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Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells
Accepted Manuscript Title: Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells Author: Katja Matt Katharina Burger Daniel Gebhard J¨org Bergemann PII: DOI: Reference:
S0047-6374(16)30013-6 http://dx.doi.org/doi:10.1016/j.mad.2016.02.008 MAD 10817
To appear in:
Mechanisms of Ageing and Development
Received date: Revised date: Accepted date:
28-7-2015 5-2-2016 10-2-2016
Please cite this article as: Matt, Katja, Burger, Katharina, Gebhard, Daniel, Bergemann, Jddotorg, Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.Mechanisms of Ageing and Development http://dx.doi.org/10.1016/j.mad.2016.02.008 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.
Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells
Katja Matta, Katharina Burgera, Daniel Gebharda, Jörg Bergemanna
a
Department of Life Sciences, Albstadt-Sigmaringen University of Applied Sciences,
Sigmaringen, Germany
Corresponding author: Katja Matt Albstadt-Sigmaringen University of Applied Sciences, Anton-Günther-Strasse 51, 72488 Sigmaringen, Germany. Tel.: +49 7571 732 8375 e-mail: [email protected]
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Highlights Successful adaption of HCRA to an application using human PBMCs ex vivo. Inter-individual differences in DNA repair ability of test persons. Calorie reduction can influence DNA nucleotide excision repair capacity positively.
Abstract Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging.
Abbreviations: CR, calorie reduction; HCRA, host cell reactivation assay; NER, nucleotide excision repair; PBMC, peripheral blood mononuclear cell; EDTA, ethylene diamine tetraacetic acid; PBS, phosphate buffered saline; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; SEM, standard error of the mean; UV, ultraviolet
Keywords: DNA repair capacity; modified host cell reactivation assay; calorie reduction; F. X. Mayr therapy; human peripheral blood mononuclear cells
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1. Introduction As a non-pharmacological intervention, calorie reduction (CR) is assumed to prolong lifespan and reduce age-related diseases in a variety of model organisms like rodents or invertebrates (Weindruch, 2003). As early as in 1935, McCay researched the effect of caloric restriction in rats, stating that caloric restriction not only retards the growth of rats, but also increases their lifespan (McCay et al., 1989). In non-human primates caloric restriction even reduces agerelated mortality as well as overall-mortality (Colman et al., 2014). Furthermore it has already been shown that caloric restriction reduces oxidative stress (Qiu et al., 2010), increases base excision repair in the nucleus (Stuart et al., 2004), has protective effects on age related diseases like atherosclerosis (Fontana et al., 2004) and protects many model organisms like non-human primates and rats from aging (Cabelof et al., 2003; Roth et al., 2001). It can be stated that caloric restriction and its effect on aging and age-related diseases is extensively researched in model organisms, but it is not so easy to find studies involving humans in these field of research, probably due to ethical reasons. If humans are involved in studies of DNA repair and aging, experiments are often conducted using cultured human cells. In 1974 for example, Hart and Setlow correlated an increasing ability to repair DNA damages via unscheduled DNA synthesis with increasing maximum lifespan of various species including humans (Hart and Setlow, 1974). A significant influence of dietary restriction on DNA repair measured via unscheduled DNA synthesis was first shown in 1989 in hepatocytes and kidney cells of rats (Weraarchakul et al., 1989). F. X. Mayr therapy represents a modified form of fasting, thus a particular way of calorie reduction for humans. Aims of this therapy are to recover the digestive tract as well as the training of proper mastication and promotion of health. For reliable analyses of systemic influences like calorie reduction, an appropriate method of measuring DNA repair ability directly out of the human body is beneficial. An ideal method would require minimally invasive sampling, so that test persons can easily be enlisted. The isolation of peripheral blood mononuclear cells (PBMCs) out of human venous blood is relatively fast and simple and provides cells needed for research of DNA repair ex vivo. Using human PBMCs, Langie et al. studied the effect of dietary intervention on nucleotide excision repair capacity using a modified comet assay (Langie, S A S et al., 2010). However, this method displays the DNA damage recognition and incision ability of the individual (Langie et al., 2006), but not the functional restoration of damaged DNA. Modified host cell reactivation assay (HCRA) is a suitable method for analyzing nucleotide excision repair capacity. The advantage of this assay is that a functional restoration of a previously damaged reporter plasmid can be measured. The principle of restoration of a reporter gene 3
was first described by Protić-Sabljić et al. in 1985 (Protić-Sabljić et al., 1985) and then modified by Athas et al. for an application using human lymphocytes (Athas et al., 1991). Host cell reactivation assay was further refined in 2000 by Roguev and Russev using green and yellow fluorescent proteins as reporters (Roguev and Russev, 2000) and in 2007 by Burger et al. by adapting HCRA to flow cytometry analysis of primary human skin cells (Burger et al., 2007; Burger et al., 2010). HCRA, as we conduct it nowadays, is based on the deactivation of a reporter gene, which is inserted into a plasmid, by DNA damages introduced via UVC irradiation. After the introduction of the damaged plasmid into the cell, the ability of the cell to repair the damages leads to the functional reactivation of the reporter gene. Since UVC radiation damages DNA directly, thus leading mainly to cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs) and these DNA damages are repaired via nucleotide excision repair (NER), HCRA displays the cells’ NER capacity. Here we present the successful adaption of this modified HCRA for an application with freshly isolated human PBMCs. Using this reliable and highly reproducible method, we studied the influence of a particular way of calorie reduction, namely F. X. Mayr therapy, in an ex vivo approach. Results of this ex vivo study show a statistically significant increase of DNA repair capacity in individuals with lower DNA repair ability before Mayr therapy, lasting until the end of the study. The repeat of the study even reproduced this finding, clearly indicating an influence of CR on DNA nucleotide excision repair of human PBMCs.
2. Materials and Methods 2.1
F. X. Mayr therapy and samples
Test persons participating in F. X. Mayr therapy were recruited at the center for Traditional Chinese Medicine (TCM) in Sigmaringen. F. X. Mayr therapy aims at cleaning, rest and regeneration of the gastrointestinal tract, the learning of a healthy eating behavior (stop eating when the first feeling of satiety occurs) and a detoxification of the human body (Witasek, 1999). Procedure of a six-week Mayr therapy is as follows: one week pretreatment (eating no indigestive food), followed by 3 weeks of diet food (stale bread rolls and milk) and two weeks of convalescence (where the body is adjusted to regular food again). Through this monotonic diet the intestinal tract is rested. For each person, the diet is individually assorted. Crucial for a successful Mayr therapy is, that during fasting with stale bread rolls and milk, the persons 4
have to stop eating when the first feeling of satiety occurs resulting in a calorie reduction. The average loss of weight during a six week F. X. Mayr therapy is about five kilograms, ranging from three to eight kilograms. The study was approved by the ethics committee of the Landesaerztekammer Baden-Wuerttemberg and all experiments were in accordance with the declaration of Helsinki. The volunteers were informed before collection of the samples and gave their written consent. All data concerning the individuals under study have been pseudonymized. The two test persons for reproducibility studies were recruited within our department and were available for all three repetitions. In brief, 12 mL venous blood was collected in tubes containing EDTA as anticoagulant and stored at 4 °C in the dark until isolation of the PBMCs. Isolation was conducted not exceeding 4 h after taking of the blood samples. For reproducibility studies, three blood samples were taken on three consecutive days at the same time of day. This study was repeated three times over a period of ten months. For the study of calorie reduction and its influence on DNA repair, three samples were collected from each test person: the first one before the beginning of F. X. Mayr therapy, the second one on the eighth day of diet food intake and the third one during convalescence.
2.2
Isolation of PBMCs
Human PBMCs were isolated from 12 mL of human blood using Leucosep tubes (Greiner Bio-One GmbH, Frickenhausen, Germany) according to the manufacturer’s instructions. PBMCs were resuspended in 5 mL phosphate buffered saline (PBS) and cell count was determined using Neubauer-improved counting chamber.
2.3
Host cell reactivation assay
2.3.1 Plasmid preparation Two plasmids were used for the determination of DNA repair capacity of freshly isolated human PBMCs: pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA, USA) and a modified DsRed-Express, where the GFP cassette of pEGFP-N1 was replaced with the DsRed cassette of pDsRed-Express. Both plasmids were augmented in E. coli K12 and purified using Plasmid Plus Giga Kit and dissolved in EB Buffer (both Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. UVC irradiation (254 nm) of DsRed-Express 5
was carried out using a Stratalinker® UV Crosslinker (Model 1800, Stratagene, La Jolla, CA, USA). Drops of 40 µL of plasmid solution were irradiated with 5 kJ/m2 of UVC. Plasmid concentrations did not exceed 900 ng/µL. After irradiation, 10 µg of pEGFP and 30 µg of DsRed (either irradiated or non-irradiated) were mixed, aliquoted and stored at – 80 °C until further use. One plasmid mixture was prepared this way for the year 2014 study and the year 2015 study to ensure the use of one plasmid mixture for the entire study of calorie reduction. For the three reproducibility studies, three independent preparations of the plasmid mixture were used. More precisely, for each of the three repeats, DsRed was irradiated and mixed with GFP separately, resulting in three independent preparations of the plasmid mixture. For day 1, day 2 and day 3, aliquots of one plasmid mixture were used.
2.3.2 Transfection Human PBMCs were transfected by electroporation using 0.4 cm electroporation cuvettes (Molecular BioProducts, Inc., purchased via Thermo Fisher Scientific GmbH, Dreieich, Germany). Therefore 2 x 106 PBMCs were mixed with 150 µL RPMI 1640 without Phenol Red (Gibco® by Life Technologies, purchased via Fisher Scientific GmbH, Schwerte, Germany) and 100 µL of EB Buffer (Qiagen GmbH, Hilden, Germany) containing 10 µg pEGFP and 30 µg DsRed-Express. 250 µL of this solution was electroporated (400 kV, 500 µF) using an electroporator (Gene Pulser II, Bio-Rad Laboratories, Inc., Hercules, CA, USA) and immediately seeded into 6 well plates (Sarstedt AG & Co., Nuembrecht, Germany) containing 3 mL of PBMC media (RPMI 1640 without phenol red and 20 % FBS, both Gibco® by Life Technologies). For the determination of DNA repair capacity, cells were transfected with a plasmid mixture containing irradiated DsRed and non-irradiated GFP, for transfection control, cells were transfected with the mixture of non-irradiated DsRed and GFP. All measurements - reproducibility studies as well as the studies of calorie reduction were carried out in triplicates.
2.3.3 FACS analyses Analysis of transfected cells was carried out using FACSCalibur and CellQuest Pro software (both Becton, Dickinson and Company, Franklin Lakes, NJ, USA) 24 h after transfection. DNA repair capacity was calculated as follows: 6
𝑅𝑒𝑝𝑎𝑖𝑟 𝐶𝑎𝑝𝑎𝑐𝑖𝑡𝑦 [%] =
𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑟𝑒𝑑 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑡 𝑐𝑒𝑙𝑙𝑠 𝑖𝑟𝑟𝑎𝑑𝑖𝑎𝑡𝑒𝑑 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑔𝑟𝑒𝑒𝑛 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑡 𝑐𝑒𝑙𝑙𝑠
∗ 𝐹 ∗ 100
The factor F is defined as the ratio of percentages of green fluorescent cells to non-irradiated red fluorescent cells. It is used to correct DNA repair capacity with regard to efficacy of transfection as described by Burger et al. (Burger et al., 2007).
2.4
Data analyses
For statistical analyses as well as for graph generation GraphPad Prism 5 for Windows (Version 5.04) was used. Regarding their DNA repair capacity, test persons participating in F. X. Mayr therapy were divided into the upper 50 % of the group and the lower 50 % of the group to better point out the influence of calorie reduction on DNA repair capacity. This procedure was applied for the year 2014 study as well as for the year 2015 study. The upper 50 % of test persons were defined as the group “normal pre-existing DNA repair capacity” as in our experience, most individuals display DNA repair capacity values of about 85 % to 90 %. The lower 50 % of test persons were defined as “low pre-existing DNA repair capacity”. Statistical analyses were carried out as paired t-Tests (p* < 0.05), as well as linear regression analyses (p* < 0.05) for age dependence of DNA repair capacity as shown in supplementary figure 2.
3. Results 3.1
Reproducibility of HCRA
For testing of reproducibility, DNA repair capacity was determined using PBMCs of two test persons A and B. On three consecutive days blood samples were taken at the same time of day and HCRA was conducted. This approach was repeated independently three times throughout ten months, so every time the blood samples of the same two individuals were used. DNA repair capacities of the two persons tested are shown in table 1 as well as in figure 1. It has to be remarked that three independent preparations of the plasmid mixture were used
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for the reproducibility studies. On day 1, 2 and 3 of each repeat aliquots of the same plasmid mix preparation were used. Interestingly, test person A showed slightly higher DNA repair capacities than B when using the different plasmid mixtures as well as over the tested time points. Furthermore, SEMs of the triplicates from person A were a little bit lower on average. For test person A, variabilities were 0.96 % for reproducibility 1, 0.5 % for reproducibility 2 and 1.33 % for reproducibility 3, whereas test person B showed variabilities of 0.63 % (reproducibility 1), 2.26 % (reproducibility 2) and 0.52 % (reproducibility 3) respectively. Nonetheless, variabilities of DNA repair capacity values over the three consecutive days never exceeded 2.26 %, as shown in sample B2 and the lowest variability was displayed in A2 with a value of 0.5 %. Neither in test person A nor in test person B significant changes in DNA repair capacity were detectable over the three consecutive days, as demonstrated by p-values in table 1. Figure 1 displays the results of all reproducibility studies. Both test persons showed a high reproducibility over the three days. Although independent preparations of the plasmid mixtures were used, the proportion of the values stayed the same. In all approaches, test person A showed higher DNA repair capacity values than test person B, but all experiments were highly reproducible.
3.2
Influence of calorie reduction on DNA repair capacity
Modified HCRA was used to research the influence of calorie reduction (F. X. Mayr therapy) on DNA repair capacity of freshly isolated human PBMCs. As Mayr therapy includes a defined nutrition with a lower food intake, it can be defined as calorie reduction. For every individual, three blood samples were required: one before the beginning of pretreatment, the second one on the eighth day of diet and the last one during convalescence. Eight test persons were included in the year 2014 study and in the year 2015 study, 18 individuals participated in the study. Summarized results of both studies are displayed in figure 2. There is an increase in DNA repair capacity from the first sampling before pretreatment to the last sampling during convalescence. To better point out the significant influence of this kind of calorie reduction on DNA repair capacity, the year 2014 study and the year 2015 study are analyzed separately, as shown in figure 3 and 4. In addition, two groups were formed for each study, depending on the preexisting DNA repair capacities of the participants: one group including the bottom 50 % and the other group including the top 50 % of the test persons. Results of the year 2014 study are 8
displayed in figure 3. Four test persons with low pre-existing DNA repair capacity showed a significant increase (p=0.0216) in DNA repair capacity from the beginning to the eighth day of therapy, indicating an influence of the treatment on DNA repair in these persons. This increase persisted until the end of the study (p=0.0013), elevating DNA repair capacity of test persons with low pre-existing DNA repair capacity to the level of DNA repair capacity of test persons with normal DNA repair capacity. DNA repair capacity of the remaining four test persons with normal pre-existing DNA repair capacity did not change significantly throughout the study. Before pretreatment, there was no difference in DNA repair capacity between female and male test persons detectable, but on the eighth day of fasting, male test persons displayed a significantly lower DNA repair capacity. However, this effect was not detectable anymore during convalescence (supplementary figure 1). Furthermore there was no significant influence of age on DNA repair capacity (supplementary figure 2). Within the scope of the year 2015 study, more individuals than in the year 2014 study were included. PBMCs of 18 individuals were analyzed with regard to changes in DNA nucleotide excision repair capacity during a period of calorie reduction. This repeat of the first study took place at the same time of the year, excluding seasonal influences on DNA repair. In test persons with low pre-existing DNA repair capacity, a significant increase (p=0.0193) in DNA repair capacity was measured on the eighth day of therapy compared to DNA repair capacity at the beginning of the study, as shown in figure 4 (a). At the end of the study, this increase was still significantly detectable (p=0.0125), again indicating a positive impact of calorie reduction on DNA repair and thus confirming the results of the year 2014 study. It can be stated that DNA repair capacity values of individuals with low pre-existing DNA repair capacity approached the DNA repair capacity values of the normal pre-existing DNA repair group. The remaining individuals with normal pre-existing DNA repair capacity showed no significant changes regarding DNA repair, as displayed in figure 4 (b). Neither gender nor age of the test persons showed an influence on DNA repair capacity (data not shown). Finally, results of the year 2015 study strongly supported the results of the year 2014 study.
4. Discussion For years it has been known that caloric restriction can influence oxidative stress, aging as well as age related diseases and lifespan in model organisms positively (Colman et al., 2014; Qiu et al., 2010; Smith et al., 2007). Licastro et al. showed that dietary restricted elderly mice 9
have repair ability for UV induced DNA damages comparable to that of younger mice on a minimally restricted diet (Licastro et al., 1988). Furthermore, a positive correlation of the ability of a species to repair UV-induced DNA damages and their expected maximum lifespan was described by Hall et al. and Hart et al. (Hall et al., 1984; Hart and Setlow, 1974). Few studies research calorie reduction exceeding hours or days of fasting involving humans (Fontana et al., 2006; Racette et al., 2006; Walford et al., 2002). One of these few studies deals for example with the occurrence of centenarians above the average on the Japanese island Okinawa and attributes this to the lower caloric intake of these people (Kagawa, 1978). None of the studies mentioned above researched DNA repair capacity in a functional assay like HCRA. However, we presented a further refined HCRA using human PBMCs to study calorie reduction as a systemic influence on DNA repair capacity. Interestingly, there are inter-individual differences in ex vivo DNA repair capacities of different test persons. This applies for the reproducibility studies as well as for the first DNA repair capacity value measured during Mayr therapy. These finding is in accordance with the results of others, who found inter-individual variations in DNA repair efficiency up to 20-fold (Bykov et al., 1999). In context of variations, it has to be remarked that three different mixtures of plasmid were used for the three reproducibility studies. This circumstance leads to the differences in DNA repair capacity over the three independently conducted reproducibility studies but underlines the reproducibility of the assay, as proportions of DNA repair capacity from person A to B stay the same. Furthermore, volume and concentration of the plasmid solution might play a role during UVC irradiation. In previous studies we found that the DNA concentration of pDsRed-Express plays a role regarding the percentage of DNA repair capacity. Using the same test person, UVC irradiation for the induction of DNA damages of a higher concentrated plasmid solution leads to higher repair capacity values than irradiation of lower concentrated plasmid solution (unpublished data). The more plasmids are in a solution, the more they can lie above each other and the less UVC does reach the plasmids below, thereby irradiation becomes less effective. Thus it is important to use one plasmid mix for the entire study, as we did for the year 2014 and 2015 study. Nevertheless, we proved this modified HCRA of being reliable, reproducible and therefore suitable for studying DNA repair of human PBMCs in an ex vivo approach. For surveying the influence of calorie reduction on DNA repair capacity, we conducted two studies involving eight and 18 participants respectively. In half of the volunteers we could show a significant increase of DNA repair, which lasted until the end of the study, approaching normal DNA repair capacity values of about 85 % to 90 %. In the second study we strongly supported that result. Either some individuals can’t be affected by 10
this kind of dietary intervention, or the assay itself leads to that result. In both of the studies as well as in reproducibility studies, DNA repair capacity never exceeded about 90 %. Taking that into account, persons displaying higher DNA repair capacity values might be at the upper limit of detection of this assay. A previous study correspondingly showed an influence of dietary intervention on nucleotide excision repair capacity in human lymphocytes using a modified comet assay (Langie et al., 2010). However, as far as we know, our results are the first ones indicating an influence of calorie reduction on DNA repair ability in humans, measured using a functional ex vivo assay. Mendez at al. used a modified HCRA to measure DNA repair in cryopreserved lymphocytes, though in an in vitro approach (Mendez et al., 2011). For some time, the correlation of fasting and the efficacy of chemotherapy is researched (Safdie et al., 2009), as cancer is known to be a frequently occurring age-related disease. Raffaghello et al. for instance found that short-term starvation previous to chemotherapy increased the effectiveness of chemotherapy on cancer cells in mice, oncogeneexpressing yeast and mammalian cancer cell lines, probably due to the low glucose level resulting from fasting and the dependency of cancer cells on glucose (Lee et al., 2012; Raffaghello et al., 2008). Besides that, patients undergoing fasting cycles reported reduced side effects of chemotherapy (Safdie et al., 2009). Accumulation of DNA mutations is acknowledged as the first step in cancer formation (Hanahan and Weinberg, 2000). Supporting DNA repair ability via calorie reduction may contribute to reverse this first step and thus contribute to a healthier aging. There are different mechanisms probably leading to the positive effect of CR on health and aging, like reducing inflammation, reducing IGF-1 blood level or increasing insulin sensitivity, for review see (Longo and Fontana, 2010) and (Ribarič, 2012). It is also known that CR reduces oxidative stress and that oxidative stress can modulate the expression of genes involved in NER, precisely, oxidative stress leads to a decrease of NER (Langie et al., 2007). These findings provide a possible mechanism explaining our results: in individuals with low DNA repair capacity in the beginning the increase of their DNA repair ability occurs due to a decrease of oxidative stress caused by CR. Our study is the basis for further research in the field of calorie reduction and its impact on DNA repair. More studies involving human test persons have to be conducted to further illuminate the underlying mechanisms leading to our results. Additionally, more biomarkers like measuring oxidative stress, mitochondrial function and gene expression of DNA repair associated genes have to be analyzed in the following studies. In conclusion, we could demonstrate a first prove of the influence of calorie reduction on DNA nucleotide excision repair measured using a functional ex vivo assay. Further studies need to be conducted for 11
researching whether other kinds of calorie reduction like fasting, dietary restriction or alternate day fasting have the same positive effect of DNA repair capacity as it seems for Mayr therapy.
Conflict of interest The authors declare no conflicts of interest.
Acknowledgements We thank Dr. med. Karin Rupprecht from the center for Traditional Chinese Medicine (TCM) in Sigmaringen for taking of the blood samples and providing information about Mayr therapy. The authors also thank Prof. Dr. med. Alexander Bürkle and Dr. Aswin Mangerich for their scientific advice.
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10.1155/2012/741468. Roguev, A., Russev, G., 2000. Two-wavelength fluorescence assay for DNA repair. Analytical biochemistry 287 (2), 313–318. 10.1006/abio.2000.4865. Roth, G.S., Ingram, D.K., Lane, M.A., 2001. Caloric restriction in primates and relevance to humans. Ann. N. Y. Acad. Sci 928, 305–315. 14
Safdie, F.M., Dorff, T., Quinn, D., Fontana, L., Wei, M., Lee, C., Cohen, P., Longo, V.D., 2009. Fasting and cancer treatment in humans: A case series report (12). Smith, D.L., McClure, J.M., Matecic, M., Smith, J.S., 2007. Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins. Aging cell 6 (5), 649–662. 10.1111/j.1474-9726.2007.00326.x. Stuart, J.A., Karahalil, B., Hogue, B.A., Souza-Pinto, N.C., Bohr, V.A., 2004. Mitochondrial and nuclear DNA base excision repair are affected differently by caloric restriction. FASEB J 18 (3), 595–597. 10.1096/fj.03-0890fje. Walford, R.L., Mock, D., Verdery, R., MacCallum, T., 2002. Calorie restriction in biosphere 2: alterations in physiologic, hematologic, hormonal, and biochemical parameters in humans restricted for a 2-year period. J. Gerontol. A Biol. Sci. Med. Sci 57 (6), B211-24. Weindruch, R., 2003. Caloric restriction: life span extension and retardation of brain aging. Clinical Neuroscience Research 2 (5-6), 279–284. 10.1016/S1566-2772(03)00004-5. Weraarchakul, N., Strong, R., Wood, W., Richardson, A., 1989. The effect of aging and dietary restriction on DNA repair (1). 10.1016/0014-4827(89)90193-6. Witasek, A., 1999. Diagnostik und Therapie nach Dr. F. X. Mayr. Forschende Komplementärmedizin 6 (1), 45–46.
Figure Captions Figure 1: Reproducibility of modified HCRA. A1 and B1 represent the first three day reproducibility study, A2 and B2 the second and A3 and B3 the third one. Bars represent percentages of DNA repair capacity with error bars displaying corresponding SEMs. All test persons showed a high reproducibility of DNA repair capacity measurements, but test person A always exhibited slightly higher values than B. Furthermore, DNA repair capacities of test person B varied slightly more throughout the three days than DNA repair capacities of test person A. Highest variability was detected in B2 (2.26%) whereas lowest variability was detected in A2 (0.5 %). Despite of the use of three independently prepared plasmid mixtures, the proportion of DNA repair capacity values stayed the same. In comparison to B, test person A always showed higher DNA repair capacities as well as slightly lower SEMs of the triplicates.
15
Figure 2: Influence of calorie reduction on DNA repair capacity of human PBMCs – summarized results of the year 2014 and the year 2015 study. There is a significant increase (p=0.0276) in DNA repair capacity from the beginning of the study to the last sampling during convalescence.
Figure 3: Influence of calorie reduction on DNA repair capacity of human PBMCs – study 2014. Eight test persons participated in this study. (a) The four persons with low pre-existing DNA repair capacity showed an increase in DNA repair capacity from the beginning of the study to the eighth day of therapy. In these individuals, DNA repair capacity was elevated significantly (p=0.0216) on the eighth day of therapy and stayed at about the same normal level during convalescence resulting in a significantly increased DNA repair capacity (p=0.0013) at the end of the study. (b) The remaining test persons with normal pre-existing DNA repair capacity showed no significant changes in DNA repair capacity throughout the study.
Figure 4: Influence of calorie reduction on DNA repair capacity of human PBMCs – year 2015 study. 18 individuals participated in the second study. (a) The nine test persons with low pre-existing DNA repair capacity showed a significant increase in DNA repair capacity from the first measurement (before pretreatment) to the second sampling on the eighth day of therapy (p=0.193), lasting until the end of the study (p=0.0125). (b) DNA repair capacity of the remaining nine individuals with normal pre-existing DNA repair capacity did not change significantly throughout the study.
16
Fig. 1
Fig. 2
17
Fig. 3
Fig. 4
18
Table 1: Three days reproducibility study of DNA repair capacity was conducted three times in triplicates. Results of the two test persons A and B are shown in percentages ± SEM. Reproducibility 1, 2 and 3 represent the three independently conducted approaches. Test person A showed slightly higher DNA repair capacities when using the three different plasmid mixtures as well as over the tested time points. There were no significant changes in DNA repair capacity throughout the three consecutive days, as displayed by the p-values. Test person A
Reproducibility Reproducibility Reproducibility 3 2 1
Repair Capacity [%]
Test person A p-values Day 1Day 1Day 2Day 2 Day 3 Day 3
Test person B Repair Capacity [%]
Day 1
89.64 ± 1.95
Day 2
89.18 ± 0.47
Day 3
90.08 ± 0.87
87.35 ± 0.16
Day 1
84.19 ± 0.67
83.38 ± 1.70
Day 2
84.26 ± 0.70
Day 3
84.69 ± 0.94
83.98 ± 0.67
Day 1
90.80 ± 0.48
89.79 ± 0.71
Day 2
90.42 ± 1.07
Day 3
89.47 ± 0.78
Test person B p-values Day 1Day 1Day 2Day 2 Day 3 Day 3
87.63 ± 1.59 0.8267
0.9537
0.7033
0.8487
0.6890
0.2213
0.4148
0.7648
0.3694
87.98 ± 0.75
81.72 ± 0.80
89.27 ± 0.44
0.6734
0.8678
0.2200
0.4697
0.8062
0.1916
0.5657
0.5778
0.7286
89.61 ± 2.88
19
S0047-6374(16)30013-6 http://dx.doi.org/doi:10.1016/j.mad.2016.02.008 MAD 10817
To appear in:
Mechanisms of Ageing and Development
Received date: Revised date: Accepted date:
28-7-2015 5-2-2016 10-2-2016
Please cite this article as: Matt, Katja, Burger, Katharina, Gebhard, Daniel, Bergemann, Jddotorg, Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.Mechanisms of Ageing and Development http://dx.doi.org/10.1016/j.mad.2016.02.008 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.
Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells
Katja Matta, Katharina Burgera, Daniel Gebharda, Jörg Bergemanna
a
Department of Life Sciences, Albstadt-Sigmaringen University of Applied Sciences,
Sigmaringen, Germany
Corresponding author: Katja Matt Albstadt-Sigmaringen University of Applied Sciences, Anton-Günther-Strasse 51, 72488 Sigmaringen, Germany. Tel.: +49 7571 732 8375 e-mail: [email protected]
1
Highlights Successful adaption of HCRA to an application using human PBMCs ex vivo. Inter-individual differences in DNA repair ability of test persons. Calorie reduction can influence DNA nucleotide excision repair capacity positively.
Abstract Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging.
Abbreviations: CR, calorie reduction; HCRA, host cell reactivation assay; NER, nucleotide excision repair; PBMC, peripheral blood mononuclear cell; EDTA, ethylene diamine tetraacetic acid; PBS, phosphate buffered saline; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; SEM, standard error of the mean; UV, ultraviolet
Keywords: DNA repair capacity; modified host cell reactivation assay; calorie reduction; F. X. Mayr therapy; human peripheral blood mononuclear cells
2
1. Introduction As a non-pharmacological intervention, calorie reduction (CR) is assumed to prolong lifespan and reduce age-related diseases in a variety of model organisms like rodents or invertebrates (Weindruch, 2003). As early as in 1935, McCay researched the effect of caloric restriction in rats, stating that caloric restriction not only retards the growth of rats, but also increases their lifespan (McCay et al., 1989). In non-human primates caloric restriction even reduces agerelated mortality as well as overall-mortality (Colman et al., 2014). Furthermore it has already been shown that caloric restriction reduces oxidative stress (Qiu et al., 2010), increases base excision repair in the nucleus (Stuart et al., 2004), has protective effects on age related diseases like atherosclerosis (Fontana et al., 2004) and protects many model organisms like non-human primates and rats from aging (Cabelof et al., 2003; Roth et al., 2001). It can be stated that caloric restriction and its effect on aging and age-related diseases is extensively researched in model organisms, but it is not so easy to find studies involving humans in these field of research, probably due to ethical reasons. If humans are involved in studies of DNA repair and aging, experiments are often conducted using cultured human cells. In 1974 for example, Hart and Setlow correlated an increasing ability to repair DNA damages via unscheduled DNA synthesis with increasing maximum lifespan of various species including humans (Hart and Setlow, 1974). A significant influence of dietary restriction on DNA repair measured via unscheduled DNA synthesis was first shown in 1989 in hepatocytes and kidney cells of rats (Weraarchakul et al., 1989). F. X. Mayr therapy represents a modified form of fasting, thus a particular way of calorie reduction for humans. Aims of this therapy are to recover the digestive tract as well as the training of proper mastication and promotion of health. For reliable analyses of systemic influences like calorie reduction, an appropriate method of measuring DNA repair ability directly out of the human body is beneficial. An ideal method would require minimally invasive sampling, so that test persons can easily be enlisted. The isolation of peripheral blood mononuclear cells (PBMCs) out of human venous blood is relatively fast and simple and provides cells needed for research of DNA repair ex vivo. Using human PBMCs, Langie et al. studied the effect of dietary intervention on nucleotide excision repair capacity using a modified comet assay (Langie, S A S et al., 2010). However, this method displays the DNA damage recognition and incision ability of the individual (Langie et al., 2006), but not the functional restoration of damaged DNA. Modified host cell reactivation assay (HCRA) is a suitable method for analyzing nucleotide excision repair capacity. The advantage of this assay is that a functional restoration of a previously damaged reporter plasmid can be measured. The principle of restoration of a reporter gene 3
was first described by Protić-Sabljić et al. in 1985 (Protić-Sabljić et al., 1985) and then modified by Athas et al. for an application using human lymphocytes (Athas et al., 1991). Host cell reactivation assay was further refined in 2000 by Roguev and Russev using green and yellow fluorescent proteins as reporters (Roguev and Russev, 2000) and in 2007 by Burger et al. by adapting HCRA to flow cytometry analysis of primary human skin cells (Burger et al., 2007; Burger et al., 2010). HCRA, as we conduct it nowadays, is based on the deactivation of a reporter gene, which is inserted into a plasmid, by DNA damages introduced via UVC irradiation. After the introduction of the damaged plasmid into the cell, the ability of the cell to repair the damages leads to the functional reactivation of the reporter gene. Since UVC radiation damages DNA directly, thus leading mainly to cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs) and these DNA damages are repaired via nucleotide excision repair (NER), HCRA displays the cells’ NER capacity. Here we present the successful adaption of this modified HCRA for an application with freshly isolated human PBMCs. Using this reliable and highly reproducible method, we studied the influence of a particular way of calorie reduction, namely F. X. Mayr therapy, in an ex vivo approach. Results of this ex vivo study show a statistically significant increase of DNA repair capacity in individuals with lower DNA repair ability before Mayr therapy, lasting until the end of the study. The repeat of the study even reproduced this finding, clearly indicating an influence of CR on DNA nucleotide excision repair of human PBMCs.
2. Materials and Methods 2.1
F. X. Mayr therapy and samples
Test persons participating in F. X. Mayr therapy were recruited at the center for Traditional Chinese Medicine (TCM) in Sigmaringen. F. X. Mayr therapy aims at cleaning, rest and regeneration of the gastrointestinal tract, the learning of a healthy eating behavior (stop eating when the first feeling of satiety occurs) and a detoxification of the human body (Witasek, 1999). Procedure of a six-week Mayr therapy is as follows: one week pretreatment (eating no indigestive food), followed by 3 weeks of diet food (stale bread rolls and milk) and two weeks of convalescence (where the body is adjusted to regular food again). Through this monotonic diet the intestinal tract is rested. For each person, the diet is individually assorted. Crucial for a successful Mayr therapy is, that during fasting with stale bread rolls and milk, the persons 4
have to stop eating when the first feeling of satiety occurs resulting in a calorie reduction. The average loss of weight during a six week F. X. Mayr therapy is about five kilograms, ranging from three to eight kilograms. The study was approved by the ethics committee of the Landesaerztekammer Baden-Wuerttemberg and all experiments were in accordance with the declaration of Helsinki. The volunteers were informed before collection of the samples and gave their written consent. All data concerning the individuals under study have been pseudonymized. The two test persons for reproducibility studies were recruited within our department and were available for all three repetitions. In brief, 12 mL venous blood was collected in tubes containing EDTA as anticoagulant and stored at 4 °C in the dark until isolation of the PBMCs. Isolation was conducted not exceeding 4 h after taking of the blood samples. For reproducibility studies, three blood samples were taken on three consecutive days at the same time of day. This study was repeated three times over a period of ten months. For the study of calorie reduction and its influence on DNA repair, three samples were collected from each test person: the first one before the beginning of F. X. Mayr therapy, the second one on the eighth day of diet food intake and the third one during convalescence.
2.2
Isolation of PBMCs
Human PBMCs were isolated from 12 mL of human blood using Leucosep tubes (Greiner Bio-One GmbH, Frickenhausen, Germany) according to the manufacturer’s instructions. PBMCs were resuspended in 5 mL phosphate buffered saline (PBS) and cell count was determined using Neubauer-improved counting chamber.
2.3
Host cell reactivation assay
2.3.1 Plasmid preparation Two plasmids were used for the determination of DNA repair capacity of freshly isolated human PBMCs: pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA, USA) and a modified DsRed-Express, where the GFP cassette of pEGFP-N1 was replaced with the DsRed cassette of pDsRed-Express. Both plasmids were augmented in E. coli K12 and purified using Plasmid Plus Giga Kit and dissolved in EB Buffer (both Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. UVC irradiation (254 nm) of DsRed-Express 5
was carried out using a Stratalinker® UV Crosslinker (Model 1800, Stratagene, La Jolla, CA, USA). Drops of 40 µL of plasmid solution were irradiated with 5 kJ/m2 of UVC. Plasmid concentrations did not exceed 900 ng/µL. After irradiation, 10 µg of pEGFP and 30 µg of DsRed (either irradiated or non-irradiated) were mixed, aliquoted and stored at – 80 °C until further use. One plasmid mixture was prepared this way for the year 2014 study and the year 2015 study to ensure the use of one plasmid mixture for the entire study of calorie reduction. For the three reproducibility studies, three independent preparations of the plasmid mixture were used. More precisely, for each of the three repeats, DsRed was irradiated and mixed with GFP separately, resulting in three independent preparations of the plasmid mixture. For day 1, day 2 and day 3, aliquots of one plasmid mixture were used.
2.3.2 Transfection Human PBMCs were transfected by electroporation using 0.4 cm electroporation cuvettes (Molecular BioProducts, Inc., purchased via Thermo Fisher Scientific GmbH, Dreieich, Germany). Therefore 2 x 106 PBMCs were mixed with 150 µL RPMI 1640 without Phenol Red (Gibco® by Life Technologies, purchased via Fisher Scientific GmbH, Schwerte, Germany) and 100 µL of EB Buffer (Qiagen GmbH, Hilden, Germany) containing 10 µg pEGFP and 30 µg DsRed-Express. 250 µL of this solution was electroporated (400 kV, 500 µF) using an electroporator (Gene Pulser II, Bio-Rad Laboratories, Inc., Hercules, CA, USA) and immediately seeded into 6 well plates (Sarstedt AG & Co., Nuembrecht, Germany) containing 3 mL of PBMC media (RPMI 1640 without phenol red and 20 % FBS, both Gibco® by Life Technologies). For the determination of DNA repair capacity, cells were transfected with a plasmid mixture containing irradiated DsRed and non-irradiated GFP, for transfection control, cells were transfected with the mixture of non-irradiated DsRed and GFP. All measurements - reproducibility studies as well as the studies of calorie reduction were carried out in triplicates.
2.3.3 FACS analyses Analysis of transfected cells was carried out using FACSCalibur and CellQuest Pro software (both Becton, Dickinson and Company, Franklin Lakes, NJ, USA) 24 h after transfection. DNA repair capacity was calculated as follows: 6
𝑅𝑒𝑝𝑎𝑖𝑟 𝐶𝑎𝑝𝑎𝑐𝑖𝑡𝑦 [%] =
𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑟𝑒𝑑 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑡 𝑐𝑒𝑙𝑙𝑠 𝑖𝑟𝑟𝑎𝑑𝑖𝑎𝑡𝑒𝑑 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑔𝑟𝑒𝑒𝑛 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑡 𝑐𝑒𝑙𝑙𝑠
∗ 𝐹 ∗ 100
The factor F is defined as the ratio of percentages of green fluorescent cells to non-irradiated red fluorescent cells. It is used to correct DNA repair capacity with regard to efficacy of transfection as described by Burger et al. (Burger et al., 2007).
2.4
Data analyses
For statistical analyses as well as for graph generation GraphPad Prism 5 for Windows (Version 5.04) was used. Regarding their DNA repair capacity, test persons participating in F. X. Mayr therapy were divided into the upper 50 % of the group and the lower 50 % of the group to better point out the influence of calorie reduction on DNA repair capacity. This procedure was applied for the year 2014 study as well as for the year 2015 study. The upper 50 % of test persons were defined as the group “normal pre-existing DNA repair capacity” as in our experience, most individuals display DNA repair capacity values of about 85 % to 90 %. The lower 50 % of test persons were defined as “low pre-existing DNA repair capacity”. Statistical analyses were carried out as paired t-Tests (p* < 0.05), as well as linear regression analyses (p* < 0.05) for age dependence of DNA repair capacity as shown in supplementary figure 2.
3. Results 3.1
Reproducibility of HCRA
For testing of reproducibility, DNA repair capacity was determined using PBMCs of two test persons A and B. On three consecutive days blood samples were taken at the same time of day and HCRA was conducted. This approach was repeated independently three times throughout ten months, so every time the blood samples of the same two individuals were used. DNA repair capacities of the two persons tested are shown in table 1 as well as in figure 1. It has to be remarked that three independent preparations of the plasmid mixture were used
7
for the reproducibility studies. On day 1, 2 and 3 of each repeat aliquots of the same plasmid mix preparation were used. Interestingly, test person A showed slightly higher DNA repair capacities than B when using the different plasmid mixtures as well as over the tested time points. Furthermore, SEMs of the triplicates from person A were a little bit lower on average. For test person A, variabilities were 0.96 % for reproducibility 1, 0.5 % for reproducibility 2 and 1.33 % for reproducibility 3, whereas test person B showed variabilities of 0.63 % (reproducibility 1), 2.26 % (reproducibility 2) and 0.52 % (reproducibility 3) respectively. Nonetheless, variabilities of DNA repair capacity values over the three consecutive days never exceeded 2.26 %, as shown in sample B2 and the lowest variability was displayed in A2 with a value of 0.5 %. Neither in test person A nor in test person B significant changes in DNA repair capacity were detectable over the three consecutive days, as demonstrated by p-values in table 1. Figure 1 displays the results of all reproducibility studies. Both test persons showed a high reproducibility over the three days. Although independent preparations of the plasmid mixtures were used, the proportion of the values stayed the same. In all approaches, test person A showed higher DNA repair capacity values than test person B, but all experiments were highly reproducible.
3.2
Influence of calorie reduction on DNA repair capacity
Modified HCRA was used to research the influence of calorie reduction (F. X. Mayr therapy) on DNA repair capacity of freshly isolated human PBMCs. As Mayr therapy includes a defined nutrition with a lower food intake, it can be defined as calorie reduction. For every individual, three blood samples were required: one before the beginning of pretreatment, the second one on the eighth day of diet and the last one during convalescence. Eight test persons were included in the year 2014 study and in the year 2015 study, 18 individuals participated in the study. Summarized results of both studies are displayed in figure 2. There is an increase in DNA repair capacity from the first sampling before pretreatment to the last sampling during convalescence. To better point out the significant influence of this kind of calorie reduction on DNA repair capacity, the year 2014 study and the year 2015 study are analyzed separately, as shown in figure 3 and 4. In addition, two groups were formed for each study, depending on the preexisting DNA repair capacities of the participants: one group including the bottom 50 % and the other group including the top 50 % of the test persons. Results of the year 2014 study are 8
displayed in figure 3. Four test persons with low pre-existing DNA repair capacity showed a significant increase (p=0.0216) in DNA repair capacity from the beginning to the eighth day of therapy, indicating an influence of the treatment on DNA repair in these persons. This increase persisted until the end of the study (p=0.0013), elevating DNA repair capacity of test persons with low pre-existing DNA repair capacity to the level of DNA repair capacity of test persons with normal DNA repair capacity. DNA repair capacity of the remaining four test persons with normal pre-existing DNA repair capacity did not change significantly throughout the study. Before pretreatment, there was no difference in DNA repair capacity between female and male test persons detectable, but on the eighth day of fasting, male test persons displayed a significantly lower DNA repair capacity. However, this effect was not detectable anymore during convalescence (supplementary figure 1). Furthermore there was no significant influence of age on DNA repair capacity (supplementary figure 2). Within the scope of the year 2015 study, more individuals than in the year 2014 study were included. PBMCs of 18 individuals were analyzed with regard to changes in DNA nucleotide excision repair capacity during a period of calorie reduction. This repeat of the first study took place at the same time of the year, excluding seasonal influences on DNA repair. In test persons with low pre-existing DNA repair capacity, a significant increase (p=0.0193) in DNA repair capacity was measured on the eighth day of therapy compared to DNA repair capacity at the beginning of the study, as shown in figure 4 (a). At the end of the study, this increase was still significantly detectable (p=0.0125), again indicating a positive impact of calorie reduction on DNA repair and thus confirming the results of the year 2014 study. It can be stated that DNA repair capacity values of individuals with low pre-existing DNA repair capacity approached the DNA repair capacity values of the normal pre-existing DNA repair group. The remaining individuals with normal pre-existing DNA repair capacity showed no significant changes regarding DNA repair, as displayed in figure 4 (b). Neither gender nor age of the test persons showed an influence on DNA repair capacity (data not shown). Finally, results of the year 2015 study strongly supported the results of the year 2014 study.
4. Discussion For years it has been known that caloric restriction can influence oxidative stress, aging as well as age related diseases and lifespan in model organisms positively (Colman et al., 2014; Qiu et al., 2010; Smith et al., 2007). Licastro et al. showed that dietary restricted elderly mice 9
have repair ability for UV induced DNA damages comparable to that of younger mice on a minimally restricted diet (Licastro et al., 1988). Furthermore, a positive correlation of the ability of a species to repair UV-induced DNA damages and their expected maximum lifespan was described by Hall et al. and Hart et al. (Hall et al., 1984; Hart and Setlow, 1974). Few studies research calorie reduction exceeding hours or days of fasting involving humans (Fontana et al., 2006; Racette et al., 2006; Walford et al., 2002). One of these few studies deals for example with the occurrence of centenarians above the average on the Japanese island Okinawa and attributes this to the lower caloric intake of these people (Kagawa, 1978). None of the studies mentioned above researched DNA repair capacity in a functional assay like HCRA. However, we presented a further refined HCRA using human PBMCs to study calorie reduction as a systemic influence on DNA repair capacity. Interestingly, there are inter-individual differences in ex vivo DNA repair capacities of different test persons. This applies for the reproducibility studies as well as for the first DNA repair capacity value measured during Mayr therapy. These finding is in accordance with the results of others, who found inter-individual variations in DNA repair efficiency up to 20-fold (Bykov et al., 1999). In context of variations, it has to be remarked that three different mixtures of plasmid were used for the three reproducibility studies. This circumstance leads to the differences in DNA repair capacity over the three independently conducted reproducibility studies but underlines the reproducibility of the assay, as proportions of DNA repair capacity from person A to B stay the same. Furthermore, volume and concentration of the plasmid solution might play a role during UVC irradiation. In previous studies we found that the DNA concentration of pDsRed-Express plays a role regarding the percentage of DNA repair capacity. Using the same test person, UVC irradiation for the induction of DNA damages of a higher concentrated plasmid solution leads to higher repair capacity values than irradiation of lower concentrated plasmid solution (unpublished data). The more plasmids are in a solution, the more they can lie above each other and the less UVC does reach the plasmids below, thereby irradiation becomes less effective. Thus it is important to use one plasmid mix for the entire study, as we did for the year 2014 and 2015 study. Nevertheless, we proved this modified HCRA of being reliable, reproducible and therefore suitable for studying DNA repair of human PBMCs in an ex vivo approach. For surveying the influence of calorie reduction on DNA repair capacity, we conducted two studies involving eight and 18 participants respectively. In half of the volunteers we could show a significant increase of DNA repair, which lasted until the end of the study, approaching normal DNA repair capacity values of about 85 % to 90 %. In the second study we strongly supported that result. Either some individuals can’t be affected by 10
this kind of dietary intervention, or the assay itself leads to that result. In both of the studies as well as in reproducibility studies, DNA repair capacity never exceeded about 90 %. Taking that into account, persons displaying higher DNA repair capacity values might be at the upper limit of detection of this assay. A previous study correspondingly showed an influence of dietary intervention on nucleotide excision repair capacity in human lymphocytes using a modified comet assay (Langie et al., 2010). However, as far as we know, our results are the first ones indicating an influence of calorie reduction on DNA repair ability in humans, measured using a functional ex vivo assay. Mendez at al. used a modified HCRA to measure DNA repair in cryopreserved lymphocytes, though in an in vitro approach (Mendez et al., 2011). For some time, the correlation of fasting and the efficacy of chemotherapy is researched (Safdie et al., 2009), as cancer is known to be a frequently occurring age-related disease. Raffaghello et al. for instance found that short-term starvation previous to chemotherapy increased the effectiveness of chemotherapy on cancer cells in mice, oncogeneexpressing yeast and mammalian cancer cell lines, probably due to the low glucose level resulting from fasting and the dependency of cancer cells on glucose (Lee et al., 2012; Raffaghello et al., 2008). Besides that, patients undergoing fasting cycles reported reduced side effects of chemotherapy (Safdie et al., 2009). Accumulation of DNA mutations is acknowledged as the first step in cancer formation (Hanahan and Weinberg, 2000). Supporting DNA repair ability via calorie reduction may contribute to reverse this first step and thus contribute to a healthier aging. There are different mechanisms probably leading to the positive effect of CR on health and aging, like reducing inflammation, reducing IGF-1 blood level or increasing insulin sensitivity, for review see (Longo and Fontana, 2010) and (Ribarič, 2012). It is also known that CR reduces oxidative stress and that oxidative stress can modulate the expression of genes involved in NER, precisely, oxidative stress leads to a decrease of NER (Langie et al., 2007). These findings provide a possible mechanism explaining our results: in individuals with low DNA repair capacity in the beginning the increase of their DNA repair ability occurs due to a decrease of oxidative stress caused by CR. Our study is the basis for further research in the field of calorie reduction and its impact on DNA repair. More studies involving human test persons have to be conducted to further illuminate the underlying mechanisms leading to our results. Additionally, more biomarkers like measuring oxidative stress, mitochondrial function and gene expression of DNA repair associated genes have to be analyzed in the following studies. In conclusion, we could demonstrate a first prove of the influence of calorie reduction on DNA nucleotide excision repair measured using a functional ex vivo assay. Further studies need to be conducted for 11
researching whether other kinds of calorie reduction like fasting, dietary restriction or alternate day fasting have the same positive effect of DNA repair capacity as it seems for Mayr therapy.
Conflict of interest The authors declare no conflicts of interest.
Acknowledgements We thank Dr. med. Karin Rupprecht from the center for Traditional Chinese Medicine (TCM) in Sigmaringen for taking of the blood samples and providing information about Mayr therapy. The authors also thank Prof. Dr. med. Alexander Bürkle and Dr. Aswin Mangerich for their scientific advice.
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Figure Captions Figure 1: Reproducibility of modified HCRA. A1 and B1 represent the first three day reproducibility study, A2 and B2 the second and A3 and B3 the third one. Bars represent percentages of DNA repair capacity with error bars displaying corresponding SEMs. All test persons showed a high reproducibility of DNA repair capacity measurements, but test person A always exhibited slightly higher values than B. Furthermore, DNA repair capacities of test person B varied slightly more throughout the three days than DNA repair capacities of test person A. Highest variability was detected in B2 (2.26%) whereas lowest variability was detected in A2 (0.5 %). Despite of the use of three independently prepared plasmid mixtures, the proportion of DNA repair capacity values stayed the same. In comparison to B, test person A always showed higher DNA repair capacities as well as slightly lower SEMs of the triplicates.
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Figure 2: Influence of calorie reduction on DNA repair capacity of human PBMCs – summarized results of the year 2014 and the year 2015 study. There is a significant increase (p=0.0276) in DNA repair capacity from the beginning of the study to the last sampling during convalescence.
Figure 3: Influence of calorie reduction on DNA repair capacity of human PBMCs – study 2014. Eight test persons participated in this study. (a) The four persons with low pre-existing DNA repair capacity showed an increase in DNA repair capacity from the beginning of the study to the eighth day of therapy. In these individuals, DNA repair capacity was elevated significantly (p=0.0216) on the eighth day of therapy and stayed at about the same normal level during convalescence resulting in a significantly increased DNA repair capacity (p=0.0013) at the end of the study. (b) The remaining test persons with normal pre-existing DNA repair capacity showed no significant changes in DNA repair capacity throughout the study.
Figure 4: Influence of calorie reduction on DNA repair capacity of human PBMCs – year 2015 study. 18 individuals participated in the second study. (a) The nine test persons with low pre-existing DNA repair capacity showed a significant increase in DNA repair capacity from the first measurement (before pretreatment) to the second sampling on the eighth day of therapy (p=0.193), lasting until the end of the study (p=0.0125). (b) DNA repair capacity of the remaining nine individuals with normal pre-existing DNA repair capacity did not change significantly throughout the study.
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Fig. 1
Fig. 2
17
Fig. 3
Fig. 4
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Table 1: Three days reproducibility study of DNA repair capacity was conducted three times in triplicates. Results of the two test persons A and B are shown in percentages ± SEM. Reproducibility 1, 2 and 3 represent the three independently conducted approaches. Test person A showed slightly higher DNA repair capacities when using the three different plasmid mixtures as well as over the tested time points. There were no significant changes in DNA repair capacity throughout the three consecutive days, as displayed by the p-values. Test person A
Reproducibility Reproducibility Reproducibility 3 2 1
Repair Capacity [%]
Test person A p-values Day 1Day 1Day 2Day 2 Day 3 Day 3
Test person B Repair Capacity [%]
Day 1
89.64 ± 1.95
Day 2
89.18 ± 0.47
Day 3
90.08 ± 0.87
87.35 ± 0.16
Day 1
84.19 ± 0.67
83.38 ± 1.70
Day 2
84.26 ± 0.70
Day 3
84.69 ± 0.94
83.98 ± 0.67
Day 1
90.80 ± 0.48
89.79 ± 0.71
Day 2
90.42 ± 1.07
Day 3
89.47 ± 0.78
Test person B p-values Day 1Day 1Day 2Day 2 Day 3 Day 3
87.63 ± 1.59 0.8267
0.9537
0.7033
0.8487
0.6890
0.2213
0.4148
0.7648
0.3694
87.98 ± 0.75
81.72 ± 0.80
89.27 ± 0.44
0.6734
0.8678
0.2200
0.4697
0.8062
0.1916
0.5657
0.5778
0.7286
89.61 ± 2.88
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