (%) in OB vs. STD (1.6-fold)
10 (M)
[111]
n=36♂, 6 wks old, HFD: n=18, STD: n=18 fed either with STD (6% fat) or HFD (22% fat, 0.15% cholesterol) weight after 9 wks: HFD=30.0g, STD=29.4 g
↑ sperm DNA damage (%) in OB vs. STD (9.4-fold)
10 (M)
↑ mitochondrial ROS in OB vs. STD (1.1-fold) ↓ sperm mobility in HFD vs. STD (11%) ↑ abnormal sperm tail in OB vs. STD (1.2-fold) ↔ free fatty acid in OB vs. STD ↑ glucose in OB vs. C in OB vs. STD (1.0-fold) ↑ cholestrol in OB vs. C in OB vs. STD (1.6fold) ↑ triglyceride in OB vs. C in OB vs. STD (1.2fold) ↑ mitochondrial (ROS) in OB vs. STD (1.6fold) ↑ intracellular ROS in OB vs. STD (1.9-fold)
M
A
Animal/method/p arameter (indicator cells) Comet assay
C57BL/6J mice
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comet assay, tail length, tail extent moment, olive tail moment and tail DNA (testes) Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL assay)
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TUNEL assay, cell detection kit (testes)
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C57BL/6J mice
TUNEL assay, cell detection kit (testes)
Table 12. Continued
87
[110]
I N U SC R
Abbreviations: AU, arbitrary unit; BMI, body mass index; DFI, DNA fragmentation index; N, number; n.s. , not specified; OB, obese; OW, over weight; SCSA, sperm chromatin structure assay; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; UW, underweight; Y, year; high (H), medium (M) and low (L) refer to the classification
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SCSA, DNA fragmentation index (testes) C57BL/6J mice
♂, fed either STD (10% fat) or HFD (45 % fat) weight after 11 wks: HFD=33.6g, STD=29.4g n=9 to 14 per group
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comet assay (testes, blood and liver) SCSA, flow cytometry (sperm)
Results
QS
Remarks
Ref
↑ DFI in HFD vs. STD (1.1fold)
11 (M)
(+) correlation BMI and DFI (+) correlation between blood glucose and DFI
[112]
↑ DNA damage (single strand breaks and alkali-labile sites) in STD vs. HFD in liver ↔ DNA damage in testes and blood ↑ FPG in white blood cells and liver in STD vs. HFD (1.1-fold) ↔ DFI in HFD vs. STD
11 (M)
↑ haemoglobin glycidamide adduct levels in HFD vs. STD ↑ weight gain HFD vs. STD (1.5-fold)
[93]
M
C57BL/6J (wild type mice), C57BL/6J-ob/+ (leptin heterozygous mice) and C57BL/6J-ob/ob (Lepob; leptin- deficient mice)
A
Animal/method/parameter Experimental design (indicator cells) Sperm chromatin structure assay (SCSA)
n=6 per group ♂, 5-6 wks fed either STD (10% fat) or HFD (45 % fat) weight after 6 wks: HFD=31.7g, STD=28.1g
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on the basis of quality scoring (QS) (For details see supplementary Table).
88
S1383-5742(17)30096-0 https://doi.org/10.1016/j.mrrev.2018.07.001 MUTREV 8246
To appear in:
Mutation Research
Received date: Revised date: Accepted date:
27-10-2017 3-5-2018 6-7-2018
Please cite this article as: Setayesh T, Nersesyan A, Miˇs´ık M, Ferk F, Langie S, Andrade VM, Haslberger A, Knasmuller ¨ S, Impact of obesity and overweight on DNA stability: Few facts and many hypotheses, Mutation Research-Reviews in Mutation Research (2018), https://doi.org/10.1016/j.mrrev.2018.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Impact of obesity and overweight on DNA stability:
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few facts and many hypotheses
Tahereh Setayesh1, Armen Nersesyan1, Miroslav Mišík1, Franziska Ferk1, Sabine Langie2,
1.
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Vanessa M. Andrade3, Alexander Haslberger4, Siegfried Knasmüller1*
Institute of Cancer Research, Department of Medicine I, Medical University of Vienna,
Environmental Risk and Health Unit, Flemish Institute for Technological Research
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2.
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Vienna, Austria
Laboratório de Biologia Celulare Molecular, Programa de Pós-Graduação em Ciências da
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3.
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(VITO), Mol, Belgium
Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul
Department of Nutritional Sciences, University Vienna, Vienna, Austria
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4.
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Catarinense (UNESC), Brazil
* Corresponding author: Siegfried Knasmüller, Institute of Cancer Research, Department
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of Medicine I, Medical University of Vienna, Vienna, Austria, Tel: +43 1 4016057562; Fax: +43 1 40160957500; E-mail: [email protected]
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Abstract: Health authorities are alarmed worldwide about the increase of obesity and overweight in the last decades which lead to adverse health effects including inflammation, cancer, accelerated aging and infertility. We evaluated the state of knowledge concerning the impact of elevated body mass on genomic instability. Results of investigations with humans (39 studies) in which
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DNA damage was monitored in lymphocytes and sperm cells, are conflicting and probably as
a consequence of heterogeneous study designs and confounding factors (e.g. uncontrolled
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intake of vitamins and minerals and consumption of different food types). Results of animal studies with defined diets (23 studies) are more consistent and show that excess body fat
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causes DNA damage in multiple organs including brain, liver, colon and testes. Different
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molecular mechanisms may cause genetic instability in overweight/obese individuals. ROS
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formation and lipid peroxidation were found in several investigations and may be caused by
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increased insulin, fatty acid and glucose levels or indirectly via inflammation. Also reduced DNA repair and formation of advanced glycation end products may play a role but more data
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are required to draw firm conclusions. Reduction of telomere lengths and hormonal imbalances are characteristic for overweight/obesity but the former effects are delayed and
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moderate and hormonal effects were not investigated in regard to genomic instability in obese
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individuals. Increased BMI values affect also the activities of drug metabolizing enzymes which activate/detoxify genotoxic carcinogens, but no studies concerning the impact of these alterations of DNA damage in obese individuals are available. Overall, the knowledge
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concerning the impact of increased body weight and DNA damage is poor and further research is warranted to shed light on this important issue. Abbreviations: AGEs, advanced glycation end products; AP-1, activating protein -1; APE1, apurinic/apyrimidinic endonuclease 1; BER, base excision repair; BMI, body mass index; CAF, cafeteria diet; CAT, Catalase; CBMN-Cyt, cytokinesis block micronucleus cytome assay; CEL, carboxyl ester lipase; CML, Nε-carboxymethyllysine; CRP-1, C-reactive protein; DDR, DNA damage 2
response; DFI, DNA fragmentation index; DMBA, 7,12-dimethylbenz(α)anthracene; DNA, deoxyribonucleic acid; DNMTs, DNA methyltransferases; DSBs, double strand breaks; EGCG, Epigallocatechin gallate; ELISA, enzyme linked immunosorbent assay; ENDO III, endonuclease III; ER, estrogen receptor; FA, fatty acid; FACS, fluorescence-activated cell sorting; FPG, formamidopyrimidine DNA glycosylase; GPx, glutathione peroxidase; GSH/GSSG, glutathione/ oxidised glutathione disulphide; GSK-3β, glycogen synthase kinase-3β; GST, glutathione-Stransferase; HFD, high fat diet; Hras, harvey rat sarcoma virus oncogene 1; HPLC, high-performance
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liquid chromatography; 5hmU, 5-hydroxymethyluracil;4-HNE, 4-hydroxy-2-nonenal; HO-1, haem oxygenase-1; HUVEC, human umbilical vein endothelial cells; IGF, insulin-like growth factor; IL-6,
interleukin-6; IR, insulin receptor; IRAC, international agency for research cancer; JNK, Jun N-
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terminal kinase; LC-MC, liquid chromatography-mass spectrometry; LP, lipid peroxidation; MAPK,
mitogen activated protein kinase; MDA, malondialdehyde; MetS, metabolic syndrome; MLH-1, MutL homolog 1; MMR, mismatch repair; MN, micronuclei; NAT, N-acetyltransferase; NBs, nuclear bridges; Nbuds, nuclear buds; NER, nucleotide excision repair; NF-kB, nuclear factor kappa-light-
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chain-enhancer of activated B cells; NOX1, NADPH oxidase 1; 8-OHdG, 8-hydroxy-2'– deoxyguanosine; 8-oxodG, 8-oxo-7,8-dihydro-2' –deoxyguanosine; 8-OH-Gua, 8-OH-guanosine; 8-
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oxoGuo, 8-oxo-7,8-dihydroguanosine; 16α-OHE1, 16α-hydroxyestrone; PAHs, polycyclic aromatic
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hydrocarbons; PCNA, proliferating cell nuclear antigen; PDK1, phosphatidylinositol-4,5-
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bisphosphate 3-kinase; PI3K, phosphatidylinositol-3-kinase; PKC, proteinkinase C; PTEN, phosphatase and tensin homolog; RAGE, receptor advanced glycation end products; ROS, reactive oxygen species; SAM, S-adenosyl-L-methionine; SCGE, single cell gel electrophoresis assay; SDH,
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sorbitol dehydrogenase; SOD, superoxide dismutases; SSBs, single strand breaks; SCSA, sperm chromatin structure assay; TBARS, thiobarbituric reactive substances; T2D, type 2 diabetes; TdT,
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terminal deoxynucleotidyl transferase; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor; TP53, tumor suppressor gene p53; TUNEL, terminal deoxynucleotidyl transferase dUTP
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nick end labelling UGT, uridine diphosphate glucuronosyltransferase; YAP, yes-associated protein
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Keywords: overweight; obesity; DNA damage; genomic instability; somatic cells; germ cells
Contents 1.
Introduction ................................................................................................................... 4
2.
Data collection .............................................................................................................. 7 3
3.
Methods used in investigations concerning associations between genetic instability and overweight/obesity ................................................................................................ 8
4.
Human studies ............................................................................................................... 9 4.1. Studies with overweight/obese individuals ............................................................... 9 4.2. Studies with MetS patients ...................................................................................... 11 4.3. Impact of body weight on DNA damage in human sperm cells .............................. 12 4.4. Comprehensive evaluation of human studies .......................................................... 13 Results of animal studies ............................................................................................ 15
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5.
5.1. DNA damage in somatic cells ................................................................................. 15
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5.2. DNA damage in germ cells...................................................................................... 17 5.3. Comprehensive evaluation of animal studies .......................................................... 18 Impact of obesity/overweight on mutations in oncogenes .......................................... 18
7.
Results of in vitro studies............................................................................................ 19
8.
Molecular mechanisms ............................................................................................... 21
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6.
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8.1. Direct formation of ROS ......................................................................................... 22 8.2. Obesity induced lipid peroxidation and DNA damage ............................................ 25
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8.3. Impact of overweight on sex hormones ................................................................... 26
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8.4. Glycation end products ............................................................................................ 29 8.5. Impact of obesity on DNA repair ............................................................................ 30
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8.6. Obesity and telomere lengths................................................................................... 33 8.7. Impact of obesity/overweight on the activities of enzymes that activate and detoxify mutagens .................................................................................................... 35
9.
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8.8. Inflammation and DNA damage .............................................................................. 37 Consequences of obesity induced DNA damage ........................................................ 38
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10. Conclusions and knowledge gaps ............................................................................... 40 Conflict of interest statement ............................................................................................. 43
Acknowledgments.................................................................................................................. 43
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References .............................................................................................................................. 44
1.
Introduction Obesity and overweight are characterized by abnormal accumulation of body mass and
are associated with increased health risks, in particular with diseases such as type 2 diabetes 4
(T2D), cardiovascular diseases, polycystic ovary syndrome, infertility and cancer [1]. Scientists and health authorities are alarmed by the fact that the increase of overweight/obesity is a worldwide phenomenon. A global estimate indicates that about 640 million adults were obese in 2014, this number corresponds to a 6-fold increase since 1975 [2]. Obesity has a prevalence of 10.8 % among men and 14.9 % among women [3]. The worldwide incidence
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of childhood overweight and obesity increased from 4.2% in 1990 to 6.7% in 2010. This trend is expected to reach 9.1% or 60 million, in 2020 [4].
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A typical feature of excess body weight is the formation of ROS and of cytokines which are characteristic for inflammation. These processes lead to damage of the genetic material
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which in turn plays a key role in the induction of cancer and infertility; these disorders are
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increased in overweight/obese humans [5, 6]. Aim of this article is to describe the current
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state of knowledge of the relation between excess body weight and genomic instability.
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Recently, several studies have been published which concern the association between MetS and DNA damage [7-9], however they are based on mechanistic considerations but not on a
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systematic evaluation of experimental data. The first part of the present paper focuses on the description of results of animal and human studies in which different parameters of DNA
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instability and chromosomal damage were measured in overweight/obese individuals in somatic and germ cells. Furthermore we describe also measurements which were performed
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in these studies concerning biological processes that lead to DNA stability such as inhibition of DNA repair processes, decrease of the telomere lengths, changes of the redox status and of
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the activities of antioxidant enzymes, lipid peroxidation and of the immune status. The association between overweight/obesity and cancer is well documented; it is, apart
from cardiovascular diseases, the most important health consequence of excess body weight. In 2016, the IARC published a report which is based on the evaluation of more than 1000 individual epidemiological studies. The Agency comes to the conclusion that the absence of 5
excess body fatness lower the risks of most cancer [2]. A comprehensive study from the US states that 14% of all cancer cases in man and 20% in women are due to obesity [10]. Numerous investigations showed that gene mutations as well as chromosomal aberrations play a key role in malignant transformation [11, 12]. Nevertheless, it is apparent
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that most studies which concerned the mechanisms by which excess body weight leads to cancer focused on other modes of action such as alterations of signaling pathways, increased
production of growth factors and hormones and activation of transcription factors (for reviews
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see [13-15]).
In the last decades, a variety of methods was developed which enable the investigation
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of DNA stability in human and animal models [16]. These approaches were frequently used
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to study occupational, lifestyle and dietary factors [16-20]. Furthermore, in vitro assays [21]
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and rodent mutagenicity tests [22, 23] are conducted worldwide for routine monitoring of
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chemicals. These models can be also used to identify drugs and dietary components which protect humans against DNA damage and its consequences [16, 24, 25] .
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This review describes human and animal studies which concern the impact of overweight/obesity on DNA integrity. The results of these investigations may contribute to a
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better understanding of the mechanisms by which excess body weight causes adverse health
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consequences such as cancer and infertility and can also contribute to the development of strategies to prevent these effects. We included also investigations concerning the MetS, which is the most important consequence of abdominal obesity. Studies concerning T2D and
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the polycystic ovary syndrome were excluded; these disorders are related to obesity but are characterized by severe additional pathological changes, which may have an impact on DNA stability but are not attributable to excess body weight [26, 27]. The following chapter defines the search strategy (chapter 2) and the methods which were used (chapter 3). The subsequent paragraphs describe investigations with humans 6
(chapter 4) and animals (chapter 5), the impact of obesity on mutations in oncogenes (chapter 6) and in vitro studies (chapter 7). Furthermore we describe the molecular mechanisms by which overweight/obesity may cause DNA damage (chapter 8), the consequence of obesity induced DNA damage and the links between DNA damage and cancer (chapter 9) and define
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knowledge gaps and future research strategies (chapter 10).
2. Data collection
Library
of
Medicine,
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The literature search was performed by using the MedLine/PubMed data-base (National http://www.ncbi.nlm.nih.gov/PubMed),
SCOPUS
(Elsevier,
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http://www.scopus.com), Thomson ISI’s Web of Science (Thomson Reuters Corporation,
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http://apps.webofknowl-edge.com), Google Scholar (http://www.google.scholar.-com) and
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covered the period between December 1961 and August 2017.
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Additionally, a manual search of the reference list of studies and of reviews was conducted. References of retrieved articles were analysed to identify further publications
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which may have been missed. Only articles in English were considered. The keywords and combinations of search terms were: body mass index, chromosomal aberrations,
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chromosomal damage, comet assay, DNA damage, DNA adducts, DNA repair, DNA
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instability, γ-H2AX, genomic instability, metabolic syndrome, micronuclei, mutation, obesity, 8-OHdG, 8-oxodG; overweight, single cell gel electrophoresis (SCGE), sister chromatid exchange, sperm DNA integrity, telomere length and TUNEL assay.
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The quality of the individual studies was assessed by a quality score (QS) system, which
is similar to a model which was used for MN- studies with occupationally exposed humans [28]. A detailed description of this system is described in the section “supplementary information”. According to the criteria, the studies were classified and grouped in different
7
categories (Low-, Medium- and High- quality). The score and the categories are indicated in the supplementary tables S1 and S2. Exclusion criteria for human studies were as follows (i) an insufficient number of participants (less than 20 individuals), (ii) lack of information concerning the number of
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individuals per group, (iii) lack of information concerning the number of scored cell, (iv) an insufficient number of evaluated cells (<1000 per experimental point in MN studies, in comet
assays <100 cells per experimental point, in TUNEL tests <200 and in SCSA experiments
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<5000 cells per experimental point respectively and (v) small differences in the BMI-values of different study group (≤0.5%). Exclusion criteria for animal studies were (i) an insufficient
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number of animals per group (less than 3) and (ii) an inadequate number of scored cells
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(<1000 per experimental point in MN studies with bone marrow and lymphocytes, in comet
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assays <100 cells per experimental point, in TUNEL tests <200 and in SCSA experiments
Methods used in investigations concerning associations between genetic instability
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3.
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addition of the individual scores).
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<5000 cells per experimental point respectively The overall QS values were determined by
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and overweight/obesity
Figure 1 gives an overview of the methods which are used in studies with overweight/
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obese humans and laboratory animals.
Figure 1
Table. 1 describes different methods which were used to study impact of overweight/obesity on DNA stability. Most of these approaches are also used to investigate the effect of 8
chemicals, lifestyle factors and chemical exposure in somatic cells, for studies with sperm cells additional test systems such as TUNEL assay and SCS assays were employed.
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The most frequently method in human studies are comet assay experiments with and without restriction enzymes (in total 12 studies were published) followed by measurement of
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8-OHdG (7 studies), MN (8 studies), only two publications concern γ-H2X measurements.
Also in animal studies, comet assays are dominating (8 investigation), followed by 8-
Human studies
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4.
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OHdG or 8-oxodG (4 studies) and by MN and γ-H2X (3 studies per endpoints).
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The following chapters describe results which were obtained with somatic cells of healthy individuals (chapter 4.1) and MetS patients (chapter 4.2), chapter 4.3 summarizes the
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human studies.
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results with sperm cells and the last paragraph contains a comprehensive evaluation of the
4.1. Studies with overweight/obese individuals
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Only relatively few studies (in total 22) were realized which focused on the impact of
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body weight on genomic instability in somatic cells (Table. 2 to 5).
In five out of six comet assay trials with parallel design, increased DNA migration was
seen in lymphocytes of overweight/obese individuals [41-45]. In MN experiments lymphocytes, positive effects were found only in two of five studies with parallel design [47, 50]. Other markers of genomic instability (Nbuds and NBs) which were additionally scored
9
in a MN study by Donmez-Altuntas et al. [47] were also increased while no association was found between MN induction and formation of oxidised guanine (8-OHdG) levels in plasma [47]. Another endpoint which was elevated in the study by Scarpato et al. was γ-H2AX [50] Two studies were conducted with buccal cells. Unexpectedly the authors found in Malaysian farmers, which were exposed to pesticides and fertilizers, higher MN-rates in
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individuals with normal BMI compared to participants with higher body weights [51]. A second trial was realized with school children in Mexico and no differences were seen
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between normal, obese and overweight participants [52]. In the latter study a Giemsa based
stain was used which may leads to false positive results [60] and in both trials [51, 52] only
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1000 instead of 2000 cells were scored per participants which is not in agreement with the
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standard protocol [61].
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Table 2. describes also the results of several intervention trials. Clear reduction of DNA
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damage was seen in a study with calorie restriction [38]. No clear effect was seen in an intervention trial in which comparisons were made between consumption of red meat and a
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high carbohydrate diet [46]. A clear difference of the rate of Nbuds was observed at the end of this study but no differences between meat and carbohydrate consumption were detected.
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It is mentioned that the weight loss at the end of intervention was significant but the extent is not specified [46]. The most recent study was conducted with bariatric surgery patients
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(n=56); the authors found no alteration of the comet formation in lymphocytes after 6 months after the surgery, but reduced DNA damage was observed after 1year [39].
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Table 5. concerns findings from experiments in which phosphorylation of H2X (which
is indicative for DSBs) was monitored. Seven studies concerned formation of oxidised guanosin in plasma, whole blood or urine. In three trials a positive association between base oxidation and overweight was found [54, 56, 57], three studies reported an inverse relation [47, 53, 55]. No effect was seen in an Italian study; but clear evidence for formation of FPG
10
sensitive sites was detected in the same trial [41]. Two γ-H2AX studies were performed with lymphocytes and significant correlations were found in both trials [50, 58]. The last line of Table 5. summarizes the results of a study concerning associations between telomere lengths and damage of DNA bases before and after calorie restriction. The authors found increased telomere lengths and in parallel reduced formation of abasic sites
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which were monitored with a commercial test kit in rectal biopsy samples. This assay was rarely used and not standardized [59].
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It is notable, that changes which were seen in intervention trials are not necessarily due to reduction of the BMI but may be caused by other factors, i.e. by alterations of the intake of
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vitamins and macronutrients which have an impact on genomic instability [62].
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4.2. Studies with MetS patients
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Table 6. summarizes the results of currently available studies. Five investigations concerned comparisons between overweight MetS patients and healthy individuals. Karaman
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et al. [63] and Demiberg et al. [64] found evidence for increased comet formation in lymphocytes with higher BMI values. Furthermore, also evidence for increased MN
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frequencies and higher levels of malondialdehyde (MDA) and decreased activities of
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antioxidant enzymes was found in one of the studies [63]. On the contrary, no elevated comet formation was seen in a trial which was realized in Croatia [65]; the authors hypothesized that the lack of an effect may be due to the fact that the study was performed with young
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participants who had a high repair capacity. The most recent study concerned DNA damage in morbidly obese women [45]. The
authors found no evidence for increased DNA damage in lymphocytes, also oxidised purines (FPG-sensitive lesions) were not increased. Interestingly, the number of individuals with gene
11
polymorphism in the fat mass and obesity associated gene (AA-FTO) was higher in the obese group, while another polymorphism in the same gene (TT-FTO) was found less frequently. Results of 8-OHdG measurements were performed in two MetS studies. Abdilla and co-workers [66] monitored several endpoints in different MetS groups and in healthy controls and detected no differences while Milić et al. [65] found even lower levels of 8-OHdG in the
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urine of patients compared to the controls; they hypothesized that this unexpected result may
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be due an adaptive response.
4.3. Impact of body weight on DNA damage in human sperm cells
The findings of different studies are listed in Table 7. The results of comet experiments
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are controversial. No effects were seen in a trial in which the neutral version (which detects
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mainly DSBs and cross-links) was used [67], while clear differences were found in a study
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from Brazil with the standard protocol i.e. under alkaline conditions which detect DSBs, SSBs
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and apurinic sites [68].
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The results of TUNEL assays indicate that differences exist between obese and normal weight individuals while no effects were found between overweight and lean participants [69,
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70]. Vignera et al. [69] analysed additional parameters and found a higher rate of sperm cells
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with increased chromatin compactness in the former group. The authors postulated that this is a sign of early uncontrolled apoptosis. This assumption was supported by the observation of increased externalization of phosphatidylserine and reduced mitochondrial membrane
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potential as a consequence of elevated body weight. These processes lead to programmed cell death and DNA fragmentation [78]. Interestingly, an increase of the DFI by 50% was observed in obese but not in overweight individuals in a large study (n=330) by Dupont et al. [70].
12
The results of 7 studies in which the effects of body weight were analysed in SCSA assays are controversial. The findings of Bandel et al. [79] are unexpected as the authors found an inverse association between BMI values and sperm DNA integrity, also the motility of the cells increased with the body weight. No such effects were found in several other trials [71, 73-76].
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Two further studies are notable; one is an intervention trial in which the DFI of the
participants was compared after a weight loss period of 14 weeks in three groups. Several
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sperm parameters were improved after weight reduction, also the DFI values were lower at
the end of the trial. The strongest reduction of this parameter (from 18 to 10) was found in the
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group with the highest BMI values (46.1-60.9), however the effect did not reach significance
N
[71].
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An interesting study from Denmark concerned the impact of the BMI of mothers on the
M
DFI in sperm of the sons. Higher values were found in the offspring of overweight mothers compared to mothers with normal weight [72].
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4.4. Comprehensive evaluation of human studies
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The main methods which were used in human studies are MN assays and the comet experiments. It is notable that MN reflect persisting chromosomal aberration, while “comets”
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are caused by SSBs and DSBs and apurinic sites and may disappear as a consequence of repair processes. For MN studies a standard protocol is available and MN can be easily identified [80]. As described above, MN experiments yielded conflicting findings, only 3 out of 8
A
investigations found positive correlations with the BMI. In comet experiments different parameters (tail intensity (synonymous with % DNA in tail), tail moment and olive tail moment) can be measured by automated scoring systems while other authors use “manual scoring” and determine DNA migration by definition of arbitrary units. All these parameters were found acceptable, however it was emphasised by various expert groups that tail intensity 13
(%DNA in tail) is the most reliable one [23, 81, 82]. While studies which used this marker yielded strongly controversial results (6 negative, 9 positive) consistently positive findings were obtained in all manual scoring experiments which may be indicative for a biased evaluation.
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Also the results of studies which concerned the induction of DNA damage in sperm are conflicting. It is difficult to assess which endpoints are the most reliable ones (SCSA, TUNEL assay and comet assay). It was emphasized that the different techniques reflect different
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aspects i.e. the TUNEL assay and the comet assay are indicative for “real” damage while the
SCSA reflects the susceptibility of the DNA to denaturation [83]. Only for the later assay a
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standardized protocol is available [84].
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In the case of experiments which concern the quantification of the oxidation of DNA
A
bases not only differences of antioxidants in the diet but also methodological aspects may
M
play a role. It was emphasize that HPLC measurements are more reliable as compared to
ED
ELISA based measurements [85]. Furthermore, 8-oxodG and oxoGuo were monitored either in urine, plasma or in different target organs which may add to heterogeneity of the results.
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The calculation of the QS shows that the quality of the most studies is in a relatively
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narrow range, therefore it is not possible to explain the conflicting results by shortcomings of the study design of the individual studies. As described in more detail in chapter 10 none of the investigations which were published so far was controlled in regard to the mineral and
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vitamin intake of the participants which may have a strong impact on the results. It is conceivable that the composition of the foods in lean and overweight human has a strong impact on the outcome of the studies, also weight loss programs are based on substantial changes of the dietary patents.
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5. Results of animal studies 5.1. DNA damage in somatic cells Different models were used in experiments with laboratory rodents. In most trials, the
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animals were fed with a high fat diet (HFD) or a cafeteria diet (CAF) for several weeks which
lead to an increase of the body fat and also to excess body weight. These studies were mainly
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conducted with C57BL/6j mice; these animals develop symptoms which are characteristic for
obese humans namely hyperinsulinemia, hyperglycemia, and hypertension [86]. In some
U
studies Zucker rats were used; obesity is in these animals an autosomal recessive trait and
N
resembles the early onset of human obesity [87] i.e. the animals have hyperlipidaemia,
A
hyperinsulinemia and peripheral insulin resistance. In one study, WNIN/Ob rats were used
M
which are hyperglycaemic and insulin–resistant and reflect the metabolic syndrome in humans [88].
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The first paper which described chromosomal aberrations as a consequence of obesity was published by the Swedish scientist Karl Fredga [89] more than 50 years ago. He observed
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in a pilot study (2 animals per group) that a specific strain of hyperglycaemic mice had
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aberrant numbers of chromosomes in epithelial cells from the cornea. Our literature search identified 18 further studies which concerned the effects of
overweight on DNA stability in somatic cells (Table 8. to 11.). Results of all comet trials
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(n=8) indicate that excess body fat causes genomic instability in a variety of inner organs [88, 90-96]. Tenorio and co-workers [94] analysed DNA migration in different tissues of rats and found larger comets in liver and lymphocytes but not in kidneys, brain and heart. Also in a Brazilian study [96] clear evidence for increased DNA damage was seen in obese mice which were fed with a carbohydrate and fat rich cafeteria diet (consisting of various supermarket
15
products). The consumption of this chow induced glucose intolerance and DNA damage in lymphocytes, kidneys, liver and in the brain [96]. A positive result was also obtained in MN experiments with bone marrow cells in the same study [96]. Also in HFD fed mice induction of DNA–migration was detected in liver and colon in a number of studies [90-93]. Remely et al. [90, 91] found an effect in both organs; Setayesh et al. [92] detected additionally increased
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damage in brain, liver and colons while no significant induction of DNA migration was
observed in blood cells and adipose tissue. Gutzkow et al. [93], analysed in a more recent
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study the extent of damage in different cell types (liver, blood and testes) of HFD fed mice.
They found in standard comet assays only in the liver an effect; furthermore an increase of
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formation of oxidised purines was detected in hepatocytes and also in white blood cells. Table
N
8. describes the results of a study with genetically modified rats, which are a model for the
A
MetS. Clear evidence for increased damage was observed in different regions of the brain.
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The authors state that the effects which they observed in 3-months old obese rats are similar to those seen in 15-months old parental control animals and conclude that obesity leads to
ED
premature ageing [88].
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Also in γ-H2AX measurements which are indicative for DSBs, clear effects were seen in livers and colons of obese animals [101]. Furthermore, alterations of the urinary excretion
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of 8-oxodG were monitored in the same trial. All these markers of DNA damage were decreased as a consequence of weight loss after gastric bypass surgery [101]. Another γ-
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H2AX study was published with obese Zucker rats; the authors found clear induction of DSBs in the lungs of the animals when they conducted the measurements after intraperitoneal injection of glucose, which leads to hyperglycemia, but not in fasting animals [105]. A further γ-H2AX study concerned the relation between DSB induction in ovaries and alterations of DNA repair proteins [106]. Also in a study with obese rats in which 5-hydroxymethyluracil
16
was measured in the livers and in the mammary glands, a protective effect was detected after calorie restriction [107]. Most studies, which are available so far were carried out with nuclear DNA; only few investigations concerned on mitochondrial DNA [103, 104, 108]. In one of them, the authors measured damage in liver and skeletal muscles of mice and detected pronounced differences
in liver, heart, kidney and testes of HFD mice were detected [103].
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between obese and normal weight animals [108]. In another study, higher levels of 8-oxodG
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Table 10. describes also findings of a study in which obese and lean rats were treated
with the breast carcinogen 7,12-dimethylbenz(α)anthracene (DMBA). The authors found in
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obese animals increased oxidative damage (8-OH-guanosine) in the liver and in parallel an
N
altered ratio of reduced to glutathione/oxidised glutathione disulphide (GSH/GSSG), which
A
is indicative for oxidative stress. This effect was was paralleled by changes of the overall
DNA damage in germ cells
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5.2.
M
DNA methylation status [104].
We found in total five animal studies which concerned the effects of obesity on sperm
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integrity. In four of them clear associations were observed, regardless if the damage was
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quantified in comet assay, TUNEL assays or SCSA experiments [93, 109-112]. Only in one study, the authors did not find an increase of DNA damage in testes of HFD mice; it is notable that the differences of the body weights of the experimental groups were only marginal in this
A
study [93].
17
5.3.
Comprehensive evaluation of animal studies In contrast to the conflicting findings which were obtained in the human studies in
lymphocytes and sperm, consistent results were found in animal studies with rodents, i.e. most of the comet experiments which have been published so far with somatic and sperm cells yielded positive results. The reasons for the unequivocal results of experiments with rodents
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are probably due the use of indicator organisms with a similar genetic background and feeding
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of standardized diets.
In contrast to the findings of comet assay studies, several MN experiments with cells from the haematopoietic system failed to detect an association between DNA damage and
U
increased body weight. The reason for the discrepancy to the comet results may be differences
N
in the sensitivity of the tissues which were analyzed in comet assay and MN experiments and
A
not differences in the sensitivity of the endpoints. Sasaki et al. [114] stressed that 54 out of
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165 proven rodent carcinogens were not detected in MN experiments with haematopoietic
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cells and that 49 of these chemicals (which gave false negative results) were positive in comet
Impact of obesity/overweight on mutations in oncogenes
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6.
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assays in at least one or more organs in mice in comet experiments.
Only few studies concerned mutations in cancer associated genes of laboratory rodents
and humans. These investigations are important as they enable to establish direct causal links
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between overweight induced genomic instability and malignant transformation. Shen et al. [115] found in whole genome sequencing experiments with tissue from
hepatocellular carcinomas of obese mice increased rates of mutations in the carboxyl ester lipase (Cel) gene and in the Harvey rat sarcoma virus oncogene 1 (Hras). In Cel, multiple mutations were detected which lead to functional loss, while in Hras single mutations were 18
found in a specific codon (61). Mutant Cel causes accumulation of cholesteryl ester in liver cells which leads to induction of endoplasmic reticulum stress and consequently activates IRE1 α/c-Jun N-terminal kinase (JNK)/c-Jun/activating protein -1 (AP-1) signalling cascade to promote liver cell growth. In the case of Hras, gain of function mutations promote liver cell growth through activation of mitogen-activated protein kinase (MAPK) and
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phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt pathways [116].
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A study on Kras mutations in colon cancer found in colon tissue of obese and lean
patients differences in the mutation rates in several genes [117]. The most striking observation was a lower number of mutations in patients with high BMI. The authors hypothesised that
N
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the reduced number found in “driver genes” of overweight individuals is due to the fact that mutations in genes which lead to malignant cells are not required in obese patients as in this
M
A
case “other alterations of signalling pathways” may substitute them [117]. The impact of overweight on tumor suppressor gene p53 mutations was analysed by
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Ecke et al. [118] in bladder cancer patients and no impact of the BMI on the mutation
Results of in vitro studies
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7.
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frequencies was detected.
It is not possible to investigate biochemical changes which are caused by excessive
calorie intake and elevated body weight under in vitro conditions. Nevertheless, results of
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studies were published with mammalian and human cells which reflect physiological conditions that are characteristic for obese individuals such as increased concentrations of glucose, fatty acids and insulin. These findings contribute to a better understanding of the mechanisms by which overweight/obesity lead to the damage of the genetic material.
19
Cavazos et al. [119] exposed human prostate cells to serum from obese and non-obese mice. They monitored induction of DSBs in γ-H2AX experiments and found increased damage in malignant and non-malignant cells with sera from overweight animals. This effect correlated with ROS formation; furthermore, several markers of aerobic glycolysis were induced. The authors found that a potent antioxidant (N-tert-butylhydroxylamine) prevents
involved in aerobic glycolysis) and formation of γ-H2AX foci [119].
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induction of hypoxia-inducible factor 1-α (a transcriptional regulator of genes which is
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The effects of insulin were investigated in several studies by Othman et al. [120-122]. They observed induction of DNA damage in colon derived cells (HT29) and also in
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lymphocytes in MN and comet experiments and explained these effects by intracellular ROS
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formation [120, 122]. Also with primary kidney cells, positive results were obtained.
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Subsequent in vivo studies with rats confirmed the assumption that insulin causes ROS
M
formation [120, 121]. Results of a further experiment in which the authors investigated the mechanisms of insulin induced DNA damage are described in chapter 7.
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Several investigations concerned the impact of glucose, which is increased in the blood of diabetic patients, but also as a consequence of consumption of carbohydrate and sugar rich
PT
diets. Lorenzi et al. [123] studied the effects of glucose on induction of DNA damage with a fluorometric technique which detects the unwinding of DNA in endothelial cells from
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umbilical veins and human fibroblasts already 30 years ago. They found positive results in the former cell type; furthermore they detected also increased hydroxyurea incorporation of
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radio-labelled thymidine which is indicative for DNA repair. Quagliaro et al. [124] used human umbilical vein endothelial cells (HUVEC) and found evidence for formation of 8OHdG as well as increased concentrations of nitrotyrosine (an indicator of NO· formation) and apoptosis as a result of high glucose levels [124]. The authors postulated that the oxidative stress is caused by protein kinase C (PKC) dependent activation of NAD(P)H oxidase. This
20
mechanism was also proposed by a Japanese group which worked with aortic smooth muscle and endothelial cells [125]. The findings of the latter studies are in contrast to results of Shimoi et al. [126], they found in HUVEC induction of oxidised purines but failed to explain these effects by formation of ROS formation and postulated that this phenomenon is caused by glyoxal.
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Increased levels of fatty acids (FA) are typical for obese and diabetic individuals.It was reported by Beeharry et al. [127] that palmitic acid (a major saturated FA in plasma) causes
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comet formation in an insulin producing cell line and also in fibroblasts. This effect was
inhibited by linoleic acid and the authors postulated that the protection is due to prevention of
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oxidative stress. The assumption of ROS formation from palmitate was confirmed by
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Inoguchi et al. [125] who found in electron spin resonance spectroscopy experiments evidence
8.
Molecular mechanisms
M
A
for the involvement of PKC.
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The following paragraphs concern mechanisms which may play a role in the association between excess body weight and genomic instability (Figure 2). Apart from enzymatic
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processes which lead to formation of ROS also other modes of action could be involved. Lipid
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peroxidation (LP) processes are induced by ROS and lead to formation of DNA reactive products. Furthermore, inhibition of DNA repair and induction of inflammation have been found in a number of studies. Also formation of advanced glycation products, hormonal
A
effects and telomere shortening are phenomena which may lead to genomic instability.
Figure 2
21
8.1.
Direct formation of ROS The evaluation of human and animal studies (Tables 2-5 and 8-11) shows that in some
of them evidence for induction of oxidative DNA damage and/or decreased total antioxidant capacity and induction of antioxidant enzymes was observed [41, 53, 54, 56, 57, 63, 101, 103, 108]; also increased LP by ROS was found in a number of investigations (see chapter 8.2).
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The results of Othman et al. [120] concerning the effects of insulin are of particular
interest as the authors measured in their experiments also genetic damage in comet assays. By
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use of specific inhibitors they developed a model for the induction of DNA migration in colon and kidney cells (Figure. 4). Insulin binds either to its receptor (IR) or to the insulin-like growth
factor-1 (IGF-1) receptor. After internalization of the receptor-insulin-complex into the cells,
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the hormone is digested by proteases. Binding to both types of receptors leads to activation
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of PI3K. Results with a specific PI3K inhibitor showed that this enzyme plays a key role in
A
signalling and also in the induction of genomic damage by insulin. The subsequent step is the
M
activation of Akt by PI3K which translocates from the membrane to the nucleus where it
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phosphorylates a wide array of downstream proteins.
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Figure 3
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The reduction of PI3K/Akt levels which was seen few hours after insulin exposure was
explained as a consequence of activation of p53 which controls the phosphatase and tensin
A
homolog (PTEN), a deactivator of Akt and PI3K. Two pathways lead subsequently to ROS formation: (i) insulin enhances the translocation of Akt into mitochondria where it causes ROS formation; (ii) another pathway is the activation of NADPH-oxidase. Different isoforms of this enzyme are involved in specific cell types. While NADPH oxidase 1 (NOX1) was found to be activated in colon cells, NOX4 is active in the kidney.
22
In this context it is notable that is known that cancer cells produce their energy by increasing the rate of anaerobic glycolysis (Warburg-Effect), therefore it is possible that elevated glucose levels may accelerate the proliferation of transformed cells [128]. This phenomenon has been demonstrated under in vitro conditions, and it is notable in this context that lower blood glucose levels in cancer patients correlated with better outcomes [129].
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Increased glucose levels in blood lead to ROS formation via different pathways. (i) In
cultured vasoendothelial cells, radical formation was observed via PKC-dependent activation
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of NADPH-oxidase (Figure 4) [124]. (ii) Glycoxidation products are formed as a consequence
of sequential glycation and oxidation reactions between proteins and reducing sugars [130]. (iii) Furthermore, an alteration of the ratio of NADH/NAD+ was found in hyperglycaemic
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tissues. This condition is also termed “pseudohypoxia” as the tissue levels of oxygen are
N
normal and is characterized by increased production of O2· and NO·[131]. (iv) Finally,
M
A
intracellular oxidation processes may cause O2· formation in mitochondria. This process starts with glycolysis in the cytoplasm which generates NADH and pyruvate. Cytoplasmic NADH
ED
can donate reducing equivalents to the transport chain in the mitochondria. Pyruvate is reduced in the cytoplasm to lactate or migrates into the mitochondria. Two main sites in these
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organelles generate O2· namely the NADH-dehydrogenase complex I and the interface
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between ubiquinone and complex III [132].
Figure 4
The assumption of a relation between insulin levels and DNA damage is supported by
the results of a calorie restriction study; the authors found lower hormone levels and in parallel less DNA damage at the end of the intervention phase. Notably, also the blood glucose levels were reduced at the end of the trial [38]. Also in a Korean study associations were detected
23
between insulin and glucose levels in blood and comet formation [42]. Furthermore, it is notable that correlations between DNA damage in sperm and insulin and glucose levels were found in the blood of mice [111]. It is well known that lipid levels are elevated as a consequence of excess consumption of fat and in metabolic diseases [133]. In this context, it is notable that palmitate, which is the
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most abundant fatty acid in plasma was shown to increase the activity of PKC and to cause ROS production [125].
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Only few studies measured ROS formation in obese and lean individuals and monitored its relation to DNA damage. Demirbag et al. [64] found reduced total antioxidant capacity in
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a study with MetS patients and in parallel increased comet formation in lymphocytes. Higher
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mitochondrial ROS levels in sperm cells were found in two studies (i.e. in a human
A
intervention trial and in an experiment with mice); in both, elevated DNA damage was
M
detected [110, 111].
Also investigations which concerned the reduction of the activities of antioxidant
ED
enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-Stransferase (GST)) and of oxidised glutathione as a consequence of excess body weight
PT
provide evidence for oxidative stress. Such effects were seen in a number of studies with humans [55, 63, 88, 108]. Also in the brain activities of SOD and catalase (CAT) were
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decreased in a rat model which reflects the MetS in humans; in parallel higher MDA levels were found. These effects correlated with induction of DNA damage in comet experiments
A
[88].
In a study with 7,12-dimethylbenzo anthracene (DMBA; a polycyclic aromatic
hydrocarbon which causes breast cancer) reduction of the ratio of oxidised/reduced glutathione (GSH/GSSG) which is indicative for oxidative stress was observed in obese rats. This effect was paralleled by increased 8-OHdG levels [104].
24
Another marker of oxidative stress are protein carbonyl concentrations in serum [134], no alterations in this marker were seen in a 6-months calorie restriction study with overweight individuals [38].
8.2. Obesity induced lipid peroxidation and DNA damage
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Oxidative stress leads to oxidation of fatty acids in biological membranes; as a consequence of this chain reaction, lipid peroxides and alkoxyl radicals as well as aldehydes
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and ketones are formed [135]. Some of these molecules attack DNA and were postulated to
play a key role in the aetiology of cancer and other diseases [136]. The formation of DNA adducts by LP products for example by MDA, acrolein, 4-hydroxy-2-nonenal (4-HNE) and
U
other compounds are described in recent reviews [137, 138]. Elevated levels of different LP
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markers were repeatedly seen in blood and muscles and also in adipose tissue of obese
M
A
individuals [42, 56, 63, 88, 95, 101, 103, 139-143].
The most commonly monitored biomarkers which are used in LP studies are MDA,
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thiobarbituric reactive substances (TBARs), hydroperoxides, 4-HNE, isoprostanes and
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conjugated dienes [134]. We found only few publications in which LP markers and DNA stability were monitored in parallel in obese/overweight and in normal weight subjects.
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Karaman et al. [63] conducted such a study in Turkey; they found increased MDA levels in individuals with MetS and also higher MN rates in peripheral lymphocytes. Also in experiments with obese rats evidence for an association between MDA formation and DNA
A
damage was observed [95]. In a study from Poland, the serum levels of MDA and 4hydroxyalkenals were measured as an index for LP, in parallel 8-oxodG concentrations were determined in blood [56]. The authors found that both parameters are associated with the BMI and with hip circumference. Furthermore, correlations between LP and 8-oxodG were observed. 25
Notably, also a clear relation between isoprostane (8-epi-prostaglandin F2α) concentrations in urine and MDA in plasma (which are both markers for LP) and comet formation in lymphocytes was seen in a study which focused on the effect of visceral body fat in non-obese Korean men [42]. Yu et al. [103] compared the association between MDA in different organs of rats which
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received a HFD and formation of oxidised DNA (8-OHdG) in mitochondria. The LP product was increased in heart, kidney, testes and liver tissue after 10 weeks but in the hepatic tissue
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even a decrease was observed after 8 weeks.
A large number of studies focused exclusively on the impact of obesity on LP markers
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but not on genomic stability. In total, about twenty papers have been published and in most
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of them evidence for increased LP as a consequence of excess body weight was found (for
A
review see [139]).
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Also mitochondrial membranes are attacked by ROS in overweight individuals and in MetS patients. This process leads to alterations of proteins which are involved in the electron
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transport chain. As a consequence, increased release of ROS formation was postulated [144].
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8.3. Impact of overweight on sex hormones
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It is well documented that excess body weight causes hormonal imbalance. One of the enzymes which plays a key role is aromatase which catalyses the conversion of androgens to estrogens [145]. Ovaries are the main site of estrogen production in pre-menopausal women,
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but peripheral tissues including body fat become the primary source in overweight postmenopausal individuals [15, 146, 147]. Cauley et al. [148] showed that estriol and estradiol levels are increased by more than 40% in obese post-menopausal women. Also in obese prepubertal girls, elevated levels of these hormones were found [149]. This effect was
26
explained by enhanced production of aromatase as a consequence of secretion of TNF-alpha and IL-6 adipocytes. Apart from enhanced production of humans also decreased levels of serum hormonebinding globulin were found in obese individuals [150]. This glycoprotein binds estrogens
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and testosterones and inhibits their activities. Alterations of the hormonal status were proposed to play a key role in the induction of breast and endometrial cancer which have an increased prevalence in post-menopausal obese
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women [151-154]. Furthermore, it was also found that excess body weight in children increases the overall cancer death rates as well as breast cancer mortality [155].
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Receptor-dependent as well as independent mechanisms are involved in estrogen
N
induced breast carcinogenesis [156]. The hormones enhance the proliferation of breast cells
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by binding to specific receptors. As a consequence of increased mitotic activity, errors in
M
DNA-replication are induced, the repair of mutations is less efficient and DNA adducts,
ED
breaks and other lesions are more frequently converted to persisting mutations [157]; furthermore, cell divisions cause mitotic recombinations which lead to errors that are passed
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to the daughter cells [157].
It is well documented that certain estrogen metabolites cause direct DNA damage.
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Estradiol (17-βE2) is converted to catechol metabolites which are hydroxylated at the 2- or 4positions to form 2-hydroxyestrogen (2-OHE) and 4-hydroxyestrogen (4-OHE). The latter
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compound is mutagenic in rat and human cells; i.e. it is converted to E(2)-3,4-quinone which forms covalent estrogen-DNA-adducts [158]. Additionally, E(2)3-4 quinoline can undergo redox cycling with E(2)3-4 hydroquinone leading to release of ROS. Quinones are backconverted via quinone reductase to create a cycle which leads to continuous release of radicals and also to formation of unstable depurination products with adenine and guanine [159].
27
Estrone undergoes a similar metabolism as estradiol but can be also converted to 16αhydroxyestrone (16α-OHE1) which binds covalently to DNA and enhances the proliferation of cells via binding to the ER [155, 160]. The genotoxic effects of estrogens in human derived breast cancer cells are well studied.
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For example, Mobley and co-workers [161-163] found evidence for induction of oxidative damage under conditions which are relevant for humans. Comparisons of effects in cells with
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and without ERs indicate that their presence increases the extent of oxidative damage.
The associations between formation of genotoxic estrogen metabolites and breast cancer have been investigated in a number of studies. It was shown that E(2)3-4 quinoline
U
causes cancer in animals [160, 164-168]. However, it is not clear, if the concentrations of
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DNA reactive estrogen metabolites are indeed increased in obese individuals. Mauras et al.
A
[149] found elevated concentrations of 16α-OHE1 in a small study in sera of obese
M
prepubertal girls (12 lean, 24 obese individuals) while no relation between the 4- and the 16α-
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OHE1 pathway and BMI values was found in a larger study (n=603) with post-menopausal women [169].
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Hormonal effects are also involved in the development of endometrial tumors [170]. Apart from enhanced cell proliferation, alterations of signalling pathways may play a role.
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However, at present experimental evidence is lacking that genomic instability is enhanced in the endometrium of obese women and further studies are required to assess the impact of
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overweight induced genomic instability on this form of cancer.
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8.4. Glycation end products Advanced glycation end products (AGEs) are formed as a consequence of reactions between sugar moieties and proteins; their biological effects are described in a number of reviews [171-173].
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The endogenous formation of these products in the human body takes place nonenzymatically via the Maillard reaction but also via the so called “polyol pathway”, a reaction
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which is catalysed by sorbitol dehydrogenase (SDH) and phosphokinase. Reactive αoxoaldehydes which are formed during these reactions (glyoxal, methylglyoxal and 3deoxyglucosone) are converted irreversibly by further reactions to products such as Nε-
U
carboxyethyllysine, arginine pyrimidine, pentosidine, pyrralin, methylglyoxal, glyoxal lysine
N
and Nε-carboxymethyllysine (CML) [171].
A
AGEs bind in the human body to specific receptors (receptor advanced glycation end
M
products (RAGE)) which are involved in different signalling pathways; the most relevant ones
ED
are shown in Figure 5. These interactions cause formation of ROS via activation of NADH-
Figure 5
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oxidase which leads to activation of various transcription factors including NF-κB [174].
A
Consumption of carbohydrate and sugar rich Western diets causes increased formation
of AGEs; also excess body fat may lead to elevated concentrations in specific tissues. Some findings indicate that the serum levels of CML are inversely related to the BMI and also to body fat, but enhanced levels were found in adipose tissue [175-178]. A different observation was made with sera from the MetS patients, i.e. the concentrations of AGEs were elevated in
29
these individuals and the concentrations correlated with the levels of markers which are indicative for insulin resistance and inflammation [179]. Also in experiments with HFD fed mice it was found that the concentrations of AGEs are increased in adipose tissue and in the liver [180].
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AGEs cause DNA damage directly and via interactions with signalling pathways. It is well documented that methylglyoxal causes gene mutations in bacterial test [181, 182] and also in yeast [182]. Furthermore, it was shown that CML accumulates in nuclear proteins and
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causes DNA strand breaks in human skin cells [183].
Stopper and co-workers [184] studied the effects of methylglyoxal and CML in comet
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assays with different cell types. They found induction of DNA damage and postulated on the
N
basis of results obtained with inhibitors that formation of ROS as a consequence of the
A
activation of the transcription factor NF-κB plays a key role (Figure 5). One of the proteins
M
which is regulated by NF-κB is angiotensin which is vasoconstrictive and causes down
ED
regulation of peroxisomes. The authors observed pronounced reduction of DNA damage when they added a specific receptor antagonist (candesartan) to the incubation mix [184, 185].
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Apart from formation of AGEs, sugars undergo also direct reactions with DNA-bases [186]. The products cause depurination [187] as well as SSBs and mutations in vitro [188]. In
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recent years, improved techniques (for example LC-MS and HPLC measurements) as well as antibodies have been developed which enable to quantify these products in humans [189-191]
A
but it is not known if increased concentrations are present in obese individuals.
8.5. Impact of obesity on DNA repair Overweight has an impact on different DNA repair pathways (Figure 6), but the tissue specificity and the underlying molecular mechanisms are only partly understood.
30
Recently, results of a study with young adults were published in which the authors found an association between the BMI and NER in a modified comet experiments with lymphocytes [192]. Langie and co-workers [102] performed interesting experiments with mice in which the mothers received a low folate diet and the offspring a HFD. They found reduction of BER in several brain regions of the progeny. Furthermore, they analysed the impact of this feeding
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is involved in repair of oxidised DNA bases in the hippocampus [102].
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scheme on the expression of BER related repair genes and observed alterations of Ogg1 which
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Figure 6
N
It is well documented that defects of BER and NER are associated with increased cancer
A
rates. Single nucleotide polymorphisms in genes that encode for proteins involved in these
M
pathways were found to cause reduced activities of repair enzymes and to increase the cancer
ED
risks in humans [193]. Furthermore, it is known that defective NER in Xeroderma pigmentosum patients leads to skin cancer and glioblastomas [194, 195].
PT
It is notable that also other repair pathways are affected by oxidation of the DNA [196198] and by LP products [199, 200]. A study with transgenic overweight mice concerned the
CC E
induction of DSBs, the synthesis of DNA repair proteins and transcription of repair associated genes after administration of phosphoramide mustard. The authors found clear evidence for
A
induction of DNA damage and activation of certain proteins (PRKDC and XRCC6) as well as increased levels of the ATM protein in ovaries of obese animals [106]. A number of investigation concerned the impact of obesity on the methylation status of genes which are directly or indirectly involved in DNA repair. The MutL homolog 1 (MLH1) is one of seven proteins which play a role in DNA mismatch repair (MMR) [201]. Remely 31
et al. [91, 98] found an inverse association between DNA damage in liver tissue of obese mice (which was monitored in comet experiments) and alterations of the methylation status of MLH1 in CpG1 in the same organ [91, 98]; on the contrary a positive association of the methylation of the same gene was seen in the liver in CpG4 [98]. In colon mucosa cells an inverse relation was seen with this gene in CpG2. These effects were reversed by
IP T
supplementation of the drinking water with the green tea constituent, EGCG and also with vitamin E. [91] On the contrary, no relation between BMI and MLH-1 methylation was
SC R
detected in a human study (n=128) with biopsies from endometrial tumors [202].
DNA methyltransferases (DNMTs) are responsible for the transfer of methyl groups
U
from the universal methyl donor S-adenosyl-L-methionine (SAM) to the 5-position of
N
cytosine residues in DNA. One of the members of this group, DNMT1 encodes for the
A
maintenance methyltransferase and plays a key role in MMR [201]. Remely et al. [91, 98],
M
found in the aforementioned studies no significant differences in the CpG methylation status in the promoter region of DNMT1 in colon and liver tissue of obese HFD mice.
ED
Hypomethylation of this gene leads to increased transcription of genes which are involved in
PT
MMR.
A recent study with lymphocytes of obese and normal weight adolescents indicated that
CC E
the capacity mitomycin C induced DNA repair is lower in the latter groups [58]. The authors measured in this investigation the time kinetics of disappearance of γ-H2AX foci in
A
lymphocytes as a parameter of the repair capacity. A further protein which may play an important role in obesity associated repair is p53.
The activation of this tumor suppressor protein as a consequence of excess body weight is well documented [7]. One of its functions is the inhibition of cell divisions which prolongs the repair phase [7]. It is known that p53 is activated by different types of DNA damage,
32
including oxidation of bases [7, 203]. Apart from beneficial effects of p53, newer findings indicate that its activation in adipocytes contributes to the development of insulin resistance [203, 204]. The impact of obesity on sex hormones is described in chapter 8.3. It was emphasized
IP T
that ER signalling leads to suppression of DNA repair and apoptosis in favour of cell proliferation [205]. The most important processes which are influenced by steroid hormones concern the regulation of DSB repair, non-homologous end-joining and homologous
SC R
recombination [206]. Most data come from in vitro experiments and it is not known at present
to which extent obesity induced alterations of the hormonal status have an impact on repair
U
function in humans.
N
Also telomeres have an impact on DNA repair; the impact of increased BMI on their
M
A
length is described in the following chapter.
ED
8.6. Obesity and telomere lengths
Telomeres are DNA sequences (tandem repeats of the sequence TTAGGG) which are
PT
localized at the end of chromosomes [207]. It was shown in vitro that shortening leads to
CC E
cellular senescence and that overexpression of the enzyme telomerase (which maintains the levels of telomeres) leads to immortalization [208]. Also results of animal experiments
A
support the assumption of the involvement of these structures in senescence and ageing [209]. The impact of obesity on these structures may be an important factor which affects DNA
stability. The mechanisms which cause this phenomenon are partly known [210]. One of the modes of action which may be involved is the dependency of DSB repair on telomeric regions. Chromosomal fusion in mammalian cells involves homologues end joining which is one of the major forms of DSB repair. Chromosome fusion initiates chromosome instability 33
involving breakage fusion cycles in which dicentric chromosomes form bridges and breaks when the cells attempt to divide [211]. One of the mechanisms which could lead to telomere shortening in overweight/obese individuals is oxidative stress as it was shown in in vitro that telomeres are highly sensitive to damage by ROS [212, 213].
IP T
Only one intervention study has been conducted which concerned the correlation between alterations of telomere lengths and DNA base damage in overweight humans [59]. The authors monitored alterations of these parameters after weight loss (for details see Table
SC R
5) and found an increase of telomere lengths and in parallel reduction of apurinic/apyrimidinic
sites. In another study with a parallel design, no connections were detected between obesity
U
and telomere length and DNA damage [40].
N
A large number of studies was published which concerned the effects of body weight
A
on telomeres. The results are reviewed in two papers, which contain meta-analyses [214, 215].
M
In the first, the authors evaluated 16 studies. Only in cross-sectional investigations (n=14), a
ED
inverse relation was observed [214]. A second analysis [215] comprised 63 studies (119.439 subjects); in 39 an inverse correlation between obesity and telomere lengths was observed.
PT
The authors found in a pooled analysis a trend for an inverse association. However, they emphasize the need for further longitudinal studies and stress that the assumption of a
CC E
relationship between obesity and telomere lengths remains open due to the heterogeneity of the results.
A
Several studies indicate that the relation between telomere lengths and adiposity is more
pronounced in younger subjects; this observation was also confirmed in a meta-analysis of Müezzinler [214]. Another aspect which was addressed in a study by Martens et al. [216] concerns the impact of the maternal BMI on telomere integrity in new-borns. The authors found evidence for shorter telomeres in babies of overweight/obese mothers.
34
The impact of weight loss on telomere lengths was investigated in several trials; overall the findings are controversial. Moreno-Navarrete et al. [217] concluded that shorter telomere length in obese subjects is an established irreversible feature which cannot be improved by weight loss while O’Callaghan et al. [59] state that weight loss as a consequence of consumption of a calorie restricted diet prevents telomere shortening. Also results of studies
IP T
with bariatric surgery patients are inconsistent. No effect was seen in a trial with 97 subjects
[218] one year after the operation, while an increase was found in a 10 year prospective study
SC R
[219].
N
U
8.7. Impact of obesity/overweight on the activities of enzymes that activate and detoxify mutagens
A
Many dietary and environmental carcinogens such as polycyclic aromatic
M
hydrocarbons, heterocyclic aromatic amines, aflatoxins and nitrosamines are converted in the body to DNA reactive metabolites by specific phase I enzymes and partly also by phase II
ED
enzymes [220]. The latter ones catalyse also the detoxification of DNA reactive intermediates and of directly active compounds [221]. A number of human and animal studies addressed
PT
the question if obesity affects the activities of these enzymes. In experiments with humans,
CC E
the clearance rates of enzyme specific drugs were studied with chemical analytical methods while in animal models mainly hepatic enzyme activities were monitored. A comprehensive review of human studies was published by Brill et al. [222]; the
A
authors state that there is evidence that the clearance of CYP3A4 substrates it reduced in individuals with high BMI while increased excretion was observed with substrates for CYP2E1 and CYP1A2. Also in children, increased activities of the later isozymes was found [223]. These observations are partly in agreement with animal data; increased i.e. CYP2E1 was observed for example in genetically modified obese female mice but not in males [224],
35
in a trial with monosodium glutamate treated overweight male mice an increase of this enzyme and also of CYP2A5 and 3A were detected in hepatic tissue and in colon mucosa [225]; furthermore, there is also evidence for CYP2E1 induction in obese rats [226]. This isozyme plays a key role in the activation of genotoxic nitrosamines. In regard to phase II enzymes, increased clearance of N-acetyltransferase (NAT) and
IP T
uridine diphosphate glucuronosyltransferase (UGT) substrates was found in humans [222]; the later effects were also seen in overweight children [223]. UGT activity was also increased
SC R
in livers of obese mice with steatosis [227].
Data concerning the impact of obesity on GST are controversial. In diabetic mouse
U
model, decreased activity was found in the liver [224]; no such effects were seen in genetically
N
modified (ob/ob) mice in females but a higher activity was detected in animals in the same
A
study [228].
M
It is conceivable that alterations of the activities of the different drug metabolizing enzymes have a strong impact on the genotoxic properties of chemicals; however we are not
ED
aware of studies in which the consequences of overweight on the genotoxic effects of dietary, occupationally and environmentally relevant groups of DNA reactive carcinogens were
PT
investigated and related to alterations of drug metabolizing enzymes. An interesting study was published by Johnson et al. [229], who quantified cyclophosphamide DNA crosslinks in blood
CC E
cells of overweight and lean patients who underwent hematologic cell transplantation and found no evidence for BMI associated differences despite the fact that the overweight patients
A
received substantially higher doses of the drug. This cytostatic is activated by CYP2B6 and the authors hypothesized that their finding may be due to altered metabolism of the drug in obese individuals but no enzyme measurements were conducted to confirm this assumption [229]. Data concerning the impact of polycyclic aromatic hydrocarbon (PAH) exposure on oxidative DNA damage are controversial [55]. In a large study with PAH exposed coke oven
36
workers, an inverse relation with the BMI was observed in lymphocytes, while elevated levels of 8-OHdG were detected in livers of obese mice which were treated with PAH in comparison to lean controls [104].
Inflammation and DNA damage
IP T
8.8.
Inflammations are defence mechanisms which protect the host against infections and
SC R
other insults (for reviews see [230, 231]). It was found in many investigations that overweight/obesity are associated low grade inflammation [232, 233].
Adipose tissue plays a central role in this reaction. Adipocytes release so-called
U
“adipokines” into the blood stream which exert systemic effects [234]. These inflammatory
N
mediators belong to several groups including hormones (e.g. leptin which is pro-inflammatory
A
and adiponectin which is anti-inflammatory), chemokines and pro-inflammatory cytokines
M
(e.g. interleukins 1β, 6, 10 and TNF-α), acute phase proteins and growth factors such as IGF-
ED
1 [235]. Apart from adipocytes also macrophages and other cell types accumulate in adipose tissue and are important contributors to the production of substances which trigger
PT
inflammation [236].
Pro-inflammatory proteins which are produced in adipose tissue including the enzyme
CC E
haem oxygenase-1 (HO-1) cause substantial alterations of several pathways which activate transcription factors [237]. Probably the most relevant one is NF-kB which is activated by ROS [174] and adipokines [238] and causes increased transcription of genes encoding for
A
inflammatory molecules including COX-2. This enzyme catalyzes the formation of proinflammatory eicosanoids from arachidonic acid [239]. Inflammations cause DNA damage which in turn induces further inflammatory reactions [240] and it is likely that genomic instability plays a key role in the aetiology of
37
diseases which are associated with inflammations, in particular with cancer [241, 242]. However, there is only limited evidence for formation of oxidised DNA bases in obese humans by ROS as a consequence of inflammation (see Tables 2-6). In a study by Scarpato et al. the levels of inflammation markers (IL-6 and C-reactive protein) were found to be
9.
IP T
significantly higher in individuals with increased body weight [50].
Consequences of obesity induced DNA damage
SC R
Overweight/obesity are associated with increased prevalence of different forms of cancer in humans [243, 244]. The assumption of an association between excess body weight and cancer is also supported by results of rodent experiments [2, 90-92]. We are not aware of
U
any targeted studies in which correlations between DNA damage and cancer rates were
N
investigated in obese humans and animals; however, direct links can be postulated on the
A
basis of mechanistic studies.
M
Another relevant issue is the impact of obesity on infertility which was seen in a number
ED
of studies. As described in chapter 4.3 there is some evidence for increased DNA damage in sperm cells of obese infertile men, but further investigations concerning this relation are
PT
warranted. It is also conceivable that obesity causes mutations in germ cells which lead to heritable diseases in the offspring but this issue has never been investigated according to our
CC E
knowledge.
The term “DNA damage response” (DDR) concerns the consequences of genomic
A
instability [7, 205, 245, 246]. It describes pathways by which cells react to damaged DNA to prevent adverse health effects such as initiation of mitotic arrest, senescence, repair and cell death [205]. It was postulated that overweight and the MetS cause processes which prevent malignant transformation by activation of processes which lead to senescence and programmed cell death. The induction of inflammation via DNA damage and the activation
38
of transcription factors as well as telomere dysfunctions play a key role in these response mechanisms. A detailed description of this concept can be found in the papers of Shimizu et al. [246] and Erol [7]. The results of comet experiments with rodents indicate that excess body weight causes
IP T
genomic instability in multiple organs. The extent of damage which we measured in experiments with overweight mice in different tissues was similar (e.g. in colon, brain and liver) and no pronounced gender specific differences was observed [92]. It is conceivable that
SC R
the formation of tumors involve different organs specific tumors promoting mechanisms
which cause clonal expansion of damaged cells. These mechanisms are described in several
U
reviews [14, 247]. In regard to breast and endometrial cancer hormonal effects play an
N
important role, whereas in the liver the key mechanisms include lipogenesis and insulin
A
resistance which cause inflammation. In regard to formation of pancreatic tumors,
M
dysfunctional autophagy and toxic effects are discussed while obesity associated colorectal cancer is probably a consequence of dysbiosis and thinning of the intestinal mucosa layer
ED
resulting in increased permeability of the intestinal epithelial to microbial products.
PT
Apart from these organ specific effects, several mechanisms have been identified in obese individuals which may favour the development of tumors at multiple sites from initiated
CC E
cells including sustained angiogenesis, tissue invasion and metastasis and specific metabolic disturbances. These effects are described in detail in a recent working report of the IRAC [2,
A
248].
39
10. Conclusions and knowledge gaps Obesity and metabolic diseases are among the most relevant health risk factors for humans worldwide. The evaluation of the literature show that the impact of overweight/obesity on DNA stability which plays a key role in the etiology of cancer, infertility and ageing is not adequately investigated. We found in total 27 human studies
IP T
concerning the effects of obesity/overweight individuals and MetS patients on genomic instability in somatic cells, 12 human and 5 animal investigations concerning on DNA damage
SC R
in sperm and 18 investigations with rodents in which DNA damage was studied in blood cells
and in inner organs. It is interesting that a substantially higher number of human studies has been published which concern the genotoxic effects of chemicals. On the basis of a computer
U
aided search we estimate that about 220 articles deal with the effects of selected heavy metals
N
(Cr, Ni, Pb, As and Hg), the impact of radioactivity was monitored in more than 700 trials.
A
The quality of studies concerning the impact of overweight/obesity on DNA stability is
M
in general low since potential confounding factors such as intake of vitamins, minerals and
ED
the effects of plant derived DNA protective components were not taken into consideration in the most trials. Only in two studies the intake of micronutrients were recorded [53, 63]. It is
PT
known from a number of human studies that vitamins such as folate, B6, B12 and minerals such as Se, Zn and Fe have a strong impact on genomic instability [249, 250]. Furthermore it
CC E
is notable that most animal trials were conducted with diets which differ in regard to their macronutrient levels, this leads to differences of the body weight and to accumulation of fat
A
but the differences of the composition of the chows may cause results which do not necessarily reflect the effects of excess body weight. The literature provides only limited evidence for an association between overweight/obesity and damage of the genetic material in men. The evaluation of human studies on the basis of the QS values shows that in most comet trials with medium quality (no 40
study reached a high score) evidence for an association was detected. Four of six investigations with medium quality found that increased BMI values are associated with comet formation while results of MN studies with medium quality are conflicting (50% positive, 50% no relation). Also 8-OHdG measurements in humans do not allow to draw firm conclusions (positive, as well as negative correlations were detected); notably the study with
IP T
the highest QS found even a significant inverse association [53]. Notable, significant positive
correlations between induction of DSBs and excess body weight were detected in all γ-H2AX
SC R
experiments. Overall, 24 human studies with somatic and sperm cells found evidence for an
increase of DNA damage with the BMI while 22 trials reported no or even inverse
U
associations. It is probable that the highly conflicting results are due to the differences in the
N
diets, i.e. it is likely that normal and slim individuals consume higher amounts of vegetables,
A
fruits and full grain products which contain a broad variety of DNA protective compounds
M
while obesity is associated with increased intake of fats and foods which contain sugars and purified flour.
ED
The results of animal studies in which standardized diets were fed under controlled conditions provide clearer answers; i.e. positive associations between excess body weight and
PT
comet formation were seen in all studies with a medium quality (no high quality trials were
CC E
published so far) in somatic cells. Also the findings of sperm experiments with medium quality are in good agreement (two TUNEL and two SCSA trials, found consistently positive associations). Results of MN studies with animals are conflicting (one positive and two
A
negative were reported) but all of them had only low quality scores. The assumption of a causal relation between excess body weight and DNA damage is
strongly supported by results of mechanistic studies which indicate that several modes of action may cause genetic instability in overweight/obese individuals. Many investigations indicate that oxidative stress may play an important role; the formation of ROS may be a 41
consequence of increased insulin and glucose levels. ROS may cause either direct oxidative DNA damage (which was seen not in all studies), or via formation of DNA reactive LP products. As mentioned above, pronounced LP was seen in many trials with animals and humans. Also inflammation may be casually related as it leads to formation of oxygen and nitrogen radicals. Increased levels of pro-inflammatory cytokines and activation of NF-kB are
IP T
characteristic features of increased excess body fat; however, further investigations are warranted to allow firm conclusion. As described in chapters 8.3 to 8.6 also hormonal effects,
SC R
formation of AGEs, inhibition of DNA repair process and reduction of telomere lengths may play a role in the association between increased BMI values and genomic instability.
U
However, further experimental data are required which address these issues. In the case of
N
hormonal effects it is conceivable that they are organ specific, but similar DNA damage was
A
seen in multiple organs in a number of studies and no gender specific effects were observed.
M
The impact of excess body weight on telomere lengths was quite moderate and was only observed after longer time periods while DNA protective effects were seen in obese humans
ED
and laboratory rodents after short intervention periods with dietary components indicating that the impact of the BMI on telomeres may be of minor relevance. The effects of AGEs are
PT
difficult to assess at present as no studies are available in which their formation was correlated
CC E
with DNA stability in humans and animals. Many important knowledge gaps exist at present. It is not known exactly which BMI
values lead to a significant increase of genomic instability and if the effects increase linear or
A
exponentially. Further research is also warranted to elucidate the consequences of overweight/obesity induced DNA damage in regards to malignant transformation of cells. So far, research concerning the induction of cancer by excess body weight focused mainly on inflammation, signaling pathways and hormonal effects [251-253]. Other relevant topics for further studies concern the consequences of DNA damage on cognitive dysfunctions and 42
investigations concerning induction of mutations in germ cells. As mentioned above, some animal studies indicate that increased BMI leads to DNA damage in different regions of the brain [88, 92, 96], furthermore some animal studies found elevated levels of DNA damage in sperm cells as a consequence of excess body weight.
IP T
Genotoxicity studies will also enable to assess if and to which extent weight loss strategies as well as therapeutic interventions with drugs and bioactive natural dietary components lead to an improvement of the stability of the genetic material. Weight loss diets
SC R
which are advocated worldwide are generally not designed on the basis of scientific data and
it is conceivable that drastic reduction of the caloric intake and imbalanced consumption of
U
vitamins and minerals (which are required to maintain the stability of genetic material) will
N
lead to adverse health effects including destabilization of the DNA. Also this important issue
ED
Conflict of interest statement
M
A
should be addressed in future investigations.
PT
There are no conflict of interest.
CC E
Acknowledgments This works was supported by COST Action CA15132 (hCOMET); the authors are
A
also thankful to participants of this research initiative for discussions.
43
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22.
40.
A
41. 42.
43. 44.
45
52. 53.
54. 55.
56. 57. 58. 59.
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60.
SC R
51.
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251. 252. 253.
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Legends for figures
55
Figure 1. Use of different methods for the detection of DNA damage in obesity studies. DNA damage was analysed in animal experiments mainly in comet assays (in different organs) which were also used in human studies with lymphocytes. Micronucleus (MN) experiments are conducted in bone marrow cells of rodents and in human studies with lymphocytes and with exfoliated epithelial cells. For the analysis of sperm cells, terminal deoxynucleotidyl-
IP T
transferase dUTP nick end labelling (TUNEL) and sperm chromatin structure assays (SCSA) are conducted. Oxidative damage of the DNA and alterations of DNA repair processes (BER,
SC R
NER) can be monitored with a modified protocol of the comet assay (not shown) and by
U
determination of 8-OHdG and/or 8-oxodG in urine, plasma and bloods cells.
N
Figure 2. Induction of DNA damage as a consequence of obesity/overweight. DNA damage
A
can be induced directly via estrogen metabolites, formation of advanced glycation end
and
alterations
of
the
activation
of
enzymes
which
catalyse
the
ED
shortening
M
products, LP products and radicals or indirectly via increased mitotic activity, telomere
activation/detoxification of mutagens and by inhibition of repair pathways. Phenomenona
PT
which were observed in overweight/obese humans (H) or animals (A) are indicated in circles. Solid lines indicate that experimental evidence shows that they lead to DNA stability or ROS
CC E
formation in rodents or men. Dotted lines are indicative for effects which were not demonstrated so far in overweight/obese subjects were observed in in vitro experiments.
A
Numbers indicate chapters in which the different effects are described.
Figure 3. Induction of ROS by insulin. The hormone binds to receptors (insulin receptor and insulin growth factor receptor) which leads to activation of phosphatidylinositol-3-kinase (PI3K) and Akt, the subsequent steps are activation of NADPH-oxidase and translocation of 56
Akt into mitochondria. Both processes lead to formation of ROS. Most findings come from in vitro experiments with mammalian cells but it was also shown that increased insulin levels cause DNA damage in rodents.
IP T
Figure 4. Induction of ROS by increased glucose levels. ROS can be formed by different molecular mechanisms, namely via activation of PKC or as a consequence of glycolysis in
SC R
the cytosol. AGEs are formed as a consequence of reactions of sugars with proteins, they activate transcription factors and cause also direct DNA damage. Another pathway of ROS
formation is caused by pseudohypoxia. All findings come from in vitro experiments with
A
N
U
mammalian cells but it was shown that increased insulin levels cause DNA damage in rodents.
M
Figure 5. Formation of AGEs and activation of NF-kB via RAGEs. Dotted line: intermediate processes which are not described in detail. AGEs are formed via Maillard reactions or via
ED
the polyol pathway and cause activation of transcription factors via interactions with signalling pathways. All findings come from in vitro experiments with mammalian cells but
CC E
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it was shown that increased insulin levels cause DNA damage in rodents.
Figure 6. Impact of obesity on DNA repair processes. Inflammation and estrogens increase
A
the mitotic activity of cells which results in errors of DNA replication and reduces the duration of the repair phase. Hormones have also a direct impact on different forms of repair. Telomere shortening was found in a number of investigations with overweight humans and leads to chromosomal aberrations as a consequence of inhibition of repair pathways. It is documented that ROS have an impact on repair functions (e.g. on NER); they also activate p53 which results in inhibition of cell divisions. Few studies are available indicating that 57
overweight/obesity have an impact on the methylation of DNA repair enzymes. Phenomenon which were observed in overweight/obese humans (H) or animals (A) are indicated in circles. Solid lines indicate that experimental evidence shows that they lead to DNA stability or ROS formation in rodents or men. Dotted lines are indicative for effects which were not
A
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ED
M
A
N
U
SC R
IP T
demonstrated so far in overweight/obese subjects but were observed in in vitro experiments.
58
59
A ED
PT
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IP T
SC R
U
N
A
M
60
A ED
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61
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Table. 1. Overview of genotoxicity methods which are used in studies concerning the impact of overweight/ obesity on DNA stability Endpoints
Principle
BER/NER
cells are exposed to ROS generating chemicals e.g. (hydrogen peroxide or radiation) [30] detection of alterations of DNA repair activities by induction of BER and NER specific DNA damage [31] MN are formed as a consequence of structural and numerical chromosomal aberrations, additional markers in MN cytome studies are nuclear buds and nuclear bridges which reflect genomic instability, furthermore acute toxicity markers are evaluated i.e. karyolysis, karyorrhexis, pyknotic nuclei, binucleated cells and condensed chromatin [32] determination of oxidised deoxyguanosine by use of HPLC, ELISA or calorimetric assays [30]
ED
FPG/Endo sensitive sites ROS sensivity
A
detection of DNA migration in an electric field. in humans: lymphocytes and whole reflect formation of single and double strand breaks blood in rodents: different inner organs (SSBs and DSBs) and apurinic sites [29] *the methods can be also used to detect DNA damage in sperm cells III detection of oxidised purines and pyrimidines
M
Comet formation Standard conditions
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PT
Micronuclei
A
8-OXOdG/ 8-OHdG/
γ-H2X
Target cells/ tissues and body fluids
in humans: lymphocytes and exfoliated cells from the mouth in rodents: bone marrow and erythrocytes
Remarks the predictive value of comet formation in regard to human cancer risks is not known.
MN in lymphocytes of humans are a reliable marker for cancer [33]
in humans: lymphocytes, plasma and relation to urine human cancer in rodents: plasma and urine risks not known. Induced by inflammatory diseases detection of double strand breaks by measurement of in humans: lymphocytes relation to human phosphorylation of a specific histone as a consequence of in rodents: different inner organs cancer risks not double strand DNA repair [34] known.
62
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Table. 1. continued Principle
Terminal deoxynucleotidyltransferase dUTP nick end labelling (TUNEL) assay
based on the ability of the enzyme terminal sperm cells in humans and animals deoxynucleotidyl- transferase (TdT) to incorporate labelled dUTP into free 3hydroxyl termini generated by fragmentation of genomic DNA into low molecular weight double stranded DNA and high molecular weight single stranded DNA [35] based on heat treatment or acidic denaturation sperm cells in humans and animals of sperm DNA and subsequent staining with acridine orange which enables to discriminate between DSBs and SSBs (double strands stain green, single strands red) [36]
M
ED
A
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Sperm chromatin structure assay (SCS)
Target cells/ tissues
A
Endpoints
63
Remarks in some animal and human studies results correlated with reduced fertility.
in some animal and human studies results correlated with reduced fertility.
I N U SC R
Table 2. Results of comet assay in overweight/obese subjects1 Study design
Parameter (indicator cells)
Participants and treatments
Intervention
tail intensity (lymphocytes)
48♂/23♀, BMI=18.0–23.9, n=21 BMI=24.0–26.9, n=26 BMI=27.0–39.9, n=24 age (all groups) 35-64
Intervention
arbitrary units (leukocytes)
Results
QS
Remarks
Ref
↔ DNA damage ↔ FPG in groups with different BMI
22 (M)
↑ oxidative DNA damage after consumption of sugar (1.8-fold) ↔ gender ↔ age
[37]
calorie restriction (CR): n=6♂/6♀, CR + exercise: n=6♂/6♀, very low- calorie diet: n=6♂/6♀, C: n=6♂/6♀, baseline BMI (all groups)=27.8, age (all groups) 26-49 9♂/47♀, BMI=51.1 n=56 before surgery 6 and 12 months after surgery age (all groups) 44.70 ± 1.4
↓ DNA damage after CR and after CR+exercise and very low-calorie diet groups at month 6 vs. baseline (1.03-fold)
18 (M)
weight loss after very low-caloric diet (12 kg) weight loss after CR + exercise and CR (8 kg) ↓ fasting insulin level after 3 and 6 months in CR + exercise and CR ↔ fasting glucose ↔ serum protein carbonyl
[38]
↓ DNA damage after 12 months (38%) ↔ DNA damage after 6 months
20 (M)
[39]
OB: n=20, BMI ≥30 OB+ type 2 diabetes (T2D): n=21, BMI ≥30 C: n=20, BMI ≤30 age (all groups) 44-55
↔ DNA damage OB vs. C
19 (M)
↓ ferric reducing antioxidant power assay after 6 and 12 months ↓ weight (22% after 6 months) and (29% after 12 months) ↑ total glutathione after 6 months (1.1-fold) ↑ total oxidised glutathione after 6 months (1.6-fold) ↔ total glutathione and oxidised glutathione after 12 months ↔MDA ↓ MDA in T2D vs. C (75%) ↓ telomere length in OB+T2D vs. OB
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ED
M
A
Comet assay2
tail intensity (whole blood)
A
Intervention
Parallel
tail intensity (visceral adipose tissue)
64
[40]
I N U SC R
Study design
Parameter (indicator cells)
Participants and treatments
Parallel
tail intensity (lymphocytes)
OB: n=20♀, BMI ≥25 C: n=20♀, BMI ≤25 normal weight obese syndrome3 (NWO): n=20♀, BMI ≤25 age (all groups) 25-35
Parallel
tail intensity (lymphocytes)
ED
M
A
Comet assay
QS
Remarks
Ref
↑ DNA damage OB vs. C (1.5-fold) ↑ DNA damage NWO vs. C (1.5-fold) ↑ FPG NWO vs. C (1.7fold) ↑ FPG OB vs. C (1.6-fold) ↑ DNA damage in high visceral fat vs. normal visceral fat (1.6-fold)
18 (L)
↔ 8-OHdG in whole blood
[41]
↑ MDA in high visceral fat vs. normal visceral fat (1.419 (M) fold)
[42]
↑ DNA damage in OB vs. C (5.0-fold)
↔ WC 21 (M) ↔ waist to hip ratio ↔ gender ↔ age
[43]
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healthy, non-obese Koreans, BMI=23.8, high visceral fat ≥100 cm2, n=27♂ normal visceral fat,<100 cm2, n=27♂ age (all groups) 36.5±0.8 OB: n=18♂/17♀, BMI= 31.9 (age <20: n=12, age 25-53: n=23) C: n= 8♂/7♀, BMI= 23.3 (age<20: n=9 and age 2553: n=6) OB: n=80♂/80♀, BMI=29 C: n=80♂/80♀, BMI =16.022.0 age (all groups) young and old (n.s)
Results
A
Parallel
Parallel
arbitrary units (leukocytes)
length of DNA migration (μm) (peripheral blood)
↑ DNA damage in ♀ young 17 OB vs. C (2.5-fold) (L) ↑ DNA damage in ♂ young OB vs. C (2.2-fold) ↑ DNA damage in ♀ old OB vs. C (2.6-fold) ↑ DNA damage in ♂ old OB vs. C (2.3-fold)
65
alterations of DNA damage and plasma homocysteine correlated with triglycerides, LDL-cholesterol, systolic blood pressure, cholesterol, MDA and total oxygen stress in OB. (-) association DNA damage and plasma homocysteine with total oxidant status and globulin.
[44]
I Study design
Parameter (indicator cells)
Participants and treatments
tail intensity (lymphocytes)
OB-MetS pts.: n= 30♀, BMI=45.7, age 32.4±6.8 OB-without MetS: n= 30♀, BMI=21.9 age 32.4±6.5
ED
M
A
Comet assay Parallel
N U SC R
Table 2. Continued
1
Results
QS
Remarks
Ref
↑ DNA damage in OB vs. C (1.7-fold) ↑ FPG in OB vs. C (1.4fold) ↑ endonuclease (ENDO) in OB vs. C (1.5-fold)
19 (M)
↑ AA-FTO genotype and allele A OB♀ (1.2-fold) ↓ TT-FTO genotype and allele T in morbidly OB♀ (1.2-fold)
[45]
3
Unless otherwise indicated comet assays were conducted under standard condition (SC), alkaline version which detects single/ double and apurinic sites.
CC E
2
PT
Abbreviations: BMI, body mass index; C, control; FPG, formamido pyrimidine glycosylase; LPO, lipid peroxidation; MN, micronuclei; MDA, malondialdehyde; NMO, normal weight obese; n, number of participants; n.s., not specified; OB, obese; OW, overweight; 8-OHdG, 8-hydroxy-2’-deoxyguanosine; SC, standard conditions; T2D, type 2 diabetes; wk, week; WC, waist circumference; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table)
A
Normal weight obese (NWO): according to the article by Tomasello et al [41], NWO known as a metabolic condition which observed in women with normal index but fat mass (FM) >30%.
66
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Table 3. Results of cytokinesis – block micronucleus cytome (CBMN-Cyt) assay in overweight/obese subjects1 Parameter Participants and treatments (indicator cells) Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay
MN (lymphocytes)
CC E
M
PT
Parallel
OB: n=33♂, 2 dietary interventions (both diets: 7000 kJ): either high protein-high red meat group (n=16), BMI=32.0 or high carbohydrate-low red meat diet group (n=17), BMI=31.0 age (all groups) 20-65 OB: n=36♂/47♀, BMI= 38.0, age 37.9±10.5 OW: n=10♂/11♀, BMI= 26.0, age 40.5±10.6 C: n=7♂/14♀, BMI= 22.0, age 34.8±11.5
ED
Intervention MN (randomized (lymphocytes) trial)
A
Study design
A
Parallel
Parallel
MN (lymphocytes)
OW/OB: n=14♀/♂, BMI>25 C: n=23, 20
MN (lymphocytes)
OB: n=33♂, BMI= 33.6 OW: n=55♂, BMI= 27.9, age elderly population (all groups) 68.0±6.8
Results
QS
Remarks
Ref
↔ MN (after weight loss) ↔ MN (by diet and time) ↑ nuclear buds during intervention (specific diet)
17 (M)
only 1000 lymphocytes scored for MN ↓ weight loss by both interventions after 12 wks (12%)
[46]
↑ MN in OB vs. C (1.7-fold) ↑ MN in OB vs. OW (1.5-fold) ↑ nucleoplasmic bridges in OB vs. C (1.7-fold) ↑ nucleoplasmic bridges in OB vs. OW (1.5-fold) ↑ nuclear buds in OB vs. C (2.2-fold) ↑ nuclear buds in OW vs. C (1.6fold) ↔ MN in OW/OB vs. C
16 (M)
only 1000 lymphocytes scored for MN ↓ 8OHdG in OB vs. C (7%) ↔ correlation waist to hip ratio and CBMN assay parameters and 8-OHdG (+) correlation between MN and BMI in OW and OB (+) correlation between BMI and nuclear buds in OW and OB
[47]
15 (M)
↔ gender ↔ age ↔ smoking ↔ effect of mobile phone
[48]
↔ MN in OB vs. OW
14 (M)
↔ smoking ↔ age ↔ BMI lower DNA damage in former uranium workers who received X-ray examination
[49]
67
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Table 3. Continued
Study Parameter Participants and treatments design (indicator cells) Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay
Parallel
MN (buccal cells)
Crosssectional
MN (buccal cells)
OB: n= 31♂/30♀, BMI= 29, age: 10.8±3.4 OW: n= 9♂/11♀, BMI= 24.4 age: 12.2±2.6 C: n=16♂/22♀, BMI= 17.5 age:10.8±2.6 Malaysian farmers ♀/♂ OB: n=3, 35≤BMI OB: n=5, 30≤BMI<34.9 OW: n=35, 25≤BMI<29.9 normal: n=21, 18.5≤BMI<24.9 UW: n=4, BMI <18.5 age (all groups) 26-65 OB: n=20, BMI=22.4 OW: n=20, BMI=18.3 C: n=20, BMI=16.2 age (all groups) 7-11
A
MN (lymphocytes)
QS
↑ MN in OW vs. C (2.5fold) ↑ MN in OB vs. C (2.7fold)
17 (M)
↑ MN in normal vs. OW (1.25-fold) ↔ MN in normal vs. OB
17 (M)
↔ MN in different groups
18 (M)
A
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PT
ED
M
Parallel
Results (fold)
1
Remarks
Ref
↑ γ-H2AX foci in OW vs. C (5.6-fold) ↑ γ-H2AX foci in OB vs. C (8-fold) ↔ tumor necrosis factor-alpha in all groups ↑ interleukin 6 in OW and OB vs. C (9- fold) ↑ C-reactive protein in OW and OB vs. C (17.7-fold) only 1000 cells scored for MN ↔ binucleated between different groups ↑ MN in pesticide exposed farmers vs. C ↔ age
[50]
only 1000 cells scored for MN ↔ binucleated cells ↔ pycnosis cells ↔ broken-eggs cells ↔ karyorrhexis cells ↔ condensed chromatins ↔ total nuclear abnormalities
[52]
[51]
Abbreviations: BMI, body mass index; C, control; CBMN-Cyt, cytokinesis block micronucleus cytome assay; MN, micronuclei; n, number of participants; n.s., not specified; OB, obese; OW, overweight; SC, standard conditions; UW, under-weight; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table)
68
I N U SC R
Table 4. Results of 8-hydroxy-2' -deoxyguanosine (8-OHdG) in overweight/obese subjects1 Parameter (indicator cells)
Participants and treatments
8-hydroxy-2' -deoxyguanosine (8-OHdG)
A
Study design
HPLC (urine)
n=31♂/52♀, BMI= 26.0, age (all groups) 40-64
Parallel
colorimetric assay (whole blood)
OB: n=20♀, BMI ≥25 C: n=20♀, BMI <25 NWO2: n=20♀, BMI ≤25 age (all groups) 25-35 OB: n=12♂/8♀, BMI= 46.8, age 44.2±12.0 and laparoscopic gastric banding C: n=10♂/10♀, BMI= 22.5, age 40.7±12.3
ED
ELISA (plasma & urine)
CC E
PT
Parallel
M
Intervention (randomizedtrial)
HPLC (plasma)
A
Parallel
Parallel
HPLC (plasma)
coke oven workers ♂ BMI ≤ 24: n=18 exposed, 12 non exposed BMI > 24: n=29 exposed, 19 non exposed, age according to BMI (n.s) OW and OB: n=6♂/52♀, BMI=31.9 C: n=3♂/17♀, BMI=22.3 age (all groups) 46-54
Results
QS
Remarks
↓ 8-OHdG in high BMI (4%) ↑ 8-OHdG in ♂ vs. ♀(1.3fold) ↔ 8-OHdG
18
↔ intake of vitamin C, E, A on DNA damage [53] ↑ 8-OHdG in smokers (2.0-fold)
(H) 14
(M) ↑ urinary 8-OHdG in OB vs. C (2.6-fold) ↔ plasma 8-OHdG ↓ urinary 8-OHdG after surgery (59%) ↓ plasma 8-OHdG after surgery (50%) ↓8-OHdG with BMI (not significant)
14
↑ 8-oxodG in OW and OB vs. C (2.0-fold)
17 (M)
69
(M)
15
(M)
Ref
↑ FPG NWO vs. C (1.7-fold) ↑ FPG OB vs. C (1.6-fold) ↑ DNA damage NWO vs. C (1.5-fold) ↑ DNA damage OB vs. C (1.5-fold) ↓ BMI after surgery (20%) ↓ body weight after surgery (19%)
[41]
↑ 8-OHdG and glutathione S-transferase in the exposed group (not significant)
[55]
↑ LPO in OW and OB vs. C (3.0-fold) (+) correlation 8-oxodG and BMI, WC, hip circumference, triglycerides (+) correlation LPO and BMI
[56]
[54]
I Study design
Parameter (indicator cells)
Participants and treatments
ELISA (plasma)
Parallel
ELISA (urine)
OB: n=36♂/47♀, BMI= 38.0, age 37.9±10.5 OW: n=10♂/11♀, BMI= 26.0, age 40.5±10.6 C: n=7♂/14♀, BMI= 22.0, age 34.8±11.5
↓ 8-OHdG in OB vs. C (7%)
QS
Remarks
Ref
17 (M)
↔ association waist to hip ratio and CBMN assay parameters and 8-OHdG ↑ MN in OB vs. C (1.7-fold) ↑ MN in OB vs. OW (1.5-fold) ↑ nucleoplasmic bridges in OB vs. C (1.7-fold) ↑ nucleoplasmic bridges in OB vs. OW (1.5-fold) ↑ nuclear buds in OB vs. C (2.2-fold) ↑ nuclear buds in OB vs. C (1.6-fold) (+) correlation between MN and BMI in OW and OB (+) correlation between BMI and nuclear buds in OW and OB association between 8-oxoGuo and waist-hip ratio association between 8-oxodG and adrenaline (↔) no association between 8-oxoGuo and 8-oxodG with leptin, adiponectin and high sensitive-Creactive protein
[47]
CC E
1
PT
ED
M
Parallel
Results
A
8-hydroxy-2' -deoxyguanosine (8-OHdG)
N U SC R
Table 4. Continued
OB hypertensive (OB-H): n=63♂, BMI≤30 OB normotensive (OB-N): n=40♂, BMI≤30 lean normotensive (L-N): n=27♂, 20< BMI≤25 age (all groups) 39-58
↔ 8-oxodG ↑ 8-oxoGuo in OB-H vs OB-N (1.1-fold) ↑ 8-oxoGuo in OB-N vs LN (1.2-fold) ↑ 8-oxoGuo in OB-H vs LN (1.3-fold)
14 (M)
[57]
A
Abbreviations: BMI, body mass index; C, control; ELISA, enzyme linked immunosorbent assay, FACS, fluorescence-activated cell sorting; FPG, formamido pyrimidine glycosylase; HPLC, high-performance liquid chromatography, LPO, lipid peroxidation; MN, micronuclei; MDA, malondialdehyde; NWO, normal weight obese; n, number of participants; n.s., not specified; OB, obese; OW, overweight; 8-OHdG, 8-hydroxy-2’-deoxyguanosine; SC, standard conditions; wk, week; WC, waist circumference; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table) 2
Normal weight obese (NWO): according to the article by Tomasello et al [41], NWO known as a metabolic condition which observed in women with normal index but fat mass (FM) >30%.
70
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Table 5. Results of Gamma-H2AX (γ-H2AX) in overweight/obese subjects1 Participants and treatments
Parallel
FACS (lymphocytes)
OB: n= 11♂/12♀, BMIZscore2=2.8,age:11.0±0.8 C: n=7♂/8♀, BMIZ-score =0.7 age: 12.4±0.4
Parallel
FACS (lymphocytes)
M
A
Parameter (indicator cells) Gamma-H2AX (γ-H2AX)
ED
Study design
QS
Remarks
Ref
↑ γ-H2AX foci in OB vs. C after 2 hours inducing DNA damage by mitomycin C (2.0-fold) ↑ γ-H2AX foci in OB vs. C after 4 hours inducing DNA damage by mitomycin C (1.7-fold) ↑ γ-H2AX foci in OW vs. C (5.6-fold) ↑ γ-H2AX foci in OB vs. C (8.0-fold)
14 (M)
↑ expression of the high mobility group box-1 protein (HMGB1) involved in doublestrand breaks repair in OB
[58]
15 (M)
↑ MN in OW vs. C (2.5-fold) ↑ MN in OB vs. C (2.7-fold) ↔ TNF-α in all groups ↑ IL-6 in OW and OB vs. C (9.0- fold) ↑ CRP in OW and OB vs. C (17.7-fold)
[50]
↓abasic sites in DNA at 12 wks (30%) ↓abasic sites in DNA at 52 wks (65%)
-
↑ telomere lengths after weight loss (not significant)
CC E
PT
OB: n= 31♂/30♀, BMI= 29, age: 10.8±3.4 OW: n= 9♂/11♀, BMI= 24.4 age: 12.2±2.6 C: n=16♂/22♀, BMI= 17.5 age:10.8±2.6
Results
Miscellaneous
A
Intervention
test kit for abasic sites (apyrimidinic/ apurinic) (midrectal)
OB: after weight loss for 12wks (7000 kJ/day): n=45♂, BMI=31.9, age 51±1.0 and after 52 wks weight loss: n=12, BMI=31.6, age 56±1.1
1
[59]
Abbreviations:; BMI, body mass index; C, control; CRP, c-reactive protein; FACS, fluorescence-activated cell sorting; IL-6, interleukin 6; MN, micronuclei; n, number of participants; OB, obese; OW, overweight; TNF-α, tumor necrosis factor-alpha; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table) 2
BMI Z-score: Body mass index z-scores, also called BMI standard deviation (s.d.) scores, are measures of relative weight adjusted for child age and sex.
Table 6. Genomic instability in metabolic syndrome (MetS) patients1
71
I Parameter (indicator cells) Comet assay2
tail intensity early stage of MetS pts: (lymphocytes) n=65 ♂, BMI=33.0,age 47.4±5.5 C: n=56♂, BMI=24.5 age (all groups) 46.8±4.8 arbitrary units MetS pts: n= 38♂/27♀, (lymphocytes) BMI= 27.8 C: n=42♂/23♀, BMI= 26, age (all groups) 54-57 arbitrary units MetS pts: n=20♂/32♀, (whole blood) BMI=30.3, age 32-80 C: n=♂17/♀20, BMI=25, age (all groups) 27-70
Results
QS
Remarks
Ref
↔ DNA damage ↔ hOGG1 modified comet assay ↔ neutral comet assay
19 (M)
↓ 8-OHdG in urine MetS pts. (38%)
[65]
↑ DNA damage in MetS pts (1.7fold)
18 (M)
↓ total antioxidant capacity in MetS pts (50%)
[64]
↑ DNA damage in MetS pts (3.3fold)
20 (M)
↔ DNA damage in OB-MetS pts vs. OB-without MetS ↔ FPG in OB-MetS pts vs. OBwithout MetS ↔ endonuclease (ENDO) in OBMetS pts vs. OB-without MetS
19 (M)
↑ MN in MetS pts (2.2-fold) [63] ↑ malondialdehyde (MDA) in MetS pts (2.0-fold) ↓ superoxide dismutase in MetS pts (44%) ↓ glutathione peroxidase in MetS pts (27%) ↑ AA-FTO genotype and allele A OB♀ (1.2- [45] fold) ↓ TT-FTO genotype and allele T in morbidly OB♀ (1.2-fold)
tail intensity OB-MetS pts.: n= 30♀, (lymphocytes) BMI=45.7, age 32.4±6.8 OB-without MetS: n= 30♀, BMI=21.9 age 32.4±6.5
A
CC E
Parallel
ED
Parallel
PT
Parallel
M
A
Parallel
Participants and treatment
N U SC R
Study design
72
I N U SC R
Table 6. Continued
Study Parameter Participants and treatment design (indicator cells) Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay MetS pts: n=20♂/32♀, BMI=30.3, age 32-80 C: n=17♂/20♀, BMI=25, age 27-70
A
MN (lymphocytes)
QS
Remarks
Ref
↑ MN in MetS pts (2.2-fold)
17 (M)
↑ DNA damage in MetS pts (3.3-fold) ↑ MDA in MetS pts (2.0-fold) ↓ superoxide dismutase in MetS pts (44%) ↓glutathione peroxidase in MetS pts (27%)
[63]
↔ 8-OHdG levels in different MetS groups
13 (M)
↔ oxidative stress in the different groups ↑ 8-OHdG in hypertensive pts vs. C (1.5-fold)
[66]
↓ 8-OHdG in MetS pts.(38%)
15 (M)
↔ DNA damage in lymphocytes ↔ hOGG1 modified comet assay ↔ neutral comet assay
[65]
M
Parallel
Results
HPLC (genomic and mitochondrial – mononuclear cells)
CC E
PT
Parallel
ED
8-hydroxy-2' –deoxyguanosine (8-OHdG)
A
Parallel
ELISA (urine)
MetS pts: n=108♂/77♀ number of groups of MetS: 1- n=10♂/11♀, BMI=23 2- n=28♂/12♀, BMI=28 3- n=40♂/26♀, BMI=29 4- n=24♂/20♀, BMI=32 5- n=6♂/8♀, BMI=32 C (no MetS): n=16♂/14♀, BMI= 24 age (all groups) 25-50 early stage of MetS pts: n=65 ♂, BMI=33.0,age 47.4±5.5 C: n=56♂, BMI=24.5 age (all groups) 46.8±4.8
1
Abbreviations: BMI, body mass index; C, controls; CBMN-Cyt, Cytokinesis block micronucleus cytome assay; ENDO, endonuclease; FPG, Formamidopyrimidine DNA glycosylase; HPLC, high-performance liquid chromatography; MN, micronuclei; MetS, metabolic syndrome; 8-OHdG, 8-hydroxy-2' –deoxyguanosine; n, number of participants; OB, obese; OW, overweight; 8-oxo-dG, 8-hydroxy-2' –deoxyguanosine; pts, patients; SC, standard conditions; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table) 2
Unless otherwise indicated comet assays were conducted under standard condition (SC), alkaline version which detects single/ double and apurinic sites
73
I Method/ Participants and treatments Results parameter Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL assay)
A
Study design
N U SC R
Table 7. Obesity related DNA damage in sperm
♂ in subfertile couples, n=330 OB: n=43, BMI≥30 OW: n=137, BMI=25-29.9 C: n=151, BMI=18.5-24.9 age (all groups) 37.6±6.2 Sperm chromatin structure assay (SCSA) TUNEL assay
Intervention
SCSA, DFI
Intervention
SCSA, DFI
ED
M
Parallel
PT
CC E
A
♂n=43, BMI=33-61, 14 wks residential weight loss program n=15, BMI=46.0-61.0 n=14, BMI=41.7-46.0 n=14, BMI=33.3-41.6 age (in all group) 20-59 weight of women before pregnancy: under weight (UW): n=33 BMI<18.5 normal weight (C): n=263 BMI=18.5-24.9 OW: n=22, BMI≥25.0 off spring: UW: n=11 BMI<18.5 C: n=243 BMI=18.5-24.9 OB: n=83, BMI≥25.0
QS
Remarks
Ref
↑ DFI in OB vs. C (1.5-fold) ↔ DFI in OW vs. C (1.2-fold)
16 (M)
↑ viable spermatozoa OB vs. C (30%)
[70]
↔ DFI after weight loss
16 (M)
(+) correlation DFI and BMI weight loss associated with increased testosterone levels
[71]
↑ DFI in sons of OW mothers vs. sons of C mothers (1.2-fold)
15 (M)
↑ DFI in OW vs. C (but not significant), after 20years of follow-up cohort study
[72]
Table 7. Continued
74
I N U SC R
Table 7. Continued Study design
Method/ parameter
Participants and treatments
Parallel
SCSA (flow cytometry) DFI
Parallel
SCSA DFI
Crosssectional
SCSA (flow cytometry), DFI
PT
CC E A
♂ in subfertile couples, n=153 OB: n=16, BMI≥30, age 32.5±4.0 OW: n=63, BMI=25.0-29.9, age 32.8±6.7 C: n=74, BMI<24.9, age 30.2±5.9 ♂n=287, C: n=91 OW: n=136 OB: n=59 age (all groups) 23-45 ♂n=468, n=72, BMI≥35 n=122, BMI=30-35 n=191, BMI=25-29 n=83, BMI<25 age (all groups) 31.8 ± 4.8 n=175♂, BMI=25.5 age (all groups) 25-60
QS
Remarks
Ref
↔ DFI
13 (M)
↔ levels of chromatin integrity impairment ↑ cells with defective chromatin condensationin OB vs. C ↔ BMI and basic semen parameters, chromatin integrity and chromatin condensation
[73]
↔ DFI in different BMI ↑ high DNA sustainability in OB vs OW (4-fold) ↔DFI in different BMI
15 (M)
↑ cell phone use (11-25 years) vs (6-10 years) ↑ occupational stress (work)
[74]
14 (M)
(-) association WC and sperm count no significant association body size and semen concentration , motility, vitality or morphology
[75]
↔ BMI and DFI
12 (L)
(-) significant between association DFI and age
[76]
M
SCSA (flow cytometry) DFI
ED
Parallel
A
Sperm chromatin structure assay (SCSA)
Results
75
I Study Method/ Participants and treatment design parameter Sperm chromatin structure assay (SCSA)
♂;cohort A: n=275, age 18 cohort B: n=304 Swedish military recruits, BMI=22.6, age 18 cohort C: n=724 fertile men and men without fertility problems, BMI=25.3, age 18-69 cohort D: n=200 of men without known fertility problems, BMI=24.6, age 19-40
A
SCSA DFI
ED
M
Crosssectional
N U SC R
Table 7. Continued
Results
QS
Remarks
Ref
↑ DFI in C vs. OW (when excluding cohort C, the significance was lost)
14 (M)
↑ progressive motile spermatozoa in OW vs. C (when excluding cohort C, the statistical significance was lost)
[77]
A
CC E
PT
Abbreviations: AU, arbitrary unit; BMI, body mass index; DFI, DNA fragmentation index; N, number; n.s. , not specified; OB, obese; OW, over weight; SCSA, sperm chromatin structure assay; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; UW, underweight; WC, waist circumference; Y, year; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS) (For details see supplementary Table)
76
Participants and treatments
I
Method/ parameter
Results
N U SC R
Study design Comet assay
♂ in subfertile couples, n=413 ↔ DNA damage between high obese (OB): n=40, BMI=39.4, groups age 35.7±5.6 low OB: n=87, BMI=32.0. age 37.2±5.9 overweight (OW): n=233, BMI=27.0, age 36.3±5.2 control (C): n=123, BMI=23.0, age 35.7 ±5.2 Parallel standard ♂ in subfertile couples ↑ DNA damage in OB vs. C comet assay OB: n=36, BMI≥30, age 34.3±4.9 (1.8-fold) (arbitrary OW: n=187, BMI=25.0-29.9, age ↑ DNA damage in OW vs. C units) 34.7±7.9 (1.1-fold) C: n=82, BMI<24.9, age 33.5±6.1 Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL assay) neutral comet assay (tail intensity)
Remarks
Ref
19 (M)
↑ estradiol in both OB groups vs. C (1.2-fold) ↓ total testosterone in both OB groups vs. C (36%) ↑ sex hormone-binding globulin in both OB groups vs. C (40%)
[67]
18 (M)
↓ mitochondrial activity in OB vs. C (27%) ↓ sperm motility in OB vs. C (17%)
[68]
14 (M)
↓ viable spermatozoa in OB vs. C ↑ spermatozoa with decondensed chromatin in OB and OW vs. C (2.0-fold) ↑ spermatozoa with low mitochondrial membrane potential OB vs. C (14.0-fold) ↑ spermatozoa with low mitochondrial membrane potential OW vs. C (10.0-fold)
[69]
PT
TUNEL assay, flow cytometry
♂ healthy non-smokers, n=150 OB: n=50, BMI≥30, 31.6 ± 1.7 OW: n=50, BMI=25.0-29.9, age 31.2 ± 1.2 C: n=50, BMI=19.0-24.9, age 31.5 ± 1.1
↑ DFI in OB vs. C (3.0-fold) ↔ DFI in OW vs. C (1.5fold)
A
CC E
Parallel
ED
M
A
Parallel
QS
Table 8. Results of comet assay in animal studies concerning the impact of obesity on DNA stability1
77
I Zucker rats
Results
QS
Remarks
Ref
obese (OB) and control (C), n=5 p.g.♀ 3, 6, 12 months old weight after 12 months: OB: 747g; C: 259g OB: n=40♂, C: n=10♂ fed either with a hypercaloric (weight 620g) or a standard (STD) diet (weight 450g) for 30 wks after 20 wks , half of the OB rat were fed a 25%-caloric restriction diet (CRD) of STD for 10 wks n=6 p.g.♂ fed either STD or cafeteria diet (CAF) weight after 13 wks: STD: 44g, CAF:46g
↑ DNA damage in peripheral blood cell and liver OB vs. C (2.0-fold) ↔ DNA damage in kidney, brain and heart OB vs. C
13 (M)
↑ DNA damage in old rats vs. younger rats (3.0-fold in blood cells)
[94]
↑ DNA damage in OB vs. CRD (2.1-fold) ↑ DNA damage in OB vs. STD (10.0-fold)
13 (M)
↑ malondialdehyde (MDA) in OB vs. STD (2.2-fold) ↑ MDA in OB vs. CRD (1.8-fold) ↑ body weight in OB vs. STD animal lost weight after CRD vs. OB
[95]
↑ DNA damage in kidney CAF vs. STD (3.1-fold) ↑ DNA damage in peripheral blood CAF vs. STD (5.0-fold) ↑ DNA damage in liver CAF vs. STD (3.8-fold) ↑ DNA damage in brain CAF vs. STD (5.2-fold)
14 (M)
[96]
n=5p.g. ♂ and ♀ fed with either HFD (60% fat) or STD weight after 12 wks: ♂. OB: 43g; STD: 32g ♀. OB: 30g; STD:23g
↑ DNA damage in liver OB vs. STD in ♂ (1.6fold) and ♀ (2-fold) ↑ DNA damage in colon OB vs. STD in ♂ (1.5fold) and ♀ (1.5-fold) ↑ DNA damage in brain OB vs. STD in ♂ (1.9fold) and ♀ (1.7-fold) ↔ DNA damage in blood OB vs. STD in ♂ and ♀
15 (M)
↔ ratio polychromatic: normochromatic erythrocytes from bone marrow ↑ MN in CAF vs. STD (4.0-fold) ↑ oral glucose tolerance OB vs. STD (2.0-fold) ↑ TNF-α in plasma OB vs. STD in ♂ (1.9-fold) and ♀ (2.1-fold) ↑ MCP-1 in plasma OB vs. STD in ♂ (1.9-fold) and ♀ (3.3-fold) ↑ insulin in plasma OB vs. STD in ♂ (9.0-fold) and ♀ (9.9-fold) only abstract available
M
ED
tail moment (kidney, liver, heart, brain, blood cells) Wistar rats
N U SC R
Experimental design
A
Animal/parameter (indicator cells) comet assay2
CC E
PT
tail intensity (lymphocytes)
Swiss albino mice
A
arbitrary units (peripheral blood, kidney, liver, brain) C57BL/6J mice tail intensity (liver, colon, brain, blood)
78
[92]
I Experimental design
C57BL/6J mice
A
tail intensity (liver, colon)
Remarks
Ref
n=14 per group.♂ fed with HFD (45% fat) or STD weight after 4 months: OB: 46g STD: 27g
↑ DNA damage in liver OB vs. STD (2.1-fold) ↑ DNA damage in colon OB vs. STD (2.4-fold)
14 (M)
[97]
n=6 per group ♂ 5-6 wks fed either STD (10% fat) or HFD (45 % fat) weight after 6 wks.: HFD: 32g, STD: 28g
↑ DNA damage in in liver STD vs. HFD ↔ DNA damage in testes and blood ↑ FPG in white blood cells and liver in STD vs. HFD (1.1-fold) ↔ FPG in testes
14 (M)
↓ gene expression of MLH1 in liver OB vs. STD (50%) ↔ gene expression of MLH1 in colon ↓ gene expression of IL-6 in colon OB vs. STD (70%) ↓ gene expression of Dnmt1 in colon OB vs. STD (60%) ↓ gene expression of Dnmt1 in liver OB vs. STD (50%) ↓ bacterial abundance in OB vs. STD ↑ haemoglobin glycidamide adducts in HFD vs. STD ↑ weight gain HFD vs. STD (1.5-fold) glycidamide-induced sperm DNA fragmentation higher in HFD vs. STD ↔DFI in sperm HFD vs. STD
n=14 per group ♂ fed with STD or HFD (45% fat) weight after 4 months: OB: 46g, STD : 27g
↑ DNA damage in liver OB vs. STD (2.3-fold) ↑ DNA damage in colon OB vs. STD (2.6-fold)
14 (M)
↓ gene expression of MLH1 in liver OB vs. STD (49%) ↔ gene expression of MLH1 in colon OB vs. STD ↓ gene expression of Dnmt1 in colon OB vs. STD (61%) ↓ gene expression of Dnmt1 in liver OB vs. STD (61%) ↓ bacterial abundance in OB vs. STD ↔ gene expression Nrf2 and MGMT
[98]
ED PT
CC E
tail intensity (testes, blood, liver) and SCSA (sperm)
C57BL/6J mice
QS
M
tail intensity (liver, colon)
C57BL/6J mice
Results
A
Animal/method/parameter (indicator cells) Comet assay
N U SC R
Table 8. Continued
79
[93]
I N U SC R
Table 8. Continued
Experimental design
Results
QS
Remarks
Ref
WNIN/Ob rat (Met S- model)
n=6 per group ♂, OB: WNIN/Ob-obese (3-months) C: WNIN/Ob-lean (3months)
↑ DNA damage (SSB) in hippocampus in OB vs. C (1.2-fold) ↑ DNA damage (SSB) in neocortex in OB vs. C (1.4-fold) ↑ DNA damage (double strand breaks (DSBs) in hippocampus in OB vs. C (2.0-fold) ↑ DNA damage (DSBs) in neocortex in OB vs. C (1.9-fold)
13 (M)
↑ MDA in OB vs. C (1.5-fold) ↓ superoxide dismutase in OB vs. C (50%) ↓ activity of catalyse in OB vs. C (44%) weight (n.s)
[88]
M
ED
tail moment (neocortex & hippocampus)
A
Animal/method/parameter (indicator cells) Comet assay
1
Unless otherwise indicated comet assays were conducted under standard conditions (SC), alkaline version which detects single/ double and apurinic sites
A
2
CC E
PT
Abbreviations: C, controls; CAF, cafeteria diet; CBMN-Cyt, cytokinesis block micronucleus cytome assay; CRD, calorie restricted diet; DIO, diet induced obesity FPG, formamido pyrimidine glycosylase; HFD: high fat diet; IL-6, interlukin 6; MN, micronuclei; MDA, malondialdehyde; n, number of participants; OB, obese; OGG1, 8oxoguanine DNA glycosylase; OW, overweight; 8-oxodG, 8-oxo-7,8-dihydro-2' –deoxyguanosine; p.g.: per group; RSD, restricted diet; SC, standard conditions; STD, standard diet, TNF-α, tumor necrosis factor-alpha; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS). (For details see supplementary Table)
80
I N U SC R
Table 9. Results of Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay in animal studies concerning the impact of obesity on DNA stability Animal (indicator cells)
Experimental design
Results
QS
Remarks
Ref
↑ DNA damage in kidney CAF vs. STD (3.1[96] fold) ↑ DNA damage in peripheral blood CAF vs. STD (5.0-fold) ↑ DNA damage in liver CAF vs. STD (3.8-fold) ↑ DNA damage in brain CAF vs. STD (5.2fold) ↑ oral glucose tolerance test OB vs. STD the promoters of p16 and Dapk genes were [99] methylated in the livers of C57BL/6J mice fed a HFD (irradiated and non-irradiated) Mgmt promoter was methylated in irradiated and HFD mice.
11 (M)
n=5, C57BL/6J DIO: fed for 3 wks, age=14 wks C3H/HeJ: fed for 12 wks, age=5 wks both strains fed either HFD (60% fat) or STD (10 % fat) gender (n.s) n=36♂ fed either with HFD (60% fat) or with LFD (10% fat) for 18 wks, weight 28 g treated either with N-ethylN-nitrosourea (ENU) or with the vehicle control for 3 days
↔ MN in HFD in vs. STD (in both strains) ↔ P/N ratio in HFD vs. STD (in both strains)
10 (M)
↔ MN-NCEs HFD vs. LFD ↔ MN RETs HFD vs. LFD ↔ MN after ENU treatment in HFD vs. LFD ↑ MN-NCEs after ENU treatment in HFD and LFD vs. control (1.1-fold) ↑ MN RETs after ENU treatment in HFD and LFD vs. control (4-fold)
9 (M)
CC E
PT
C57BL/6J DIO mice (prediabetic T2D) and C3H/HeJ mice
C57BL/6J mice
flow cytometry (blood)
A
↑ MN in CAF vs. STD (4.0 -fold) ↔ polychromatic: normochromatic (P/N) ratio in bone marrow
ED
(bone marrow)
(bone marrow)
n=6 per group ♂ fed either STD or cafeteria diet (CAF) weight after 13 wks: STD: 44g, cafeteria diet (CAF): 46g
M
Swiss albino mice
A
Cytokinesis – block micronucleus cytome (CBMN-Cyt) assay
81
↑ Pig-α mutant RBCs in HFD vs. LFD (2.5fold)
[100]
I N U SC R
Table 10. Results of 8-oxo-7,8-dihydro-2' -deoxyguanosine (8-oxodG) in animal studies concerning the impact of obesity on DNA stability1
C57BL/6J mice
n=10 per group ♀ mother: either folate-depleted (2 mg folic acid/kg diet) or folate-adequate (0.4 mg folic acid/kg diet) 4 wks before mating off-spring: at weaning (age: 22-25 days), either HFD (20% anhydrous milk fat) or C (5% anhydrous milk fat) for 5 months n=40 per group ♂ fed either with HFD (45 %) or C (12%) for 10 wks.
PT
OB Zucker fa/fa rat and lean Zucker fa/+ rats
M
HPLC (urine)
♂, OB: weight=440 g, lean (C): weight=350 g, weight loss via roux-en-Y gastric bypass surgery (n=15), weight 410 g or sham surgery (n=17), weight 580 g
ED
A
Method/parameter Experimental design Results QS Remarks (indicator cells) 8-oxo-7,8-dihydro-2' -deoxyguanosine (8-oxodG) and 8-OH-Guanosine (8-OH-Gua) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo)
CC E
HPLC (subcortical regions of brain)
A
Diet induced obesity (DIO) rat HPLC (in mitochondria of heart, liver, kidney and testes)
weight: HFD: 628 g, C: 570 g
↑ 8-oxodG after sham surgery vs. C (3.5-fold) ↔ 8-oxodG in OB vs. C ↑ 8-oxoGua in OB vs. C (2.5-fold) ↑ 8-oxoGuo in OB vs. C (3-fold)
11 (M)
↓ 8-oxodG in subcortical region of HFD vs. C (not significant)
11 (M)
↑ 8-oxodG in heart of HFD vs. C (1.1-fold) ↑ 8-oxodG in kidney of HFD vs. C (1.2-fold) ↑ 8-oxodG in testes of HFD vs. C (1.2-fold) ↑ 8-oxodG in liver of HFD vs. C in 10 wks.(1.1-fold) ↓ 8-oxodG in liver of HFD vs. C in 2 and 8 wks. (25%)
11 (M)
82
Ref
↑ MDA in OB vs. C (3.0-fold) ( + ) correlation between DNA damage and basal plasma insulin values ↑ DSBs in sham surgery vs. C (3.0fold) ↑ γ-H2AX in OB rats (7.0-fold) ↓ after intervention by bypass surgery and caloric restriction (3.2-fold) ↑8-OxodG levels correlated with lower BER (not significant) ↓ BER activity in off-spring HFD vs. C in cortex (40%), hippocampus (40%) and subcortical regions (45%)
[101]
↑ MDA in liver of HFD vs. C (1.3fold) ↑ MDA in heart of HFD vs. C (1.2fold) ↔ MDA in kidneys and testes of HFD vs. C
[103]
[102]
I N U SC R
Table 10. Continued
Method/parameter Experimental design Results QS Remarks (indicator cells) 8-oxo-7,8-dihydro-2' -deoxyguanosine (8-oxodG) and 8-OH-Guanosine (8-OH-Gua) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo)
A
↓ 8-OH-Gua in OB vs. C (24%)
ED
HPLC with ultraviolet (liver)
n=46♀ obese fa/fa: n=20 lean (C): n=26 mice received 7,12 dimethylbenz(α)anthracene (DMBA) and after 155 days, liver removed.
M
Zucker rat
1
11 (M)
↑ ratio of glutathione/ oxidised glutathione disulphide in OB vs. C (1.3-fold) ↓ S-adenosylmethionine/Sadenosylhomocysteine levels (45%) ↑methionine in OB vs. C (1.3-fold) ↑5-methylcytosine in OB vs. C (1.1-fold)
Ref
[104]
A
CC E
PT
Abbreviations: C, controls; CAF, cafeteria diet; CBMN-Cyt, cytokinesis block micronucleus cytome assay; CRD, calorie restricted diet; DIO, diet induced obesity; DMBA, 7,12 dimethylbenz(α)anthracene, ELISA, enzyme linked immunosorbent assay; ENU, N-ethyl-N-nitrosourea; GSH/GSSG, glutathione/ oxidised glutathione disulphide; HFD: high fat diet; HPLC, high-performance liquid chromatography; MDA, malondialdehyde; n, number of participants; OB, obese; OGG1, 8-oxoguanine DNA glycosylase; OW, overweight; 8-oxodG, 8-oxo-7,8-dihydro-2' –deoxyguanosine; 8-OH-Gua, 8-OH-Guanosine; 8-oxoGuo, 8-oxo-7,8-dihydroguanosine; RSD, restricted diet; SC, standard conditions; STD, standard diet; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS) (For details see supplementary Table).
83
I N U SC R
Table 11. Results of Gamma-H2AX (γ-H2AX) in animal studies concerning the impact of obesity on DNA stability
Method (indicator cells)
Experimental design
Results
QS
Remarks
Ref
OB: n=22♂, weight 360g C: n=18♂, weight 300g either fasting or acute hyperglycemia ♂, weight 440 g, lean Zucker fa/+ rats (12 wks), weight 350g weight loss either via Roux-en-Y gastric bypass (RYGB) surgery (n=15), weight 410g or sham surgery (n=17), weight 580g
↑ γ-H2AX in OB vs. C (4.5-fold) ↑ γ-H2AX in OB/acute hyperglycemia vs. C/acute hyperglycemia (4.5-fold)
9 (L)
↑ insulin in OB vs. C (2.6-fold)
[105]
↑ DSBs in sham surgery vs. C (3.0-fold) ↑ γ-H2AX in OB rats (7.0-fold) and decrease after intervention by RYGB surgery and caloric restriction (3.2-fold)
[101]
♀, OB: n=10, lean (c): n=10, 15 wks in both the lean and obese groups were ip dosed once with SO and phosphoramide mustard (PM)
↑ γ-H2AX in OB (PM) vs. lean (PM) (1.5-fold) ↑ γ-H2AX in OB(PM) vs. OB (1.5-fold)
↑ 8-OxodG after RYGB vs. lean 11 (M) (2.5-fold) ↔ 8-OxodG in OB vs. lean ↑ 8-oxoGua in OB vs. lean (2.5-fold) ↑ 8-oxoGuo in OB vs. lean (3.0-fold) ↑ malondialdehyde in OB vs. lean (3.0-fold) (+) correlation DNA damage and basal plasma insulin ↔ DNA repair genes Prkdc, Parp1 9 (M) and Rad51 mRNA by obesity ↓relative Xrcc6 mRNA in OB vs. C (45%) ↑ relative Atm mRNA in OB vs. C (1.8-fold) ↑relative Brca1 mRNA in OB vs. C (1.3-fold)
A
Gamma-H2AX (γ-H2AX)
M
Zucker rat
OB Zucker fa/fa rat
CC E
PT
(colon and kidney)
ED
(lung)
A
Normal nonagouti (a/a; designated lean), agouti lethal yellow (KK.Cg-Ay/J; designated obese) Western blot (ovaries)
84
[106]
I Method/parameter (indicator cells) Miscellaneous
Experimental design
n=344♀ groups: LFD (3% fat) hydroxymethyluracil, weight=136 g, gas spectrometry HFD (20 % fat) weight 146 (liver and mammary g, dietary restriction (CRD, gland) 40 %) weight 113 g, C (5% fat) weight 137 g, for 2 wks. C57BL/6J mice n=36♂ fed either with HFD (60% Pig-α mutation, flow fat) or LFD (10% fat) for 18 cytometry wks, weight 28 g (in normochromatic treated either with N-ethylerythrocytes (NCEs) and N-nitrosourea (ENU) or reticulocytes(RET)) vehicle control for 3 days C57BL/6J mice n=7-9♂ fed either with HFD (60% quantitative alkaline fat) or LFD (10% fat) for 16 Southern blot wks. (liver and skeletal weight: muscles) HFD: 38g LFD: 28g
Results (fold)
QS
Remarks
Ref
↓ 5-hydroxymethyluracil in liver in CRD vs. C (43.3%) ↓ 5-hydroxymethyluracil in mammary gland in HFD vs. LFD (31.8%)
-
↓ body weight in CRD vs. HFD (28%) ↑ body weight in HFD vs. STD (1.1-fold) ↑ body weight in HFD vs. LFD (1.1-fold) ↑ body weight in HFD vs. CRD (1.3-fold)
[107]
↑ Pig-α mutation in RBCs in HFD vs. LFD (2.5-fold)
-
[100]
↑ DNA damage (mitochondrial) in HFD in liver and muscle
-
↔ MN-NCEs HFD vs. LFD ↔ MN RETs HFD vs. LFD ↔ MN after ENU treatment in HFD vs. LFD ↑ MN-NCEs after ENU treatment in HFD and LFD vs. control (1.1-fold) ↑ MN RETs after ENU treatment in HFD and LFD vs. control (4-fold) ↓ ratio glutathione/glutathione disulfide ↔APE1 in skeletal muscles in mitochondria base excision DNA repair: ↑ apurinic/apyrymidinic Endonuclease 1 in liver (2.0-fold) and skeletal muscles (1,8-fold) ,in nucleus in HFD ↑ OGG1 (8-oxoguanine DNA glycosylase/AP lyase), in skeletal muscles in nucleus (1.6-fold) and mitochondrial (1.5fold) in HFD vs. LFD ↔ OGG1 in liver (nucleus and mitochondrial) ↓ GSH/GSSG HFD vs. LFD in liver (2.0-fold) ↓ GSH/GSSG HFD vs. LFD in muscle (2.8-fold) ↑ free fatty acid HFD vs. LFD (2.0-fold) ↑ insulin HFD vs. LFD (2.0-fold) ↑ triglyceride HFD vs. LFD (2.5-fold)
A
CC E
PT
ED
M
A
Fischer rats
N U SC R
Table 11. Continued
85
[108]
I N U SC R
A
Abbreviations: C, controls; CAF, cafeteria diet; CRD, calorie restricted diet; DIO, diet induced obesity; DMBA, 7,12 dimethylbenz(α)anthracene; ENU, N-ethyl-N-nitrosourea; GSH/GSSG, glutathione/ oxidised glutathione disulphide; HFD: high fat diet; MN, micronuclei; MDA, malondialdehyde; n, number of participants; OB, obese; OGG1, 8oxoguanine DNA glycosylase; OW, overweight; 8-oxodG, 8-oxo-7,8-dihydro-2' –deoxyguanosine; 8-OH-Gua, 8-oH-Guanosine; 8-oxoGuo, 8-oxo-7,8-dihydroguanosine; PM, Phosphoramide mustard; RET, reticulocytes; RSD, restricted diet; SC, standard conditions; STD, standard diet, TNF-α, tumor necrosis factor-alpha; wk, week; high (H), medium (M) and low (L) refer to classification on the basis of quality scoring (QS) (For details see supplementary Table).
A
CC E
PT
ED
M
Table 12. Results of animal studies concerning obesity related DNA damage in sperm
86
I Zucker rat
N U SC R
Experimental design
Results
QS
Remarks
Ref
n=6 per group ♂ after 64 and 100 days OB: weight in day 64=344g, day100=490g C: weight in day 64=233g, day 100=344g
↑ DNA damage (tail length) in OB vs. C (1.3-fold) ↑ DNA damage (tail extent moment) in OB vs. C (1.8-fold) ↔ DNA damage (tail DNA and olive tail moment)
12 (M)
↑ daily sperm production in C vs. OB (1.3-fold) ↑ testes weight in C vs. OB (1.5-fold)
[113]
n=40♂, 6 wks old, HFD: n=7, STD: n=8 fed either STD (6% fat) or HFD (21% fat) weight after 18wks: HFD=34.2g, STD=28.3g
↑ sperm DNA damage (%) in OB vs. STD (1.6-fold)
10 (M)
[111]
n=36♂, 6 wks old, HFD: n=18, STD: n=18 fed either with STD (6% fat) or HFD (22% fat, 0.15% cholesterol) weight after 9 wks: HFD=30.0g, STD=29.4 g
↑ sperm DNA damage (%) in OB vs. STD (9.4-fold)
10 (M)
↑ mitochondrial ROS in OB vs. STD (1.1-fold) ↓ sperm mobility in HFD vs. STD (11%) ↑ abnormal sperm tail in OB vs. STD (1.2-fold) ↔ free fatty acid in OB vs. STD ↑ glucose in OB vs. C in OB vs. STD (1.0-fold) ↑ cholestrol in OB vs. C in OB vs. STD (1.6fold) ↑ triglyceride in OB vs. C in OB vs. STD (1.2fold) ↑ mitochondrial (ROS) in OB vs. STD (1.6fold) ↑ intracellular ROS in OB vs. STD (1.9-fold)
M
A
Animal/method/p arameter (indicator cells) Comet assay
C57BL/6J mice
PT
ED
comet assay, tail length, tail extent moment, olive tail moment and tail DNA (testes) Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL assay)
CC E
TUNEL assay, cell detection kit (testes)
A
C57BL/6J mice
TUNEL assay, cell detection kit (testes)
Table 12. Continued
87
[110]
I N U SC R
Abbreviations: AU, arbitrary unit; BMI, body mass index; DFI, DNA fragmentation index; N, number; n.s. , not specified; OB, obese; OW, over weight; SCSA, sperm chromatin structure assay; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; UW, underweight; Y, year; high (H), medium (M) and low (L) refer to the classification
ED
SCSA, DNA fragmentation index (testes) C57BL/6J mice
♂, fed either STD (10% fat) or HFD (45 % fat) weight after 11 wks: HFD=33.6g, STD=29.4g n=9 to 14 per group
CC E
PT
comet assay (testes, blood and liver) SCSA, flow cytometry (sperm)
Results
QS
Remarks
Ref
↑ DFI in HFD vs. STD (1.1fold)
11 (M)
(+) correlation BMI and DFI (+) correlation between blood glucose and DFI
[112]
↑ DNA damage (single strand breaks and alkali-labile sites) in STD vs. HFD in liver ↔ DNA damage in testes and blood ↑ FPG in white blood cells and liver in STD vs. HFD (1.1-fold) ↔ DFI in HFD vs. STD
11 (M)
↑ haemoglobin glycidamide adduct levels in HFD vs. STD ↑ weight gain HFD vs. STD (1.5-fold)
[93]
M
C57BL/6J (wild type mice), C57BL/6J-ob/+ (leptin heterozygous mice) and C57BL/6J-ob/ob (Lepob; leptin- deficient mice)
A
Animal/method/parameter Experimental design (indicator cells) Sperm chromatin structure assay (SCSA)
n=6 per group ♂, 5-6 wks fed either STD (10% fat) or HFD (45 % fat) weight after 6 wks: HFD=31.7g, STD=28.1g
A
on the basis of quality scoring (QS) (For details see supplementary Table).
88