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Oxidative damage in mitochondrial fatty acids oxidation disorders patients and the in vitro effect of l -carnitine on DNA damage induced by the accumulated metabolites
Journal Pre-proof Oxidative damage in mitochondrial fatty acids oxidation disorders patients and the in vitro effect of l-carnitine on DNA damage induced by the accumulated metabolites Maira Silmara de Moraes, Gilian Guerreiro, Angela Sitta, Daniella de Moura Coelho, Vanusa Manfredini, Moacir Wajner, Carmen Regla Vargas PII:
S0003-9861(19)30375-3
DOI:
https://doi.org/10.1016/j.abb.2019.108206
Reference:
YABBI 108206
To appear in:
Archives of Biochemistry and Biophysics
Received Date: 17 May 2019 Revised Date:
19 November 2019
Accepted Date: 20 November 2019
Please cite this article as: M. Silmara de Moraes, G. Guerreiro, A. Sitta, D. de Moura Coelho, V. Manfredini, M. Wajner, C.R. Vargas, Oxidative damage in mitochondrial fatty acids oxidation disorders patients and the in vitro effect of l-carnitine on DNA damage induced by the accumulated metabolites, Archives of Biochemistry and Biophysics (2019), doi: https://doi.org/10.1016/j.abb.2019.108206. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Inc.
Authors statement
Maira Silmara de Moraes: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing. Gilian Guerreiro: Investigation, Validation, Writing, Methodology. Angela Sitta: Methodology, Investigation, Writing. Daniella de Moura Coelho: Methodology, Validation, Investigation. Vanusa Manfredini: Methodology, Validation. Moacir Wajner: Formal analysis, Writing, Visualization. Carmen Regla Vargas: Conceptualization, Validation, Writing, Supervision, Project administration, Funding acquisition.
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Title: Oxidative damage in mitochondrial fatty acids oxidation disorders patients and the in vitro effect of L-carnitine on DNA damage induced by the accumulated metabolites
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Authors: Maira Silmara de Moraes , Gilian Guerreiro , Angela Sitta , Daniella de Moura Coelho , Vanusa Manfredini , Moacir Wajner , Carmen Regla Vargas 1,2*
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1,2,3,4*
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Programa de Pós‐Graduação em Ciências Biológicas: Bioquímica, UFRGS, Porto Alegre, RS, Brazil
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Serviço de Genética Médica, HCPA,UFRGS, Porto Alegre, RS, Brazil
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Faculdade de Farmácia, UFRGS, Porto Alegre, RS, Brazil
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Programa de Pós‐Graduação em Ciências Farmacêuticas, UFRGS, Porto Alegre, RS, Brazil Programa de Pós‐Graduação em Bioquímica, Universidade Federal do Pampa, Uruguaiana, RS, Brazil
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Authors e-mails: [email protected]; [email protected]; [email protected], [email protected]; [email protected]; [email protected]; [email protected].
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*Corresponding authors at: Serviço de Genética Médica, HCPA, Rua Ramiro Barcelos, 2350, ZIP CODE 90.035-003, Porto Alegre, RS, Brazil.
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Telephone: +55 51 33598011
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Telefax: +55 51 33598010
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E-mail adresses: [email protected] (C.R. Vargas), [email protected] (M.S. Moraes)
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Abstract
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Background: The mitochondrial fatty acids oxidation disorders (FAOD) are inherited metabolic disorders (IMD) characterized by the accumulation of fatty acids of different sizes of chain according to the affected enzyme. Methods: This study evaluated the lipid peroxidation by the measurement of 8isoprostanes, nitrosative stress parameters by the measurement of nitrite and nitrate content and DNA and RNA oxidative damage by the measurement of oxidized guanine species in urine samples from long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and multiple acyl-CoA dehydrogenase deficiency (MADD) patients. Also, we analysed the in vitro DNA damage by comet assay induced by adipic acid, suberic acid, hexanoylglycine and suberylglycine, separated and in combination, as well as the effect of L-carnitine in human leukocytes. Results: An increase on 8-isoprostanes levels in all groups of patients was observed. The nitrite and nitrate levels were increased in LCHADD patients. DNA and RNA damage evaluation revealed increase on oxidized guanine species levels in LCHADD and MADD patients. The in vitro evaluation revealed an increase on the DNA damage induced by all metabolites, besides a potencialyzed effect. L-carnitine decreased the DNA damage induced by the metabolites. Conclusion: These results demonstrate that toxic metabolites accumulated could be related to the increased oxidative and nitrosative stress of FAOD patients and that the metabolites, separated and in combination, cause DNA damage, which was reduced by L-carnitine, demonstrating antioxidant protection. General Significance: This work demonstrated oxidative stress in FAOD patients and the genotoxic potencial of MCADD metabolites and the protective effect of Lcarnitine.
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Keywords: MCADD; LCHADD; MADD; oxidative stress; DNA damage; L-carnitine.
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Abbreviations: DI (damage index); FAOD (fatty acid oxidation disorders); IMD (inherited metabolic disorders); L-car (L-carnitine); LCHADD (long-chain 3hydroxyacyl-CoA dehydrogenase deficiency); MADD (multiple acyl-CoA dehydrogenase deficiency); MCADD (medium-chain acyl-CoA dehydrogenase deficiency); NO (nitric oxide); ); OGS (oxidized guanine species); RNS (reactive nitrogen species); ROS (reactive oxygen species). 2
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1. Introduction
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The fatty acid oxidation disorders (FAOD) are inherited metabolic disorders (IMD) caused by a deficient activity of specific enzymes of the beta-oxidation pathway that are involved in the mitochondrial catabolism of these fatty acids. Mitocondrial beta-oxidation consists of four steps, catalyzed by FAD-dependent acyl-CoA dehydrogenases, 2-enoyl-CoA hydratases, NAD-dependent L-3hydroxyacyl-CoA dehydrogenases and 3-ketoacyl-CoA thiolases. During each cycle the acyl-CoA ester that entered the cycle is shortened by two carbon atoms, which are released as acetyl-CoA. FAO disorders lead to the accumulation of fatty acids according to the damaged enzyme and compromise the use of this source of energy [1]. Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an autosomal recessive disorder leading to the accumulation of medium-chain fatty acids like suberic, adipic, sebacic, octanoic, 5-hydroxyhexanoic acids, as well as phenylpropionilglycine, hexanoylglycine, suberylglycine and their carnitine derivatives. The worldwide prevalence of this disease is 1: 14,600, being the most common disorder of FAOD, and clinical presentation of this disorder usually occurs 3 to 24 months after birth. In addition, in certain cases, adult and neonatal clinical forms may be found. The latter usually accompanied by cases of sudden death [2]. MCADD is caused by mutations in the ACADM gene, encoding the MCAD protein [3].
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Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is an autosomal recessive disorder caused by mutations in the HADHA gene, which encodes the α-subunit of the mitochondrial trifunctional protein, leading to the accumulation of long-chain fatty acids with a hydroxyl group like 3hydroxytetradecanoic, 3-hydroxyhexadecanoic, 3-hydroxyoctadecanoic, 3hydroxysebacic, 3-hydroxydehydrosebacic, 3-hydroxydodecanedioic acids and their carnitine derivatives in patients tissues. The estimated worldwide prevalence of this disease is 1:250,000. Patients affected by LCHADD present a poor prognosis and most of them exhibit a severe phenotype in neonatal period [2,4].
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Multiple acyl-CoA dehydrogenase deficiency (MADD), also known as Glutaric Aciduria type 2 (GA-II), is another autosomal recessive disorder that results from the deficiency of electron transfer flavoprotein (ETF alfa and beta) or electron transfer flavoprotein/ubiquinone oxidoreductase (ETF/QO) that transport the electrons from beta-oxidation to the respiratory chain. Several metabolites are accumulated in MADD patients including dicarboxylic acids, as for example, glutaric, ethylmalonic, 2hydroxyglutaric, adipic, suberic, sebacic acids, as well as glycine conjugates and their carnitine derivatives. The worldwide prevalence for this disease is 1:200,000. Clinical presentation includes three phenotypes: a) neonatal with congenital anomalies; b) neonatal without congenital anomalies (severe forms) whose manifestation generally occurs within the first 48 hours of life and might be fatal; c) mild and late clinical form that usually appear in the first months until adult life with broad clinical spectrum and better prognosis [2,5].
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All these three inherited disorders above may share common clinical and biochemical features like hypoketotic hypoglycemia, metabolic acidosis, hypotony, hyperammonemia and in severe cases skeletal myopathy, cardiomyopathy, hepatopathy and neuropathy. The frequency and appearance will depend on the clinical conditions and disease (MCAAD, LCHADD or MADD) presented by each patient [2]. Diagnosis of these diseases is based on the identification of urinary 3
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excretion of the fatty acids and their carnitine derivatives in blood. The confirmation can be performed by molecular analysis. Currently, the therapeutic strategy is to avoid fasting and to consume high amounts of carbohydrate and low amounts of fat and protein [2,6]. Also, in some cases, a supplementation of L-carnitine (L-car) is indicated, specially in patients with carnitine deficiency. Conjugation of carnitine with fatty acids contributes to elimination of toxic metabolites. Doses of L-car vary from 50 to 100 mg/kg/day, according to the patient’s age and its metabolic status [7]. Besides the important function on the transport of long-chain fatty acids across the inner mitochondrial membrane for beta-oxidation, L-car also has antioxidant properties, due to the capacity to scavenge hydrogen peroxide and superoxide radical and to chelate transition metal ions with protective effects against oxidative injury in others IMD like phenylketonuria, glutaric aciduria type I and maple syrup urine disease [8, 9, 10, 11, 12].
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Mitochondria are the most important organelles for cell homeostasis through several processes like energy production by the oxidative phosphorylation that generates ATP and reactive oxygen species (ROS). Biochemical and morphological alterations in tissues of FAOD patients suggest that the mitochondrial dysfunction contributes to the pathogenesis of the diseases. The accumulation of toxic metabolites can be harmful to the cell and may result in mitochondrial stress and collapse of internal membrane potential [13, 14]. Although ROS have an essential role on cell activities, an increase on its concentration can cause oxidative damage to biomolecules like lipids, proteins, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), compromising cell functions like membrane permeability, transport, signalling, enzymatic reactions, mutation repair and gene expression, leading to the cell death [15].
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Although many studies have demonstrated that the pathogenesis of FAOD is related to its toxic metabolites, there is not enough knowledge about the pathophysiology of the diseases and their mechanisms are still not completely known [16, 17, 18, 19]. In many others IMD it was demonstrated altered oxidative and nitrative indicators, with increased levels of ROS and reactive nitrogen species (RNS) [10, 11, 12].
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This study aimed to investigate oxidative and nitrosative stress parameters in patients affected by LCHADD, MCADD and MADD and also to evaluate the in vitro effect of L-carnitine on DNA damage induced by the main metabolites accumulated in MCADD: adipic acid (ADI), suberic acid (SUB), hexanoylglycine (HG) and suberylglycine (SG).
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2. Materials and Methods
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2.1 In vivo experiments
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2.1.1 Patients and biological samples
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We analyzed urine samples from 20 patients (from the Medical Genetic Service of Hospital de Clínicas de Porto Alegre) at diagnosis of FAOD, collected during a decompensation period. Mean age of patients was 6.6 ± 3.9 years and included LCHADD (n=9), MCADD (n=3) and MADD (n=8). 4
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The diagnosis were performed by the analysis of urinary organic acids by gas chromatography coupled to mass spectrometry as well as by detection of blood acylcarnitines accumulation by liquid chromatography coupled to mass spectrometry. For the control group (n=5), we utilized urine samples of age matched healthy people. All urine samples were stored in a freezer at -80ºC and quickly unfreezed for the analysis. The Ethics Committee of HCPA approved this project (2018-0549) and all patients or their parents gave informed consent about this study.
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2.1.2 8-Isoprostane determination 8-Isoprostane is a biomarker of lipid peroxidation in urine and was measured by the 8-isoprostane AChE Competitive ELISA commercial kit (Item No.516351; Cayman Chemical). This assay is based on the competition between 8-isoprostane and an 8-isoprostane-acetylcholinesterase (AChE) conjugate (Tracer) for a limited number of 8-isoprostanes-specific rabbit antiserum binding sites. The amount of bound Tracer is quantified at 412 nm, after addition of the Ellman’s reagent, a substrate of AChE. The intensity of the color is proportional to the amount of tracer bound to the well and is inversely proportional to the amount of free 8-isoprostane present in the urine. Results were expressed as ng/mg of creatinine.
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2.1.3 Reactive Nitrogen Species
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Total levels of nitrate + nitrite (NO3- + NO2-) in urine were determined using the Nitrate/Nitrite Colorimetric Assay Kit (LDH Method) from Cayman Chemical® (Item No780001; Cayman Chemical Company, Ann Arbor, MI, USA). This method is based on the reduction of NO3- to NO2- using nitrate reductase. By adding sulfanilamide to NO2-, a cationic intermediate is formed, which reacts with N-(1-naphtyl) ethylenediamine, resulting in an azo product whose absorption can be measured at 540 nm. Results were reported as µmol/mg creatinine.
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2.1.4 Oxidized Guanine Species determination
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Urinary 8-hydroxy-2’-deoxyguanosine (from DNA), 8-hydroxyguanosine (from RNA) and 8-hydroxyguanine (from either DN or RNA) levels were determined by the DNA/RNA Oxidative Damage ELISA Kit (Item No 589320; Cayman Chemical, USA). This assay is based on the competition between oxidatively damaged guanine species of the samples and an 8‐OH‐dG acetylcholinesterase conjugated. The assay was performed according the manufacture's protocol. After creatinine correction, the results were expressed as ng/mg creatinine.
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2.1.5 Creatinine determination
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Creatinine was determined using the Creatinine Kinetic kit of Bioclin® (Ref K067; Quibasa Química Básica Ltda., Belo Horizonte, MG, Brazil). Creatinine reacts 5
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with picric acid under alkaline conditions producing an orange colored derivative, whose absorbance was determined in a spectrophotometer at 510 nm. Results were expressed as mg/dL.
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2.2 In vitro experiments
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2.2.1 Comet Assay
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The alkaline comet assay was performed according to the method described by Singh et al. in accordance with general guidelines [20]. Human blood samples from four healthy volunteers were collected in heparinized vials. Leukocytes were incubated with different metabolites found in MCAD deficient patients - Adipic Acid (3 mM); Suberic Acid (3 mM); Hexanoylglycine (0.5 mM); Suberylglycine (1.5 mM) isolated and in combination, at 37ºC for 6 hours. Concentrations were chosen according to the values described in MCADD [21]. In order to determine the effects of L-car, leukocytes with each metabolite of MCADD were co-incubated with L-car 60 µM at the same conditions. The concentration of L-car used in this study were based on the supplementation recommended for patients with organic acidemias (50-100 mg/kg/day) [22,23]. Isolated leukocytes were suspended in agarose and spread into a glass microscope slide precoated with agarose and after allowed to set at 4ºC for 5 minutes. The slides were incubated in ice-cold lysis solution to remove cell proteins and to leave DNA as nucleoids. Following the lysis procedure, slides were placed on a horizontal electrophoresis unit, covered with fresh buffer (300 mM NaOH, 1 mM EDTA, pH>13) for 20 minutes at 4 ºC to allow DNA unwinding and the expression of alkali-labile-sites.
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Electrophoresis was performed for 20 minutes (25 V; 300 mA; 0.9 V/cm). The slides were neutralized, washed in bidistilled water and stained using a silver staining protocol. The gels were dried at room temperature overnight, and then analyzed using an optical microscope. One hundred cells (25 cells from each of the four replicate slides) were selected to be analyzed. The cells were visually scored according to tail length and received scored from 0 (no migration) to 4 (maximal migration) and therefore, the damage index (DI) for the cells range from 0 (all the cells with no migration) to 400 (all the cells with maximal migration). The slides were analyzed under blind conditions at least by two different individuals.
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2.3 Statistical analyses
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The results were expressed as mean ± standard error of the mean (SEM) and were statistically analyzed using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA - version 5.0). For in vivo assays with biological samples, comparisons between means were analyzed by unpaired T-test. For in vitro comet assay, comparisons were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey multiple range test (two groups; nonparametric). P values lower than 0.05 was considered statistically significant.
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3. Results
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Lipid peroxidation was determined by the measurement of 8-isoprostane levels in urine from FAOD patients and controls. It was verified significant increased 8-isoprostane levels in all groups of patients, LCHADD (119.5 ± 31.26) [t(12) = 2.607, p = 0.0229], MADD (27.47 ± 6.1) [t(6) = 15.94, p<0.0001] and MCADD (46.08 ± 2.5) [t(11) = 2.44, p = 0.0328], when compared to the control group (8.2 ± 1.13) (figure 1).
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Figure 1. 8-isoprostane levels in urine from LCHADD patients, n=9, MADD patients, n=8, and MCADD patients, n=3, compared to controls, n=5. Data are expressed as mean ± SEM. * indicates P<0.05 (Student’s t T-test for unpaired samples) compared to the control group.
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Nitrite and nitrate content, which represents RNS production, was determined in urine of FAOD patients and controls. It was observed a significant increase of the nitrite and nitrate content in samples of LCHADD patients (47.94 ± 2.7) compared to controls (31.03 ± 3.19) [t(8) = 4.012, p = 0.0039] (figure 2). However, no differences were found when comparisons were done between MADD patients (68.63 ± 25.3) and controls [t(9) = 1.095, p = 0.3020] or between MCADD patients (191.1 ± 119.4) and controls [t(5) = 1.601, p = 0.1702]. It is important to highlight that the high standard deviation found for the MCADD group can be explained by the small number of studied patients. Thus, although there was a clear tendency of increased RNS compared to the control group, no significant differences were observed between this group and the others.
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Figure 2. Nitrite and Nitrate levels in urine from LCHADD patients, n=9, MADD patients, n=8, and MCADD patients, n=3, compared to controls, n=5. Data are expressed as mean ± SEM. * indicates P<0.05 (Student’s t T-test for unpaired samples) compared to the control group.
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The DNA damage was evaluated in urine from FAOD patients and controls by the measurement of oxidized guanine species (OGS), like 8-hydroxy-2’deoxyguanosine (8-OH-dG), 8-hydroxyguanosine and 8-hydroxyguanine. When we compared LCHADD patients (14.42 ± 2.84) and control group (3.61 ± 0.8), a significant difference was found, indicating an increase of these biomarkers [t(9) = 2.781, p = 0.0214]. The same occurs when MADD patients’ samples (15.85 ± 1.36) were compared to the controls [t(8) = 6.753, p = 0.0001]. Only the MCADD patients group (7.65 ± 2.1) had no statistically significant difference in oxidized guanine species levels when compared to the control group [t(5) = 2.032, p = 0.0978] (figure 3).
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Figure 3. Oxidized guanine species levels in urine from LCHADD patients, n=9, MADD patients, n=8, and MCADD patients, n=3, compared to controls, n=5. Data are expressed as mean ± SEM. * indicates P<0.05 (Student’s t T-test for unpaired samples) compared to the control group.
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The evaluation of the in vitro DNA damage was performed by incubating different MCADD metabolites such adipic acid (ADI), suberic acid (SUB), hexanoylglycine (HG) and suberylglycine (SG) with leukocytes of healthy individuals. We observed that all metabolites induced a significant increase in the damage index when compared to the controls (figure 4) [F(2.11) = 99.8, p<0.0001, ADI] [F(2.11) = 15.97, p<0.001, SUB] [F(2.11) = 658.9, p<0.0001,HG] [F(2.11) = 1019, p<0.001, SG]. Hexanoylglycine (37±1.15) and suberylglycine (46±1.91) presented a higher increase on the damage index when compared to the adipic acid (15±0.5) and suberic acid (12±0.5), even in lower dose.
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Figure 4. DNA damage index induced by medium-chain acyl-CoA dehydrogenase deficiency metabolites and L-car effect. (A) In vitro effect of adipic acid (3 mM) co-incubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. (B) In vitro effect of suberic acid (3 mM) coincubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. (C) In vitro effect of hexanoylglycine (0.5 mM) co-incubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. (D) In vitro effect of suberylglycine (1.5 mM) co-incubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. Data are expressed as mean ± SEM of four independent experiments. One-way ANOVA followed by Tukey multiple range test. * indicates statistical difference compared to the control. # indicates statistical difference compared to the metabolite group. P<0.05.
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Also, table 1 show the number of cells found in each damage class at the tested metabolites concentration, obtained from four independent experiments, and it is observed that hexanoylglycine and suberylglycine presented a higher damage class - classes 2 and 3 -, compared to the adipic and suberic acids - only class 1. We tested, also, the co-incubation with L-car to investigate its effect on DNA damage caused by the MCADD metabolites (figure 4). Co-incubation with L-car was able to
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decrease significantly the damage index induced by the metabolites adipic acid, hexanoylglycine, suberylglycine (13±0.57; 29±0.5; 27± 0.3, respectively).
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Table 1. DNA damage index (DI) and number of cells in each DNA damage class induced in vitro by metabolites adipic acid (3 mM), suberic acid (3 mM), hexanoylglycine (0.5 mM) and suberylglycine (1.5 mM), isolated and in combination.
Metabolites
DI (mean ± SEM) n=4
Σ Damage class 0
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Control
8.5 ± 0.57
91
8
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0
0
Adipic Acid (ADI)
15 ± 0.5
82
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0
Suberic Acid (SUB)
12 ± 0.5
88
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0
Hexanoylglycine (HG)
37 ± 1.15
70
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1
0
Suberylglycine (SG)
46 ± 1.91
66
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0
ADI + SUB + HG + SG
51 ± 1.0
62
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9
2
0
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We also investigated the incubation of all metabolites in combination, in order to verify if a potentialized effect would occur. When all metabolites were incubated in combination, it was observed the highest increase on the damage index (51±1.0) when compared to the control (figure 5) and a higher damage class - classes 2 and 3. Co-incubation with L-car was able to decrease significantly the damage index induced by all metabolites in combination (30±0.5) but not to the control levels for the tested concentrations [F(2.11) = 1588, p<0.0001].
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Figure 5. In vitro effect of adipic acid (3 mM), suberic acid (3 mM), hexanoylglycine (0.5 mM), suberylglycine (1.5 mM) co-incubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. Data are expressed as mean ± SEM of four independent experiments. One-way ANOVA followed by Tukey multiple range test. * indicates statistical difference compared to the control. # indicates statistical difference compared to the metabolite group. P<0.05.
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4. Discussion
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Mitochondrial FAO is a complex cyclic pathway serving the stepwise shortening of saturated straight-chain fatty acids. Patients affected by inherited disorders in this pathway may present acute clinical presentation, especially during metabolic stress, that may lead to sudden death [2]. However, the pathogenic mechanism involved in the severe clinical manifestations and death in FAOD are poor established so far.
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Tissue fatty acid accumulation is observed in FAOD and affected patients usually present hepatopathy, cardiomyopathy and skeletal myopathy, since mitochondrial FAO is very active in liver, heart and skeletal muscle. Moreover, a recent study demonstrated that despite earlier diagnosis by expanded newborn screening, sudden deaths were not completely avoided and acute decompensations with severe clinical manifestations still occur as well [24, 25].
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Several studies have been conducted in order to evaluate the role of accumulated toxic metabolites in inducing excessive free radical production, that in addition to a reduced antioxidant status, lead to oxidative stress in different IMD such as phenylketonuria, maple syrup urine disease, glutaric acidemia type I and L-2hydroxyglutaric aciduria [10,11,12]. In this context, Tonin et al. found that LCHADD metabolites act as uncouplers of oxidative phosphorylation and metabolic inhibitors and compromise energy homeostasis in mitochondria [18]. Schuck et al. demonstrated similar findings for the MCADD metabolites, plus a permeabilization of the inner mitochondrial membrane [19]. Cornelius et al. verified inhibition of mitochondrial fusion with increased fractionation and mitophagy in MADD patients’ fibroblasts, besides a shift in the energy metabolism, indicating a general mitochondrial dysfunction as a rescue mechanism for the cells to escape apoptosis as a result of oxidative stress [26]. The intimate relationship between mitochondrial FAO, electron transport and oxidative phosphorylation justifies the study of oxidative damage in FAOD patients.
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In the current study, we demonstrated that FAOD patients affected by LCHADD, MCADD and MADD present significantly increased urinary 8-isoprostanes levels, a biomarker of lipid peroxidation, when compared to the control group. Isoprostanes are prostaglandin-like compounds formed by a free radical-catalysed peroxidation of arachidonic acid esterified in membrane phospholipids rather than by a cyclooxygenase-dependent oxidation of arachidonic acid. Then, it is possible to suppose that high isoprostanes levels are harmful and might compromise membrane integrity and fluidity causing the rupture of organelles or even the cells [27, 28]. Then, we can hypothesize that an excessive production of toxic metabolites (probably added to other causes, such as accumulation of misfolded proteins or electron leakage at ETF/ETFDH) may lead to increased production of reactive species, 12
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causing damage to biomolecules and contributing to the pathophysiology of FAOD. In addition, other studies in animal models [16,17] and in patients [29] corroborate our findings and helped us to better understand this pathological mechanism, indicating that the accumulation of toxic metabolites may be responsible for lipid peroxidation and might be related to the clinical manifestation observed in FAOD patients.
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Nitric Oxide (NO) is produced by different types of cells and has many functions like cellular signaling and defense against pathogens through oxidative toxicity. The NO final products are nitrite (NO2-) and nitrate (NO3-) and their levels correspond to total NO production. A high amount of these molecules may cause nitrosative damage through the formation of RNS like peroxynitrite (ONOO-) [30]. Nitrite and nitrate levels were measured in our study to evaluate nitrosative damage to the cells of patients affected by FAOD. Our present results suggest that nitrosative stress generated by elevated concentrations of nitrite and nitrate occurs in LCHADD patients and it is probably related to the pathophysiology of this disease, since it can lead to proteins nitration and compromise their functions, as well as to mitochondrial dysfunction. Thereby the cells processes may be disrupted, and in major scale, may cause energy deficiency, probably related to the clinical manifestations presented by LCHADD patients. Tonin et al. found no alteration on nitrite and nitrate production induced in vitro by LCHAD metabolites 3-hydroxydodecanoic, 3hydroxytetradecanoic and 3-hydroxypalmitic acids in rat brain, contrary to our results [17]. The different results between our work and the data of this study, could be attributed possibly to the limitation of the in vitro model on the ideal simulation of the complex in vivo system, observed in this analysis, or even to the concentration of the 3-OH fatty acids (10, 25, 50 and 100 µM) utilized in the study.
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Although it was not found any alteration in nitrite and nitrate content in MADD and MCADD patients when compared to the controls, we can observe a tendency to increased levels of this parameter. Perhaps in a future study with a greater number of patients it would be possible to detect a significant statistical difference. Besides, it is important to emphazise that different concentrations of accumulated metabolites in patients may have different impacts on oxidative damage, as evidenced by previous studies [17, 16], where it was found that different concentrations of accumulated metabolites in FAOD induced different alterations in the oxidative stress parameters in the tissues of the studied animals.
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DNA and RNA are important target biomolecules that can be damaged by elevated concentrations of ROS, leading to a variety of lesions such as DNA strand breaks, abasic sites, and several species of oxidized purine/pyrimidine and DNAprotein cross-links. Oxidative DNA damage can be considered the most severe damage to the biomolecules. These lesions can lead to genetic mutations, alterations in gene expression, chromosomal aberrations, loss of function and ultimately cell death. Studies have demonstrated an increase in oxidized guanine species in urine samples of L-2-hydroxyglutaric aciduria and glutaric acidemia type I patients, indicating the harmful potential of the toxic metabolites accumulated, L-2hydroxyglutaric and glutaric acids, to DNA and RNA of patients [11, 33].
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The hydroxyl radical, the most harmful ROS, as well as others free radicals, can attack nuclear and mitochondrial DNA and generate the oxidized forms of 8hydroxy-2’-deoxyguanosine (8-OH-dG), 8-hydroxyguanosine and 8-hydroxyguanine, oxidized guanine species. After ROS attacks on RNA and both nuclear and mitochondrial DNA, the adducts formed are excised and eliminated in blood and urine [32]. In this context, we detected a significant increase of oxidized guanine species in urine of LCHADD and MADD patients, which indicates DNA and RNA oxidative damage. The existence of this type of damage, probably secondary to the accumulation of toxic fatty acids (besides other causes), might be related to metabolic dysfunction and clinical presentation of LCHADD and MADD patients. This data, in association with lipid oxidative damage, indicate a possible reason for the modifications on energy homeostasis in patients affected by these disorders. MCADD patients from our study did not present significant statistical differences in urinary oxidized guanine species levels compared to the control group. However, a tendency toward to increased levels can be observed and future studies with more MCADD patients could clarify this issue.
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The alkaline comet assay, a simple and sensitive technique capable of measure single and double DNA strand breaks, was performed in order to investigate the in vitro effect of the main metabolites associated with MCADD (adipic acid, suberic acid, hexanoylglycine and suberylglycine) upon DNA damage as well as to study the effect of L-car. It was verified that adipic and suberic acids induced significantly elevated levels of DNA damage, with a DI (mean ± SEM) of 15 ± 0.5 and 12 ± 0.5 arbitrary units, respectively. These metabolites induced the presence of only slightly damaged cells (class 1, the lowest level of DNA injury). Co-incubation with Lcar at the concentration of 60 µM significantly decreased DI induced by adipic acid but not by suberic acid. Interestingly, hexanoylglycine and suberylglycine induced the presence of higher damaged cells (class 2 and 3) and significantly increased DI (37±1.15 and 46±1.91 arbitrary units, mean ± SEM, respectively), indicating greater DNA migration, and co-incubation with L-car (60 µM) also reduced significantly the DI caused by both metabolites although not reaching the control levels. The in vitro effect on DNA damage of the four metabolites in combination was the highest (51 ± 0.1 arbitrary units, mean ± SEM), representing the greatest DNA migration and demonstrating a potentialized effect of the metabolites. In fact, during fasting or other situations of metabolic decompensation in MCADD, high amounts of all these toxic metabolites can be detected in patients’ samples, as well as their derivative carnitines. Thus, DNA damage induced by the accumulation of toxic metabolites might be enhanced in patients during those events [24]. Co-incubation with L-car (60 µM) also prevented DNA damage induced by adipic acid, suberic acid, hexanoylglycine and suberylglycine in combination, but not to the control levels for the tested concentrations. If not repaired, the DNA damage caused by direct DNA mechanisms or by activation of Ca2+-endonucleases and by interfering with enzymes of DNA replication and repair, may lead to serious consequences in the genome, like loss of heterozygosity, microsatellite instability, mutations or neoplastic growth [32]. Patients affected by MCADD are probably exposed to this type of damage since their concentrations of toxic metabolites are higher than healthy individuals. Nevertheless, an in vivo evaluation in blood samples of MCADD patients during episodes of 14
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metabolic decompensation by comet assay would verify the scale of DNA damage, not evidenced statistically by the oxidized guanine species test.
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The treatment of several IMDs include L-car supplementation due to a secondary deficiency caused sometimes by the restricted diet and recent studies have demonstrated antioxidant properties of the molecule [11,12]. L-car prescription is recommended for MCADD patients exhibiting a deficiency in plasma carnitine, as well as the avoidance of prolonged fasting, according to the french consensus for neonatal screening, diagnosis and management [34]. In our study, L-car decreased in vitro the DNA damage induced by adipic acid, hexanoylglycine and suberylglycine, isolated and in combination. The protective effects of L-car are mainly attributed to its ability to scavenge ROS and to chelate transition metal ions [8]. Furthermore, L-car is capable to reduce oxidative damage to DNA caused by alkylating agents and free radicals, accelerating the disappearance of single-strand breaks and the repair action of poly (ADP-ribose) polymerase or other related mechanisms [35]. Another mechanism of L-car protection could be related to its conjugation to the toxic metabolites in IMD forming their carnitine derivatives [36]. Many studies have shown the oxidative stress contribution to the pathogenesis of IMD and the beneficial effects of compounds as L-car that presented antioxidants effects in vitro and in vivo. Besides, its supplementation is considered safe and with a low risk of adverse effects [37]. These data plus the findings of our work corroborate with the evidence of the antioxidant role of L-carnitine and thus it would be an encouraging complementary strategy for the treatment of FAOD patients in order to prevent oxidative damages.
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5. Conclusion
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In conclusion, our data indicate that LCHADD, MADD and MCADD patients presented lipoperoxidation, LCHADD patients presented nitrosative stress and LCHADD and MADD patients presented DNA and RNA oxidative damage, which is probably related to the pathophysiology of these disorders. However, more studies are necessary to understand the mechanisms by which the damage occurs. In addition, our present study demonstrates that accumulated MCADD toxic metabolites (adipic acid, suberic acid, hexanoylglycine and suberylglycine) induce, isolatedly and in combination, DNA damage, presenting a potentialized effect. L-car was capable to preserve DNA of this damage, evidencing the protective properties on DNA injury and its potential as an adjuvant therapy for FAOD.
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6. Acknowledgments
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The authors are also grateful for the collaboration of patients and their families and the SGM/HCPA staff.
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Funding: this study was supported by grants from CAPES, CNPq and FIPE/HCPA-Brazil.
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7. Conflict of Interest The authors declare that they have no conflict of interest in this work. 15
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18
Highlights:
•
Urine from LCHADD, MCADD and MADD patients presented lipid damage;
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LCHADD patients presented nitrosative stress;
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LCHADD and MADD patients presented oxidative DNA and RNA damage;
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MCADD metabolites, separated and in combination, induced in vitro DNA damage;
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Co-incubation with L-carnitine reduced the DNA damage induced by MCADD metabolites.
S0003-9861(19)30375-3
DOI:
https://doi.org/10.1016/j.abb.2019.108206
Reference:
YABBI 108206
To appear in:
Archives of Biochemistry and Biophysics
Received Date: 17 May 2019 Revised Date:
19 November 2019
Accepted Date: 20 November 2019
Please cite this article as: M. Silmara de Moraes, G. Guerreiro, A. Sitta, D. de Moura Coelho, V. Manfredini, M. Wajner, C.R. Vargas, Oxidative damage in mitochondrial fatty acids oxidation disorders patients and the in vitro effect of l-carnitine on DNA damage induced by the accumulated metabolites, Archives of Biochemistry and Biophysics (2019), doi: https://doi.org/10.1016/j.abb.2019.108206. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Inc.
Authors statement
Maira Silmara de Moraes: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing. Gilian Guerreiro: Investigation, Validation, Writing, Methodology. Angela Sitta: Methodology, Investigation, Writing. Daniella de Moura Coelho: Methodology, Validation, Investigation. Vanusa Manfredini: Methodology, Validation. Moacir Wajner: Formal analysis, Writing, Visualization. Carmen Regla Vargas: Conceptualization, Validation, Writing, Supervision, Project administration, Funding acquisition.
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Title: Oxidative damage in mitochondrial fatty acids oxidation disorders patients and the in vitro effect of L-carnitine on DNA damage induced by the accumulated metabolites
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Authors: Maira Silmara de Moraes , Gilian Guerreiro , Angela Sitta , Daniella de Moura Coelho , Vanusa Manfredini , Moacir Wajner , Carmen Regla Vargas 1,2*
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1,2,3,4*
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Programa de Pós‐Graduação em Ciências Biológicas: Bioquímica, UFRGS, Porto Alegre, RS, Brazil
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Serviço de Genética Médica, HCPA,UFRGS, Porto Alegre, RS, Brazil
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Faculdade de Farmácia, UFRGS, Porto Alegre, RS, Brazil
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Programa de Pós‐Graduação em Ciências Farmacêuticas, UFRGS, Porto Alegre, RS, Brazil Programa de Pós‐Graduação em Bioquímica, Universidade Federal do Pampa, Uruguaiana, RS, Brazil
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Authors e-mails: [email protected]; [email protected]; [email protected], [email protected]; [email protected]; [email protected]; [email protected].
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*Corresponding authors at: Serviço de Genética Médica, HCPA, Rua Ramiro Barcelos, 2350, ZIP CODE 90.035-003, Porto Alegre, RS, Brazil.
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Telephone: +55 51 33598011
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Telefax: +55 51 33598010
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E-mail adresses: [email protected] (C.R. Vargas), [email protected] (M.S. Moraes)
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1
Abstract
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Background: The mitochondrial fatty acids oxidation disorders (FAOD) are inherited metabolic disorders (IMD) characterized by the accumulation of fatty acids of different sizes of chain according to the affected enzyme. Methods: This study evaluated the lipid peroxidation by the measurement of 8isoprostanes, nitrosative stress parameters by the measurement of nitrite and nitrate content and DNA and RNA oxidative damage by the measurement of oxidized guanine species in urine samples from long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and multiple acyl-CoA dehydrogenase deficiency (MADD) patients. Also, we analysed the in vitro DNA damage by comet assay induced by adipic acid, suberic acid, hexanoylglycine and suberylglycine, separated and in combination, as well as the effect of L-carnitine in human leukocytes. Results: An increase on 8-isoprostanes levels in all groups of patients was observed. The nitrite and nitrate levels were increased in LCHADD patients. DNA and RNA damage evaluation revealed increase on oxidized guanine species levels in LCHADD and MADD patients. The in vitro evaluation revealed an increase on the DNA damage induced by all metabolites, besides a potencialyzed effect. L-carnitine decreased the DNA damage induced by the metabolites. Conclusion: These results demonstrate that toxic metabolites accumulated could be related to the increased oxidative and nitrosative stress of FAOD patients and that the metabolites, separated and in combination, cause DNA damage, which was reduced by L-carnitine, demonstrating antioxidant protection. General Significance: This work demonstrated oxidative stress in FAOD patients and the genotoxic potencial of MCADD metabolites and the protective effect of Lcarnitine.
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Keywords: MCADD; LCHADD; MADD; oxidative stress; DNA damage; L-carnitine.
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Abbreviations: DI (damage index); FAOD (fatty acid oxidation disorders); IMD (inherited metabolic disorders); L-car (L-carnitine); LCHADD (long-chain 3hydroxyacyl-CoA dehydrogenase deficiency); MADD (multiple acyl-CoA dehydrogenase deficiency); MCADD (medium-chain acyl-CoA dehydrogenase deficiency); NO (nitric oxide); ); OGS (oxidized guanine species); RNS (reactive nitrogen species); ROS (reactive oxygen species). 2
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1. Introduction
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The fatty acid oxidation disorders (FAOD) are inherited metabolic disorders (IMD) caused by a deficient activity of specific enzymes of the beta-oxidation pathway that are involved in the mitochondrial catabolism of these fatty acids. Mitocondrial beta-oxidation consists of four steps, catalyzed by FAD-dependent acyl-CoA dehydrogenases, 2-enoyl-CoA hydratases, NAD-dependent L-3hydroxyacyl-CoA dehydrogenases and 3-ketoacyl-CoA thiolases. During each cycle the acyl-CoA ester that entered the cycle is shortened by two carbon atoms, which are released as acetyl-CoA. FAO disorders lead to the accumulation of fatty acids according to the damaged enzyme and compromise the use of this source of energy [1]. Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an autosomal recessive disorder leading to the accumulation of medium-chain fatty acids like suberic, adipic, sebacic, octanoic, 5-hydroxyhexanoic acids, as well as phenylpropionilglycine, hexanoylglycine, suberylglycine and their carnitine derivatives. The worldwide prevalence of this disease is 1: 14,600, being the most common disorder of FAOD, and clinical presentation of this disorder usually occurs 3 to 24 months after birth. In addition, in certain cases, adult and neonatal clinical forms may be found. The latter usually accompanied by cases of sudden death [2]. MCADD is caused by mutations in the ACADM gene, encoding the MCAD protein [3].
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Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is an autosomal recessive disorder caused by mutations in the HADHA gene, which encodes the α-subunit of the mitochondrial trifunctional protein, leading to the accumulation of long-chain fatty acids with a hydroxyl group like 3hydroxytetradecanoic, 3-hydroxyhexadecanoic, 3-hydroxyoctadecanoic, 3hydroxysebacic, 3-hydroxydehydrosebacic, 3-hydroxydodecanedioic acids and their carnitine derivatives in patients tissues. The estimated worldwide prevalence of this disease is 1:250,000. Patients affected by LCHADD present a poor prognosis and most of them exhibit a severe phenotype in neonatal period [2,4].
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Multiple acyl-CoA dehydrogenase deficiency (MADD), also known as Glutaric Aciduria type 2 (GA-II), is another autosomal recessive disorder that results from the deficiency of electron transfer flavoprotein (ETF alfa and beta) or electron transfer flavoprotein/ubiquinone oxidoreductase (ETF/QO) that transport the electrons from beta-oxidation to the respiratory chain. Several metabolites are accumulated in MADD patients including dicarboxylic acids, as for example, glutaric, ethylmalonic, 2hydroxyglutaric, adipic, suberic, sebacic acids, as well as glycine conjugates and their carnitine derivatives. The worldwide prevalence for this disease is 1:200,000. Clinical presentation includes three phenotypes: a) neonatal with congenital anomalies; b) neonatal without congenital anomalies (severe forms) whose manifestation generally occurs within the first 48 hours of life and might be fatal; c) mild and late clinical form that usually appear in the first months until adult life with broad clinical spectrum and better prognosis [2,5].
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All these three inherited disorders above may share common clinical and biochemical features like hypoketotic hypoglycemia, metabolic acidosis, hypotony, hyperammonemia and in severe cases skeletal myopathy, cardiomyopathy, hepatopathy and neuropathy. The frequency and appearance will depend on the clinical conditions and disease (MCAAD, LCHADD or MADD) presented by each patient [2]. Diagnosis of these diseases is based on the identification of urinary 3
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excretion of the fatty acids and their carnitine derivatives in blood. The confirmation can be performed by molecular analysis. Currently, the therapeutic strategy is to avoid fasting and to consume high amounts of carbohydrate and low amounts of fat and protein [2,6]. Also, in some cases, a supplementation of L-carnitine (L-car) is indicated, specially in patients with carnitine deficiency. Conjugation of carnitine with fatty acids contributes to elimination of toxic metabolites. Doses of L-car vary from 50 to 100 mg/kg/day, according to the patient’s age and its metabolic status [7]. Besides the important function on the transport of long-chain fatty acids across the inner mitochondrial membrane for beta-oxidation, L-car also has antioxidant properties, due to the capacity to scavenge hydrogen peroxide and superoxide radical and to chelate transition metal ions with protective effects against oxidative injury in others IMD like phenylketonuria, glutaric aciduria type I and maple syrup urine disease [8, 9, 10, 11, 12].
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Mitochondria are the most important organelles for cell homeostasis through several processes like energy production by the oxidative phosphorylation that generates ATP and reactive oxygen species (ROS). Biochemical and morphological alterations in tissues of FAOD patients suggest that the mitochondrial dysfunction contributes to the pathogenesis of the diseases. The accumulation of toxic metabolites can be harmful to the cell and may result in mitochondrial stress and collapse of internal membrane potential [13, 14]. Although ROS have an essential role on cell activities, an increase on its concentration can cause oxidative damage to biomolecules like lipids, proteins, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), compromising cell functions like membrane permeability, transport, signalling, enzymatic reactions, mutation repair and gene expression, leading to the cell death [15].
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Although many studies have demonstrated that the pathogenesis of FAOD is related to its toxic metabolites, there is not enough knowledge about the pathophysiology of the diseases and their mechanisms are still not completely known [16, 17, 18, 19]. In many others IMD it was demonstrated altered oxidative and nitrative indicators, with increased levels of ROS and reactive nitrogen species (RNS) [10, 11, 12].
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This study aimed to investigate oxidative and nitrosative stress parameters in patients affected by LCHADD, MCADD and MADD and also to evaluate the in vitro effect of L-carnitine on DNA damage induced by the main metabolites accumulated in MCADD: adipic acid (ADI), suberic acid (SUB), hexanoylglycine (HG) and suberylglycine (SG).
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2. Materials and Methods
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2.1 In vivo experiments
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2.1.1 Patients and biological samples
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We analyzed urine samples from 20 patients (from the Medical Genetic Service of Hospital de Clínicas de Porto Alegre) at diagnosis of FAOD, collected during a decompensation period. Mean age of patients was 6.6 ± 3.9 years and included LCHADD (n=9), MCADD (n=3) and MADD (n=8). 4
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The diagnosis were performed by the analysis of urinary organic acids by gas chromatography coupled to mass spectrometry as well as by detection of blood acylcarnitines accumulation by liquid chromatography coupled to mass spectrometry. For the control group (n=5), we utilized urine samples of age matched healthy people. All urine samples were stored in a freezer at -80ºC and quickly unfreezed for the analysis. The Ethics Committee of HCPA approved this project (2018-0549) and all patients or their parents gave informed consent about this study.
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2.1.2 8-Isoprostane determination 8-Isoprostane is a biomarker of lipid peroxidation in urine and was measured by the 8-isoprostane AChE Competitive ELISA commercial kit (Item No.516351; Cayman Chemical). This assay is based on the competition between 8-isoprostane and an 8-isoprostane-acetylcholinesterase (AChE) conjugate (Tracer) for a limited number of 8-isoprostanes-specific rabbit antiserum binding sites. The amount of bound Tracer is quantified at 412 nm, after addition of the Ellman’s reagent, a substrate of AChE. The intensity of the color is proportional to the amount of tracer bound to the well and is inversely proportional to the amount of free 8-isoprostane present in the urine. Results were expressed as ng/mg of creatinine.
19 20
2.1.3 Reactive Nitrogen Species
21 22 23 24 25 26 27 28
Total levels of nitrate + nitrite (NO3- + NO2-) in urine were determined using the Nitrate/Nitrite Colorimetric Assay Kit (LDH Method) from Cayman Chemical® (Item No780001; Cayman Chemical Company, Ann Arbor, MI, USA). This method is based on the reduction of NO3- to NO2- using nitrate reductase. By adding sulfanilamide to NO2-, a cationic intermediate is formed, which reacts with N-(1-naphtyl) ethylenediamine, resulting in an azo product whose absorption can be measured at 540 nm. Results were reported as µmol/mg creatinine.
29 30 31
2.1.4 Oxidized Guanine Species determination
32 33 34 35 36 37 38 39
Urinary 8-hydroxy-2’-deoxyguanosine (from DNA), 8-hydroxyguanosine (from RNA) and 8-hydroxyguanine (from either DN or RNA) levels were determined by the DNA/RNA Oxidative Damage ELISA Kit (Item No 589320; Cayman Chemical, USA). This assay is based on the competition between oxidatively damaged guanine species of the samples and an 8‐OH‐dG acetylcholinesterase conjugated. The assay was performed according the manufacture's protocol. After creatinine correction, the results were expressed as ng/mg creatinine.
40
2.1.5 Creatinine determination
41 42
Creatinine was determined using the Creatinine Kinetic kit of Bioclin® (Ref K067; Quibasa Química Básica Ltda., Belo Horizonte, MG, Brazil). Creatinine reacts 5
1 2 3
with picric acid under alkaline conditions producing an orange colored derivative, whose absorbance was determined in a spectrophotometer at 510 nm. Results were expressed as mg/dL.
4 5
2.2 In vitro experiments
6
2.2.1 Comet Assay
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
The alkaline comet assay was performed according to the method described by Singh et al. in accordance with general guidelines [20]. Human blood samples from four healthy volunteers were collected in heparinized vials. Leukocytes were incubated with different metabolites found in MCAD deficient patients - Adipic Acid (3 mM); Suberic Acid (3 mM); Hexanoylglycine (0.5 mM); Suberylglycine (1.5 mM) isolated and in combination, at 37ºC for 6 hours. Concentrations were chosen according to the values described in MCADD [21]. In order to determine the effects of L-car, leukocytes with each metabolite of MCADD were co-incubated with L-car 60 µM at the same conditions. The concentration of L-car used in this study were based on the supplementation recommended for patients with organic acidemias (50-100 mg/kg/day) [22,23]. Isolated leukocytes were suspended in agarose and spread into a glass microscope slide precoated with agarose and after allowed to set at 4ºC for 5 minutes. The slides were incubated in ice-cold lysis solution to remove cell proteins and to leave DNA as nucleoids. Following the lysis procedure, slides were placed on a horizontal electrophoresis unit, covered with fresh buffer (300 mM NaOH, 1 mM EDTA, pH>13) for 20 minutes at 4 ºC to allow DNA unwinding and the expression of alkali-labile-sites.
24 25 26 27 28 29 30 31 32
Electrophoresis was performed for 20 minutes (25 V; 300 mA; 0.9 V/cm). The slides were neutralized, washed in bidistilled water and stained using a silver staining protocol. The gels were dried at room temperature overnight, and then analyzed using an optical microscope. One hundred cells (25 cells from each of the four replicate slides) were selected to be analyzed. The cells were visually scored according to tail length and received scored from 0 (no migration) to 4 (maximal migration) and therefore, the damage index (DI) for the cells range from 0 (all the cells with no migration) to 400 (all the cells with maximal migration). The slides were analyzed under blind conditions at least by two different individuals.
33 34
2.3 Statistical analyses
35 36 37 38 39 40 41
The results were expressed as mean ± standard error of the mean (SEM) and were statistically analyzed using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA - version 5.0). For in vivo assays with biological samples, comparisons between means were analyzed by unpaired T-test. For in vitro comet assay, comparisons were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey multiple range test (two groups; nonparametric). P values lower than 0.05 was considered statistically significant.
42 43
3. Results
6
1 2 3 4 5
Lipid peroxidation was determined by the measurement of 8-isoprostane levels in urine from FAOD patients and controls. It was verified significant increased 8-isoprostane levels in all groups of patients, LCHADD (119.5 ± 31.26) [t(12) = 2.607, p = 0.0229], MADD (27.47 ± 6.1) [t(6) = 15.94, p<0.0001] and MCADD (46.08 ± 2.5) [t(11) = 2.44, p = 0.0328], when compared to the control group (8.2 ± 1.13) (figure 1).
6 7 8 9
Figure 1. 8-isoprostane levels in urine from LCHADD patients, n=9, MADD patients, n=8, and MCADD patients, n=3, compared to controls, n=5. Data are expressed as mean ± SEM. * indicates P<0.05 (Student’s t T-test for unpaired samples) compared to the control group.
10 11 12 13 14 15 16 17 18 19 20
Nitrite and nitrate content, which represents RNS production, was determined in urine of FAOD patients and controls. It was observed a significant increase of the nitrite and nitrate content in samples of LCHADD patients (47.94 ± 2.7) compared to controls (31.03 ± 3.19) [t(8) = 4.012, p = 0.0039] (figure 2). However, no differences were found when comparisons were done between MADD patients (68.63 ± 25.3) and controls [t(9) = 1.095, p = 0.3020] or between MCADD patients (191.1 ± 119.4) and controls [t(5) = 1.601, p = 0.1702]. It is important to highlight that the high standard deviation found for the MCADD group can be explained by the small number of studied patients. Thus, although there was a clear tendency of increased RNS compared to the control group, no significant differences were observed between this group and the others.
7
1 2 3 4
Figure 2. Nitrite and Nitrate levels in urine from LCHADD patients, n=9, MADD patients, n=8, and MCADD patients, n=3, compared to controls, n=5. Data are expressed as mean ± SEM. * indicates P<0.05 (Student’s t T-test for unpaired samples) compared to the control group.
5 6 7 8 9 10 11 12 13 14 15
The DNA damage was evaluated in urine from FAOD patients and controls by the measurement of oxidized guanine species (OGS), like 8-hydroxy-2’deoxyguanosine (8-OH-dG), 8-hydroxyguanosine and 8-hydroxyguanine. When we compared LCHADD patients (14.42 ± 2.84) and control group (3.61 ± 0.8), a significant difference was found, indicating an increase of these biomarkers [t(9) = 2.781, p = 0.0214]. The same occurs when MADD patients’ samples (15.85 ± 1.36) were compared to the controls [t(8) = 6.753, p = 0.0001]. Only the MCADD patients group (7.65 ± 2.1) had no statistically significant difference in oxidized guanine species levels when compared to the control group [t(5) = 2.032, p = 0.0978] (figure 3).
8
1 2 3 4 5
Figure 3. Oxidized guanine species levels in urine from LCHADD patients, n=9, MADD patients, n=8, and MCADD patients, n=3, compared to controls, n=5. Data are expressed as mean ± SEM. * indicates P<0.05 (Student’s t T-test for unpaired samples) compared to the control group.
6 7 8 9 10 11 12 13 14 15
The evaluation of the in vitro DNA damage was performed by incubating different MCADD metabolites such adipic acid (ADI), suberic acid (SUB), hexanoylglycine (HG) and suberylglycine (SG) with leukocytes of healthy individuals. We observed that all metabolites induced a significant increase in the damage index when compared to the controls (figure 4) [F(2.11) = 99.8, p<0.0001, ADI] [F(2.11) = 15.97, p<0.001, SUB] [F(2.11) = 658.9, p<0.0001,HG] [F(2.11) = 1019, p<0.001, SG]. Hexanoylglycine (37±1.15) and suberylglycine (46±1.91) presented a higher increase on the damage index when compared to the adipic acid (15±0.5) and suberic acid (12±0.5), even in lower dose.
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9
1
2 3 4 5 6 7 8 9 10 11 12
Figure 4. DNA damage index induced by medium-chain acyl-CoA dehydrogenase deficiency metabolites and L-car effect. (A) In vitro effect of adipic acid (3 mM) co-incubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. (B) In vitro effect of suberic acid (3 mM) coincubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. (C) In vitro effect of hexanoylglycine (0.5 mM) co-incubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. (D) In vitro effect of suberylglycine (1.5 mM) co-incubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. Data are expressed as mean ± SEM of four independent experiments. One-way ANOVA followed by Tukey multiple range test. * indicates statistical difference compared to the control. # indicates statistical difference compared to the metabolite group. P<0.05.
13 14 15 16 17 18 19
Also, table 1 show the number of cells found in each damage class at the tested metabolites concentration, obtained from four independent experiments, and it is observed that hexanoylglycine and suberylglycine presented a higher damage class - classes 2 and 3 -, compared to the adipic and suberic acids - only class 1. We tested, also, the co-incubation with L-car to investigate its effect on DNA damage caused by the MCADD metabolites (figure 4). Co-incubation with L-car was able to
10
1 2
decrease significantly the damage index induced by the metabolites adipic acid, hexanoylglycine, suberylglycine (13±0.57; 29±0.5; 27± 0.3, respectively).
3 4 5 6
Table 1. DNA damage index (DI) and number of cells in each DNA damage class induced in vitro by metabolites adipic acid (3 mM), suberic acid (3 mM), hexanoylglycine (0.5 mM) and suberylglycine (1.5 mM), isolated and in combination.
Metabolites
DI (mean ± SEM) n=4
Σ Damage class 0
1
2
3
4
Control
8.5 ± 0.57
91
8
0
0
0
Adipic Acid (ADI)
15 ± 0.5
82
15
0
0
0
Suberic Acid (SUB)
12 ± 0.5
88
12
0
0
0
Hexanoylglycine (HG)
37 ± 1.15
70
24
5
1
0
Suberylglycine (SG)
46 ± 1.91
66
23
10
1
0
ADI + SUB + HG + SG
51 ± 1.0
62
26
9
2
0
7 8 9 10 11 12 13 14
We also investigated the incubation of all metabolites in combination, in order to verify if a potentialized effect would occur. When all metabolites were incubated in combination, it was observed the highest increase on the damage index (51±1.0) when compared to the control (figure 5) and a higher damage class - classes 2 and 3. Co-incubation with L-car was able to decrease significantly the damage index induced by all metabolites in combination (30±0.5) but not to the control levels for the tested concentrations [F(2.11) = 1588, p<0.0001].
15
16
11
1 2 3 4 5
Figure 5. In vitro effect of adipic acid (3 mM), suberic acid (3 mM), hexanoylglycine (0.5 mM), suberylglycine (1.5 mM) co-incubated with L-car (60 µM) on DNA damage in leukocytes from whole blood. Data are expressed as mean ± SEM of four independent experiments. One-way ANOVA followed by Tukey multiple range test. * indicates statistical difference compared to the control. # indicates statistical difference compared to the metabolite group. P<0.05.
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4. Discussion
9 10 11 12 13 14
Mitochondrial FAO is a complex cyclic pathway serving the stepwise shortening of saturated straight-chain fatty acids. Patients affected by inherited disorders in this pathway may present acute clinical presentation, especially during metabolic stress, that may lead to sudden death [2]. However, the pathogenic mechanism involved in the severe clinical manifestations and death in FAOD are poor established so far.
15 16 17 18 19 20
Tissue fatty acid accumulation is observed in FAOD and affected patients usually present hepatopathy, cardiomyopathy and skeletal myopathy, since mitochondrial FAO is very active in liver, heart and skeletal muscle. Moreover, a recent study demonstrated that despite earlier diagnosis by expanded newborn screening, sudden deaths were not completely avoided and acute decompensations with severe clinical manifestations still occur as well [24, 25].
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Several studies have been conducted in order to evaluate the role of accumulated toxic metabolites in inducing excessive free radical production, that in addition to a reduced antioxidant status, lead to oxidative stress in different IMD such as phenylketonuria, maple syrup urine disease, glutaric acidemia type I and L-2hydroxyglutaric aciduria [10,11,12]. In this context, Tonin et al. found that LCHADD metabolites act as uncouplers of oxidative phosphorylation and metabolic inhibitors and compromise energy homeostasis in mitochondria [18]. Schuck et al. demonstrated similar findings for the MCADD metabolites, plus a permeabilization of the inner mitochondrial membrane [19]. Cornelius et al. verified inhibition of mitochondrial fusion with increased fractionation and mitophagy in MADD patients’ fibroblasts, besides a shift in the energy metabolism, indicating a general mitochondrial dysfunction as a rescue mechanism for the cells to escape apoptosis as a result of oxidative stress [26]. The intimate relationship between mitochondrial FAO, electron transport and oxidative phosphorylation justifies the study of oxidative damage in FAOD patients.
36 37 38 39 40 41 42 43 44 45 46
In the current study, we demonstrated that FAOD patients affected by LCHADD, MCADD and MADD present significantly increased urinary 8-isoprostanes levels, a biomarker of lipid peroxidation, when compared to the control group. Isoprostanes are prostaglandin-like compounds formed by a free radical-catalysed peroxidation of arachidonic acid esterified in membrane phospholipids rather than by a cyclooxygenase-dependent oxidation of arachidonic acid. Then, it is possible to suppose that high isoprostanes levels are harmful and might compromise membrane integrity and fluidity causing the rupture of organelles or even the cells [27, 28]. Then, we can hypothesize that an excessive production of toxic metabolites (probably added to other causes, such as accumulation of misfolded proteins or electron leakage at ETF/ETFDH) may lead to increased production of reactive species, 12
1 2 3 4 5 6
causing damage to biomolecules and contributing to the pathophysiology of FAOD. In addition, other studies in animal models [16,17] and in patients [29] corroborate our findings and helped us to better understand this pathological mechanism, indicating that the accumulation of toxic metabolites may be responsible for lipid peroxidation and might be related to the clinical manifestation observed in FAOD patients.
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Nitric Oxide (NO) is produced by different types of cells and has many functions like cellular signaling and defense against pathogens through oxidative toxicity. The NO final products are nitrite (NO2-) and nitrate (NO3-) and their levels correspond to total NO production. A high amount of these molecules may cause nitrosative damage through the formation of RNS like peroxynitrite (ONOO-) [30]. Nitrite and nitrate levels were measured in our study to evaluate nitrosative damage to the cells of patients affected by FAOD. Our present results suggest that nitrosative stress generated by elevated concentrations of nitrite and nitrate occurs in LCHADD patients and it is probably related to the pathophysiology of this disease, since it can lead to proteins nitration and compromise their functions, as well as to mitochondrial dysfunction. Thereby the cells processes may be disrupted, and in major scale, may cause energy deficiency, probably related to the clinical manifestations presented by LCHADD patients. Tonin et al. found no alteration on nitrite and nitrate production induced in vitro by LCHAD metabolites 3-hydroxydodecanoic, 3hydroxytetradecanoic and 3-hydroxypalmitic acids in rat brain, contrary to our results [17]. The different results between our work and the data of this study, could be attributed possibly to the limitation of the in vitro model on the ideal simulation of the complex in vivo system, observed in this analysis, or even to the concentration of the 3-OH fatty acids (10, 25, 50 and 100 µM) utilized in the study.
26 27 28 29 30 31 32 33 34
Although it was not found any alteration in nitrite and nitrate content in MADD and MCADD patients when compared to the controls, we can observe a tendency to increased levels of this parameter. Perhaps in a future study with a greater number of patients it would be possible to detect a significant statistical difference. Besides, it is important to emphazise that different concentrations of accumulated metabolites in patients may have different impacts on oxidative damage, as evidenced by previous studies [17, 16], where it was found that different concentrations of accumulated metabolites in FAOD induced different alterations in the oxidative stress parameters in the tissues of the studied animals.
35 36 37 38 39 40 41 42 43 44
DNA and RNA are important target biomolecules that can be damaged by elevated concentrations of ROS, leading to a variety of lesions such as DNA strand breaks, abasic sites, and several species of oxidized purine/pyrimidine and DNAprotein cross-links. Oxidative DNA damage can be considered the most severe damage to the biomolecules. These lesions can lead to genetic mutations, alterations in gene expression, chromosomal aberrations, loss of function and ultimately cell death. Studies have demonstrated an increase in oxidized guanine species in urine samples of L-2-hydroxyglutaric aciduria and glutaric acidemia type I patients, indicating the harmful potential of the toxic metabolites accumulated, L-2hydroxyglutaric and glutaric acids, to DNA and RNA of patients [11, 33].
13
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
The hydroxyl radical, the most harmful ROS, as well as others free radicals, can attack nuclear and mitochondrial DNA and generate the oxidized forms of 8hydroxy-2’-deoxyguanosine (8-OH-dG), 8-hydroxyguanosine and 8-hydroxyguanine, oxidized guanine species. After ROS attacks on RNA and both nuclear and mitochondrial DNA, the adducts formed are excised and eliminated in blood and urine [32]. In this context, we detected a significant increase of oxidized guanine species in urine of LCHADD and MADD patients, which indicates DNA and RNA oxidative damage. The existence of this type of damage, probably secondary to the accumulation of toxic fatty acids (besides other causes), might be related to metabolic dysfunction and clinical presentation of LCHADD and MADD patients. This data, in association with lipid oxidative damage, indicate a possible reason for the modifications on energy homeostasis in patients affected by these disorders. MCADD patients from our study did not present significant statistical differences in urinary oxidized guanine species levels compared to the control group. However, a tendency toward to increased levels can be observed and future studies with more MCADD patients could clarify this issue.
17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
The alkaline comet assay, a simple and sensitive technique capable of measure single and double DNA strand breaks, was performed in order to investigate the in vitro effect of the main metabolites associated with MCADD (adipic acid, suberic acid, hexanoylglycine and suberylglycine) upon DNA damage as well as to study the effect of L-car. It was verified that adipic and suberic acids induced significantly elevated levels of DNA damage, with a DI (mean ± SEM) of 15 ± 0.5 and 12 ± 0.5 arbitrary units, respectively. These metabolites induced the presence of only slightly damaged cells (class 1, the lowest level of DNA injury). Co-incubation with Lcar at the concentration of 60 µM significantly decreased DI induced by adipic acid but not by suberic acid. Interestingly, hexanoylglycine and suberylglycine induced the presence of higher damaged cells (class 2 and 3) and significantly increased DI (37±1.15 and 46±1.91 arbitrary units, mean ± SEM, respectively), indicating greater DNA migration, and co-incubation with L-car (60 µM) also reduced significantly the DI caused by both metabolites although not reaching the control levels. The in vitro effect on DNA damage of the four metabolites in combination was the highest (51 ± 0.1 arbitrary units, mean ± SEM), representing the greatest DNA migration and demonstrating a potentialized effect of the metabolites. In fact, during fasting or other situations of metabolic decompensation in MCADD, high amounts of all these toxic metabolites can be detected in patients’ samples, as well as their derivative carnitines. Thus, DNA damage induced by the accumulation of toxic metabolites might be enhanced in patients during those events [24]. Co-incubation with L-car (60 µM) also prevented DNA damage induced by adipic acid, suberic acid, hexanoylglycine and suberylglycine in combination, but not to the control levels for the tested concentrations. If not repaired, the DNA damage caused by direct DNA mechanisms or by activation of Ca2+-endonucleases and by interfering with enzymes of DNA replication and repair, may lead to serious consequences in the genome, like loss of heterozygosity, microsatellite instability, mutations or neoplastic growth [32]. Patients affected by MCADD are probably exposed to this type of damage since their concentrations of toxic metabolites are higher than healthy individuals. Nevertheless, an in vivo evaluation in blood samples of MCADD patients during episodes of 14
1 2
metabolic decompensation by comet assay would verify the scale of DNA damage, not evidenced statistically by the oxidized guanine species test.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
The treatment of several IMDs include L-car supplementation due to a secondary deficiency caused sometimes by the restricted diet and recent studies have demonstrated antioxidant properties of the molecule [11,12]. L-car prescription is recommended for MCADD patients exhibiting a deficiency in plasma carnitine, as well as the avoidance of prolonged fasting, according to the french consensus for neonatal screening, diagnosis and management [34]. In our study, L-car decreased in vitro the DNA damage induced by adipic acid, hexanoylglycine and suberylglycine, isolated and in combination. The protective effects of L-car are mainly attributed to its ability to scavenge ROS and to chelate transition metal ions [8]. Furthermore, L-car is capable to reduce oxidative damage to DNA caused by alkylating agents and free radicals, accelerating the disappearance of single-strand breaks and the repair action of poly (ADP-ribose) polymerase or other related mechanisms [35]. Another mechanism of L-car protection could be related to its conjugation to the toxic metabolites in IMD forming their carnitine derivatives [36]. Many studies have shown the oxidative stress contribution to the pathogenesis of IMD and the beneficial effects of compounds as L-car that presented antioxidants effects in vitro and in vivo. Besides, its supplementation is considered safe and with a low risk of adverse effects [37]. These data plus the findings of our work corroborate with the evidence of the antioxidant role of L-carnitine and thus it would be an encouraging complementary strategy for the treatment of FAOD patients in order to prevent oxidative damages.
23 24
5. Conclusion
25 26 27 28 29 30 31 32 33 34
In conclusion, our data indicate that LCHADD, MADD and MCADD patients presented lipoperoxidation, LCHADD patients presented nitrosative stress and LCHADD and MADD patients presented DNA and RNA oxidative damage, which is probably related to the pathophysiology of these disorders. However, more studies are necessary to understand the mechanisms by which the damage occurs. In addition, our present study demonstrates that accumulated MCADD toxic metabolites (adipic acid, suberic acid, hexanoylglycine and suberylglycine) induce, isolatedly and in combination, DNA damage, presenting a potentialized effect. L-car was capable to preserve DNA of this damage, evidencing the protective properties on DNA injury and its potential as an adjuvant therapy for FAOD.
35 36
6. Acknowledgments
37 38
The authors are also grateful for the collaboration of patients and their families and the SGM/HCPA staff.
39 40
Funding: this study was supported by grants from CAPES, CNPq and FIPE/HCPA-Brazil.
41 42 43
7. Conflict of Interest The authors declare that they have no conflict of interest in this work. 15
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Highlights:
•
Urine from LCHADD, MCADD and MADD patients presented lipid damage;
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LCHADD patients presented nitrosative stress;
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LCHADD and MADD patients presented oxidative DNA and RNA damage;
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MCADD metabolites, separated and in combination, induced in vitro DNA damage;
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Co-incubation with L-carnitine reduced the DNA damage induced by MCADD metabolites.