CC BY
11
https://doi.org/10.26641/2307-0404.2022.L254312
THE LINKAGE BETWEEN MERCURY-CAUSED NEURO- AND GENOTOXICITY VIA THE INHIBITION OF DNA REPAIR MACHINERY: FISH BRAIN MODEL
Oles Honchar Dnipro National University 1 Research Institute of Biology Gagarin av., 72, Dnipro, 49010, Ukraine e-mail: nedzvetskyvictor@ukr.net Dnipro State Agrarian and Economic University 2 S. Yefremov str., 25, Dnipro, 49600, Ukraine e-mail: zoolog@ukr.net University of Nottingham 3
University Park, Nottingham, NG7 2RD, United Kingdom e-mail: Benedikt.Herrmann@ec.europa.eu Нацюнальний yHieepcumem iMeHi Олеся Гончара 1 Науково-до^дний iHcmumym бюлогИ пр. Гагарта, 72, Днтро, 49010, Украна
Днтровський державний аграрний та економiчний умверситет 2 вул. С. Ефремова, 25, Днiпро, 49600, Украша Умверситет Ноттшгема 3
Унiверситетський парк, Ноттiнгем, NG7 2RD, Великобританiя
Цитування: Медичт перспективы. 2022. Т. 27, № 1. С. 4-9 Cited: Medicniperspektivi. 2022;27(1):4-9
UDC 577.25:615.916
V.S. Nedzvetsky12 *, V.Y. Gasso \ B. Herrmann , R.O. Novitskiy 2
Key words: heavy metals, toxicity, oxidative stress, apurinic/apyrimidinic endonuclease APE1, base excision repair BER
Ключовi слова: важю метали, токсичнiсть, окиснювальний стрес, апуринова/апiримiдинова ендонуклеаза APE1, ексцизтна репарацiя основ BER
Ключевые слова: тяжелые металлы, токсичность, окислительный стресс, апуриновая/апиримидиновая эндонуклеаза APE1, эксцизионная репарация оснований BER
Abstract. The linkage between mercury-caused neuro- and genotoxicity via the inhibition of DNA repair machinery: fish brain model. Nedzvetsky V.S., Gasso V.Y., Herrmann B., Novitsky R.O. Heavy metals in model conditions as well as industrial pollution launch disturbances in neural cells of different animals and human beings. The neurotoxicity of mercury, which is one of the most toxic heavy metals, has been studied for several decades. However, its low doses chronic exposure effects for neural tissue cells are still poorly understood. Therefore, the basic molecular mechanisms of mercury should be clarified. The purpose of our research is to clarify the mechanism of mercury genotoxicity, the role of the DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1) in neural tissue cells, and the response to inorganic mercury-induced neurotoxicity. In our model, we used juvenile rainbow trout exposed to mercury chloride with a range of doses 9-36 jug/L for 60 days to study the cytotoxicity of chronic exposure. We detected the reactive oxygen species (ROS) production as an index of oxidative stress and APE1 as a marker of cellular DNA damage response in a neural cell. The ROS level was measured by using the fluorometric method based on 2',7'-dichlorofluorescein diacetate reaction. The analyses of markers of the DNA repair (APE1) and apoptosis (B cell lymphoma-2 anti-apoptotic protein - Bcl-2) were carried out with western blotting. The mercury chloride chronic exposure induced statistically significant upregulation of the ROS production in the fish brain. Contrary, the mercury low doses stimulated the downregulation of APE1 expression in the brain tissue. Furthermore, mercury chronic exposure inhibited the expression of Bcl-2 in the animals treated with 18 and 36 jg/L mercury chloride. The harmful effect of mercury could be promoted by oxidative stress generation. The downregulation of APE1 expression could lead to a lack of DNA damage response efficacy and initiate the decline in neural cell functioning. Obtained data on the APE1 expression have shown that the neurotoxic effect of mercury could be mediated, at least partially, by the decline in cellular DNA damage response in the brain. The evaluation of decrease in DNA repair response via detection of the APE1 expression can be a prospective tool to reveal the deleterious effects of toxicants in terms of their neuro- and genotoxicity.
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Mercury is confirmed as one of the most toxic environmental contaminants for all living organisms including humans. Global industrialization causes mercury pollution [10] and generates dangerous risk factor for both human and animal health [11] due to high mercury potential bioaccumulation in living organisms [3, 14]. Heavy metals entail disturbances in neural cells of different animals [4, 7]. The neurotoxicity of mercury is known [1, 15] and the fish brain is confirmed as a convenient model to study neurotoxicity mechanisms. There was demonstrated a direct interaction of inorganic mercury with the cells involved in the blood-brain barrier (BBB) functions that induces the disturbance in the BBB integrity [17]. The delayed effect of inorganic mercury in the fish model of low doses mercury exposure was recently approved [14].
The intracellular effect of mercury is multifac-torial due to many molecular targets. One of the unspecific consequences of mercury cytotoxicity is a generation of redox imbalance, which can induce lesions of all macromolecules including DNA. The genotoxic effect of mercury is known [3]. However, the effect of mercury exposure on DNA repairing machinery remains uncovered.
One of the most often occurred DNA lesions is single-strand breaks (SSB). Exposure to environmental toxicants can entail a meaningful increase in SSB of DNA [11]. The enzymatic system of the SSB restoring called the base excision repair (BER) is evolutionary conservative and stable in eukaryotic cells. The key enzyme of BER is mammalian apu-rinic/apyrimidinic endonuclease 1 (APE1). Both
upregulation and downregulation of this enzyme are associated with abnormalities in the cell functioning and viability. Taking into account that DNA repair machinery is involved in cellular response initiated by cytotoxic injury, the modulation of mechanisms of DNA lesions restore could reflect the vital adaptive potential to maintain genomic stability and cell viability. The fish brain is generally applicable to study detrimental effects of environmental contaminants including mercury. Similar models can clarify some molecular mechanisms of tissue-specific mercury cytotoxicity in mammals including humans [9].
The purpose of our research is to clarify the mechanism of mercury genotoxicity, the role of APE1 in neural tissue, and the response to inorganic mercury-induced neurotoxicity.
MATERIALS AND METHODS OF RESEARCH
Rainbow trouts (Oncorhynchus mykiss (Walbaum, 1792)) were divided into 4 groups (7 fish each) and exposed to low doses of 9, 18, and 36 ^g/L HgCl2 for 60 days. After 60 days treatment, the fish were sacrificed according to the procedures of the Oles Honchar Dnipro National University Bioethics Committee rules and to the principles outlined in the Helsinki declaration. The reactive oxygen species (ROS) level was measured by using the fluorometric method based on 2',7'-dichlo-rofluorescein diacetate (DCFHDA) reaction [5]. The analysis of markers of the DNA repair (APE1) and apoptosis (B cell lymphoma-2 anti-apoptotic protein - Bcl-2) was carried out with western blotting [13]. We use ANOVA and Duncan multiple comparison test and employ the IBM SPSS Statistics 28.0.1.0,
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License Subscription 15-Dec-2021 [8]. Results are shown as a mean ± standard error (SE) of seven independent experiments. Comparisons were considered statistically significant in the case of p<0.05. RESULTS AND DISCUSSION We measured the relative ROS content in the brain samples of fish from the control and exposed groups to mercury chloride. A concentration-dependent increase in ROS content was observed in all exposed to mercury fish groups in comparison with the untreated control (Fig. 1).
In order to investigate the possible genotoxic effect of inorganic mercury on the fish brain,
relative APE1 content was measured with western blot in the brain extracts. The decline in APE1 expression was detected in groups exposed to 18 and 36 ^g/L mercury chloride doses. The detection of anti-apoptotic protein Bcl-2 as a marker of programmed cell death has shown that exposure to inorganic mercury is potent to inhibit this apoptosis suppressor. The most representable data on APE1 and Bcl-2 expression in control and exposed to mercury chloride fish groups measured with western blot analysis presented in figure 2.
C - control group * - p < 0.05 as compared with the control group
Fig. 1. The effects of mercury chloride on the ROS level, APE1 and Bcl-2 expressions
in the brain of rainbow trout
Taking together the results obtained in our study, the dose-dependent increase of ROS and the decline the cytotoxicity of inorganic mercury is accompanied in the content of APE1 and Bcl-2 in brain tissue.
In spite of number of reports, molecular mechanisms of mercury neurotoxicity remain undiscovered. Mercury is involved in the initiation of BBB disruption and neural tissue cell abnormalities [2]. Since inorganic mercury neurotoxicity remains poorly understood, we have tested the chronic effect
of its low doses in the brain cells. Oxidative stress is confirmed to be a widespread cause of DNA damage, irreversible injury, and programmed cell death [12]. Mercury-induced mitochondrial dysfunction accompanied by the DNA oxidizing can switch cell fate to death through genome instability [2].
Fig. 2. The western blot results of APE1 and Bcl-2 in the brain of control (C) and exposed to mercury chloride fish groups
Observed in our study dose-dependent ROS production in the brain is agreed with literature data on the effect of sublethal mercury doses [14]. Besides, ROS upregulation is confirmed as the initiator of DNA breaks. In the presented study, we have investigated the APE1 as a marker of DNA repair in the brain to elucidate the possible mechanisms of inorganic mercury genotoxicity. The activity of APE1 mainly is associated with DNA repair pathways. Moreover, the APE1 is a key player in the BER pathway and provides the vital function of genome stability in the various cells including neural tissue.
Detected ROS production in the brain may induce DNA breaking. Observed in our study decline in APE1 content reflects the inhibition of BER. BER is an evolutionary conservative pathway directed to restore the SSB caused by different factors The SSB of DNA is one of the common DNA damages, which can block the transcription processes [6].
The defects in DNA repairing can directly initiate a decline in cell viability and a lack of neuronal functioning. Thus, the detection of DNA repair enzymes in the brain cells exposed to toxic agents is a prospective way to evaluate the both neurotoxic and genotoxic effects of environmental contaminants
including mercury. Total coordination of cellular response initiated by DNA lesions launches the DNA damage response and the BER pathway is the largest part of the total DNA damage response machinery.
Taking into account obtained results, APE1 could be considered as one of the important target for mercury genotoxicity. Furthermore, the decline in APE1 expression could be proposed as prospective bio-marker to evaluate mercury-induced genotoxicity.
Despite the known genotoxicity of mercury, its link with neurotoxicity is poorly understood [16]. The suppression of APE1 in neural cells can inhibit DNA repair response, increase genomic instability and trigger the cells to apoptosis. Thus, similar irreversible abnormalities disturb brain cell functions as well as their viability. Moreover, mercury-induced oxidative stress can directly initiate programmed cell death via the initiation of mitochondria-dependent regulators of apoptosis. The members of Bcl-2 family are mitochondria-associated pro-apoptotic and anti-apoptotic proteins. All of them are susceptible to intracellular signaling, which regulates cell viability as well as can trigger the damaged cells to the programmed cell death.
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As observed in our study, the decrease in anti-apoptotic protein Bcl-2 is evident that mercury exposure initiates apoptotic changes. Therefore, mercury is potent to induce programmed cell death in neural tissue cells via two independent effects: genome instability and mitochondria-associated apoptosis. Actually, the found increase in APE1 expression may be a convenient biomarker of mercury neurotoxicity.
CONCLUSIONS
1. Exposure to low doses of inorganic mercury induces oxidative stress, inhibits DNA repair response, and activates mitochondria-dependent apop-tosis in neural cells.
2. The evaluation of decrease in DNA repair response via detection of the apurinic/apyrimidinic endonuclease 1 expression can be a prospective tool to reveal the deleterious effects of environmental contaminants as well as their genotoxicity.
Contributors:
Nedzvetsky V.S. - conceptualization, investigation, writing - original draft, and supervision;
Gasso V.Y. - formal analysis, visualization, writing - review & editing;
Herrmann B. - methodology, resources;
Novitsky R.O. - investigation, validation.
Funding. The presented research was carried out in the frames of the projects 0119U100718 and 0120U102381 financially supported by the Ministry of Education and Science of Ukraine.
Conflict of interests. The authors declare no conflict of interest. Conflict of interests. The authors declare no conflict of interest.
Acknowledgements. The authors are grateful to the Ministry of Education and Science of Ukraine for the financial support of our research.
REFERENCES
1. Cariccio VL, Sama A, Bramanti P, Mazzon E. Mercury involvement in neuronal damage and in neurodegenerative diseases. Biol Trace Elem Res. 2019;187(2):341-56.
doi: http://dx.doi.org/10.1007/s12011-018-1380-4
2. Carocci A, Rovito N, Sinicropi MS, Genchi G. Mercury toxicity and neurodegenerative effects. Rev Environ Contam Toxicol. 2014;229:1-18. doi: https://doi.org/10.1007/978-3-319-03777-6_1
3. Chang Y, Lee W-Y, Yu-Jie Lin, Todd Hsu. Mercury (II) impairs nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos by targeting primarily at the stage of DNA incision. Aquat Toxicol. 2017;192:97-104. doi: https://doi.org/10.1016/j.aquatox.2017.09.001
4. Gasso VY, Hahut AN, Yermolenko SV, Has-so IA, Agca CA, Sukharenko EV, et al. Local industrial pollution induces astrocyte cytoskeleton rearrangement in the dice snake brain: GFAP as a biomarker. Biosyst Divers. 2020;28(3):250-6.
doi: https://doi.org/10.15421/012033
5. Nedzvetsky VS, Masiuk DM, Gasso VY, Yermolenko SV, Huslystyi AO, Spirina VA. Low doses of imidacloprid induce disruption of intercellular adhesion and initiate proinflammatory changes in Caco-2 cells. Regul. Mech. Biosyst. 2021;12(3):430-7.
doi: https://doi.org/10.15421/022159
6. Zhang D, O'Donnell M. The eukaryotic replication machine. Enzymes. 2016;39:191-229.
doi: https://doi.org/10.1016/bs.enz.2016.03.004
7. Kirici M, Nedzvetsky VS, Agca CA, Gasso VY. Sublethal doses of copper sulphate initiate deregulation of glial cytoskeleton, NF-kappa B and PARP expression in Capoeta umbla brain tissue. Regul Mech Biosyst. 2019;10(1):103-10. doi: https://doi.org/10.15421/021916
8. Landau S, Everitt BS. A handbook of statistical analyses using SPSS. Boca Raton: Chapman &
Hall/CRC Press LLC; 2004. p. 338. Available from: http://www.academia.dk/BiologiskAntropologi/Epidemiol ogi/PDF/SPSS_Statistical_Analyses_using_SPSS.pdf
9. Sukharenko EV, Samoylova IV, Nedzvetsky VS. Molecular mechanisms of aluminium ions neurotoxicity in brain cells of fish from various pelagic areas. Regul. Mech. Biosyst. 2017;8(3):461-6.
doi: https://doi.org/10.15421/021771
10. Meena RAA, Sathishkumar P, Ameen F, Yusoff ARM, Gu FL. Heavy metal pollution in immobile and mobile components of lentic ecosystems - a review. Environ Sci Pollut Res Int. 2017;25(5):4134-48. doi: https://doi.org/10.1007/s11356-017-0966-2
11. Meland S, Gomes T, Petersen K, Hall J, Lund E, Kringstad A, et al. Road related pollutants induced DNA damage in dragonfly nymphs (Odonata, Anisoptera) living in highway sedimentation ponds. Sci Rep. 2019;9:16002. doi: https://doi.org/10.1038/s41598-019-52207-4
12. Naija A, Kestemont P, Chenais B, Haouas Z, Blust R, Helal AN, et al. Effects of Hg sublethal exposure in the brain of peacock blennies Salaria pavo: Molecular, physiological and histopathological analysis. Chemosphere. 2017;193:1094-104.
doi: https://doi.org/10.1016/j.chemosphere.2017.11.118
13. Nedzvetsky VS, Agca CA, Baydas G. The pepti-doglycan fraction enriched with muramyl pentapeptide from Lactobacillus bulgaricus inhibits glioblastoma U373MG cell migration capability and upregulates PARP1 and NF-kB levels. Biotechnol Acta. 2020;13(2):65-79.
doi: https://doi.org/10.15407/biotech13.02.065
14. Pereira P, Raimundo J, Barata M, Araujo O, Pousäo-Ferreira P, Canario J, et al. A new page on the road book of inorganic mercury in fish body - tissue distribution and elimination following waterborne exposure and post-exposure periods. Metallomics. 2015;7(3):525-35.
doi: https://doi.org/10.1039/c4mt00291a
15. Pietz J, Sánchez-Bayo F, Tennekes HA. Dose-response analysis indicating time-dependent neurotoxicity caused by organic and inorganic mercury-Implications for toxic effects in the developing brain. Toxicology. 2016;347-349:1-5.
doi: https://doi.org/10.1016/j.tox.2016.02.006
16. Wyatt LH, Luz AL, Cao X, Maurer LL, Bla-was AM, Aballay A, et al. Effects of methyl and inor-
ganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans. DNA Repair (Amst). 2017;52:31-48. doi: https://doi.org/10.1016/j.dnarep.2017.02.005
17. Virgolini MB, Aschner M. Molecular mechanisms of lead neurotoxicity. Adv Neurotoxicol. 2021;5:159-213.
doi: https://doi.org/10.1016/bs.ant.2020.11.002
Crana Haginmga go pegaKmi' 16.09.2021
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