Protective effects of phenolic acids on mercury-induced DNA damage in precision-cut kidney slices

Document Type: Original Article


1 Departamento de Biología Celular y Ultraestructura, Centro de Investigación Biomédica, Facultad de Medicina, Universidad Autónoma de Coahuila. Torreón, Coah. México

2 Departamento de Biología Celular y Molecular, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, NL. México

3 Departamento de Química Analítica, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, NL. México

4 División de Investigación, Unidad Médica de Alta Especialidad # 34, Instituto Mexicano del Seguro Social, Monterrey, NL. México.

5 Laboratorio de Ingeniería Genética y Genómica, Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, NL. México


Objective(s): Precision-cut tissue slices are considered an organotypic 3D model widely used in biomedical research. The comet assay is an important screening test for early genotoxicity risk assessment that is mainly applied on in vitro models.  The aim of the present study was to provide a 3D organ system for determination of genotoxicity using a modified method of the comet assay since the stromal components from the original tissue make this technique complicated.
Materials and Methods: A modified comet assay technique was validated using precision-cut hamster kidney slices to analyze the antigenotoxic effect of the phenolic compounds caffeic acid, chlorogenic acid, and rosmarinic acid in tissue slices incubated with 15 µM HgCl2. Cytotoxicity of the phenolic compounds was studied in Vero cells, and by morphologic analysis in tissue slices co-incubated with HgCl2 and phenolic compounds.
Results: A modification of the comet assay allows obtaining better and clear comet profiles for analysis. Non-cytotoxic concentrations of phenolic acids protected kidney tissue slices against mercury-induced DNA damage, and at the same time, were not nephrotoxic. The highest protection was provided by 3 µg/ml caffeic acid, although 6 µg/ml rosmarinic and 9 µg/ml chlorogenic acids also exhibited protective effects.
Conclusion: This is the first time that a modification of the comet assay technique is reported as a tool to visualize the comets from kidney tissue slices in a clear and simple way. The phenolic compounds tested in this study provided protection against mercury-induced genotoxic damage in precision-cut kidney slices.


Main Subjects

1. Plazar J, Hreljac I, Pirih P, Filipic M, Groothuis GM. Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices. Toxicol In Vitro 2007; 21:1134-1142.
2. Graaf IA, Groothuis GM, Olinga P. Precision-cut tissue slices as a tool to predict metabolism of novel drugs. Expert Opin Drug Metab Toxicol 2007;3: 879-898.
3. Starokozhko V, Greupink R, van de Broek P, Soliman N, Ghimire S, de Graff IAM, et al. Rat precision-cut liver slices predict drug-induced cholestatic injury. Arch Toxicol 2007; 91:3403-3413.
4. De Kanter R, De Jager MH, Draaisma AL, Jurva JU, Olinga P, Meijer DK, et al. Drug-metabolizing activity of human and rat liver, lung, kidney and intestine slices. Xenobiotica 2002; 32:349-362.
5. Catania JM, Parrish AR, Kirkpatrick DS, Chitkara M, Bowden GT, Henderson CJ, et al. Precision-cut tissue slices from transgenic mice as an in vitro toxicology system. Toxicol In Vitro 2003; 17:201-205.
6. Baverel G, Knouzy B, Gauthier C, El Hage M, Ferrier B, Martin G, et al. Use of precision-cut renal cortical slices in nephrotoxicity studies. Xenobiotica 2013; 43:54-62.
7. de Graaf IA, Draaisma AL, Schoeman O, Fahy GM, Groothuis GM, Koster HJ. Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification. Cryobiology 2007; 54:1-12.
8. Poosti F, Pham BT, Oosterhuis D, Poelstra K, van Goor H, Olinga P, et al. Precision-cut kidney slices (PCKS) to study development of renal fibrosis and efficacy of drug targeting ex vivo. Dis Model Mech 2015; 8:1227-1236.
9. Liao W, McNutt MA, Zhu WG. The comet assay: a sensitive method for detecting DNA damage in individual cells. Methods 2009; 48:46-53.
10. Langie SA, Azqueta A, Collins AR. The comet assay: past, present, and future. Front Genet 2015; 6:266.
11. Plazar J, Filipic M, Groothuis GM. Antigenotoxic effect of Xanthohumol in rat liver slices. Toxicol In Vitro 2008; 22:318-327.
12. Switalla S, Knebel J, Ritter D, Dasenbrock C, Krug N, Braun A, et al. Determination of genotoxicity by the Comet assay applied to murine precision-cut lung slices. Toxicol In Vitro 2013; 27:798-803.
13. Jetten MJ, Claessen SM, Dejong CH, Lahoz A, Castell JV, van Delft JH, Kleinjjans JC. Interindividual variation in response to xenobiotic exposure established in precision-cut human liver slices. Toxicology 2014; 323:61-69.
14. Maser E, Schulz M, Sauer UG, Wiemann M, Ma-Hock L, Wohlleben W, Hartwig A, Landsiedel R. In vitro and in vivo genotoxicity investigations of differently sized amorphous SiO2 nanomaterials. Mutat Res Genet Toxicol Environ Mutagen 2015; 794:57-74.
15. Choi J, Chang JY, Hong J, Shin S, Park JS, Oh S. Low-level toxic metal exposure in healthy weaning-age infants: association with growth, dietary intake, and iron deficiency. Int J Environ Res Publ Health 2017; 14:388.
16. Li H, Li H, Li Y, Liu Y, Zhao Z. Blood mercury, arsenic, cadmium, and lead in children with autism spectrum disorder. Biol Trace Elem Res 2018; 181:31-37.
17. Wise JTF, Wang L, Zhang Z, Shi X. The 9th conference on metal toxicity and carcinogenesis: the conference overview. Toxicol Appl Pharmacol 2017; 331:1-5.
18. Woods JS, Calas CA, Aicher LD. Stimulation of porphyrinogen oxidation by mercuric ion. II. Promotion of oxidation from the interaction of mercuric ion, glutathione, and mitochondria-generated hydrogen peroxide. Mol Pharmacol 1990; 38:261-266.
19. Carranza-Rosales P, Said-Fernandez S, Sepulveda-Saavedra J, Cruz-Vega DE,  Gandolfi AJ. Morphologic and functional alterations induced by low doses of mercuric chloride in the kidney OK cell line: ultrastructural evidence for an apoptotic mechanism of damage. Toxicology 2005; 210:111-121.
20. Schurz F, Sabater-Vilar M, Fink-Gremmels J. Mutagenicity of mercury chloride and mechanisms of cellular defense: the role of metal-binding proteins. Mutagenesis 2000; 15:525-530.
21. Piccoli C, D’Aprile A, Scrima R, Ambrosi L, Zefferino R, Capitanio N. Subcytotoxic mercury chloride inhibits gap junction intercellular communication by a redox- and phosphorylation-mediated mechanism. Free Rad Bio Med 2012; 52:916-927.
22. Ramos Elizagaray SI, Soria EA. Arsenic immunotoxicity and immunomodulation by phytochemicals: potential relations to develop chemopreventive approaches. Recent Pat Inflamm Allergy Drug Discov 2014; 8:92-103.
23. Karakaya S. Bioavailability of phenolic compounds. Crit Rev Food Sci Nutr 2004; 44:453-464.
24. Caleja C, Ribeiro A, Barreiro MF, Ferreira IC. Phenolic compounds as nutraceuticals or functional food ingredients. Curr Pharm Des 2017; 23:2787-2806.
25. Peyrol J, Riva C, Amiot MJ. Hydroxytyrosol in the prevention of the metabolic syndrome and related disorders. Nutrients 2017; 9:306.
26. Viveros-Valdez E, Rivas-Morales C, Carranza-Rosales P, Mendoza S, Schmeda-Hirschmann G. Free radical scavengers from the Mexican herbal tea “poleo” (Hedeoma drummondii). Z Naturforsch C 2008; 63:341-346.
27. Viveros-Valdez E, Rivas-Morales C, Oranday-Cárdenas A, Castro-Garza J, Carranza-Rosales P. Antiproliferative effect from the mexican poleo (Hedeoma drummondii). J Med Food 2010; 13:740-742.
28. Petersen M, Simmonds MS. Rosmarinic acid. Phytochemistry 2003; 62:121-125.
29. Jiang RW, Lau KM, Hon PM, Mak TC, Woo KS, Fung KP. Chemistry and biological activities of caffeic acid derivatives from Salvia miltiorrhiza. Curr Med Chem 2005; 12:237-246.
30. Belkaid A, Curriecv JC, Desgagnés J, Annabi B. The chemopreventive properties of chlorogenic acid reveal a potential new role for the microsomal glucose-6-phosphate translocase in brain tumor progression. Canc Cell Int 2006; 6:12.
31. Hymer WC, Kuff EL. Isolation of nuclei from mammalian tissues through the use of Triton X-100. J Hstochem Cytochem 1964; 12:359-363.
32. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1998; 175:184-191.
33. Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Bioph Res Commun 1984; 123:291-298.
34. Aninat C, Piton A, Glaise D, Le Charpentier T, Langouet S, Morel F, et al. Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos 2006; 34:75-83.
35. Nelson LJ, Navarro M, Treskes P, Samuel K, Tura-Ceide O, Morley SD, et al. Acetaminophen cytotoxicity is ameliorated in a human liver organotypic co-culture model. Sci Rep 2015; 5:17455.
36. Olinga P, Schuppan D. Precision-cut liver slices: a tool to model the liver ex vivo. J Hepatol 2013; 58:1252-1253.
37. de Graaf IA, Olinga P, de Jager MH, Merema MT, de Kanter R., van de Kerkhof EG, et al. Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. Nat Protoc 2010; 5:1540-1551.
38. Olguín N, Müller ML, Rodríguez-Farré E, Suñol C. Neurotransmitter amines and antioxidant agents in neuronal protection against methylmercury-induced cytotoxicity in primary cultures of mice cortical neurons. Neurotoxicology 2018; pii:S0161-813X(18)30309-7.
39. Simić A, Manojlović D, Segan D, Todorović M. Electrochemical behavior and antioxidant and prooxidant activity of natural phenolics. Molecules 2007; 12:2327-2340.
40. Nemeikaite-Ceniene A, Imbrasaite A, Sergediene E, Cenas N. Quantitative structure-activity relationships in prooxidant cytotoxicity of polyphenols: role of potential of phenoxyl radical/phenol redox couple. Arch Biochem Biophys 2005; 441:182-190.
41. De La Cruz JP, Ruiz-Moreno MI, Guerrero A, López-Villodres JA, Reyes JJ, Espartero JL, et al. Role of the catechol group in the antioxidant and neuroprotective effects of virgin olive oil components in rat brain. J Nutr Biochem 2015; 26:549-555.
42. Andjelkovic M, Van Camp J, De Meulenaer B, Depaemelaere G, Socaciu C, Verloo M, et al. Iron-chelation properties of phenolic acids bearing catechol and galloyl groups. Food Chem 2006; 98:23–31.
43. Augusti PR, Conterato GM, Somacal S, Sobieski R, Spohr PR, Torres JV, et al. Effect of astaxanthin on kidney function impairment and oxidative stress induced by mercuric chloride in rats. Food Chem Toxicol 2008; 46:212-219.
44. Calabrese EJ, Iavicoli I, Calabrese V, Cory-Slechta DA, Giordano J. Elemental mercury neurotoxicity and clinical recovery of function: A review of findings, and implications for occupational health. Environ Res 2018; 163:134-148.
45. Ferrari CK. Functional foods, herbs and nutraceuticals: towards biochemical mechanisms of healthy aging. Biogerontology 2004; 5:275-289.
46. Abarikwu SO, Benjamin S, Ebah SG, Obilor G, Agbam G. Protective effect of Moringa oleifera oil against HgCl2-induced hepato- and nephro-toxicity in rats. J Basic Clin Physiol Pharmacol 2017; 28:337-345.
47. Joshi D, Mittal DK, Shukla S, Srivastav SK, Dixit VA. Curcuma longa Linn. extract and curcumin protect CYP 2E1 enzymatic activity against mercuric chloride-induced hepatotoxicity and oxidative stress: A protective approach. Exp Toxicol Pathol 2017; 69:373-382.
48. Halliwell B, Whiteman M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 2004; 142:231-255.
49. Sevgi K, Tepe B, Sarikurkcu C. Antioxidant and DNA damage protection potentials of selected phenolic acids. Food Chem Toxicol 2015; 77:33.
50. Tlili N, Feriani A, Saadoui E, Nasri N, Khaldi A. Capparis spinosa leaves extract: Source of bioantioxidants with nephroprotective and hepatoprotective effects. Biomed Pharmacother 2017; 87:171-179.
51. Janbaz KH, Saeed SA, Gilani AH. Studies on the protective effects of caffeic acid and quercetin on chemical-induced hepatotoxicity in rodents. Phytomedicine 2004; 11:424-430.
52. Neradil J, Veselsk R, Slanina J. UVC-protective effect of caffeic acid on normal and transformed human skin cells in vitro. Folia Biol. (Praha) 2003; 49:197-202.
53. Kang KA, Lee KH, Zhang R, Piao M, Chae S, Kim KN. Jeon, et al. Caffeic acid protects hydrogen peroxide induced cell damage in WI-38 human lung fibroblast cells. Biol Pharm Bull 2006; 29:1820-1824.
54. Abdallahh FB, Fetoui H, Fakhfakh F, Keskes L. Caffeic acid and quercetin protect erythrocytes against the oxidative stress and the genotoxic effects of lambda-cyhalothrin in vitro. Biol Pharm Bull 2011; 31:92-100.
55. Cariddi LN, Sabini MC, Escobar FM, Montironi I, Mañas F, Iglesias D, et al. Polyphenols as possible bioprotectors against cytotoxicity and DNA damage induced by ochratoxin A. Environ Toxicol Pharmacol 2015; 39:1008-1018.
56. Coelho VR, Vieira CG, de Souza LP, da Silva LL, Pflüger P, Regner GG, et al. Behavioral and genotoxic evaluation of rosmarinic and caffeic acid in acute seizure models induced by pentylenetetrazole and pilocarpine in mice. Naunyn Schmiedeberg Arch Pharmacol 2016; 389:1195-1203.
57. Migliori M, Cantaluppi V, Mannari C, Bertelli AAE, Medica D, Quercia AD, et al. Caffeic acid, a phenol found in white wine, modulates endothelial nitric oxide production and protects from oxidative stress-associated endothelial cell injury. PLOS One 2015; 10:21.
58. Huang MT, Smart RC, Wong CQ, Conney AH. Inhibitory effect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res 1998; 48:5941-5946.
59. Kapil A, Koul IB, Suri OP. Antihepatotoxic effects of chlorogenic acid from Anthocephalus cadamba. Phytother Res 1995; 9:189-193.
60. Tsuchiya T, Suzuki O, Igarashi K. Protective effects of chlorogenic acid on paraquat-induced oxidative stress in rats. Biosci Biotechnol Biochem 1996; 60:765-768.
61. Gugliucci A, Bastos DH. Chlorogenic acid protects paraoxonase 1 activity in high density lipoprotein from inactivation caused by physiological concentrations of hypochlorite. Fitoterapia 2009; 80:138-142.
62. Pavlica S, Gebhardt R. Protective effects of ellagic and chlorogenic acids against oxidative stress in PC12 cells. Free Radic Res 2005; 39:1377-1390.
63. Renzulli C, Galvano F, Pierdomenico L, Speroni E, Guerra MC. Effects of rosmarinic acid against aflatoxin B1 and ochratoxin-A-induced cell damage in a human hepatoma cell line (Hep G2). J Appl Toxicol 2004; 24:289-296.
64. Lee HJ, Cho HS, Park E, Kim S, Lee SY, Kim CS, et al. Rosmarinic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced apoptosis. Toxicology 2008; 250:109-115.
65. Furtado MA, de Almeida LC, Furtado RA, Cunha WR, Tavares DC. Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay. Mutat Res 2008; 657:150-154.
66. Rao MV, Sharma PS. Protective effect of vitamin E against mercuric chloride reproductive toxicity in male mice. Reprod Toxicol 2010; 15:705-712.
67. Rao MV, Chinoy NJ, Suthar MB, Rajvanshi MI. Role of ascorbic acid on mercuric chloride-induced genotoxicity in human blood cultures. Toxicol In Vitro 2001; 15:649-654.