Skip to main content

Differential methylation pattern of xenobiotic metabolizing genes and susceptibility to Balkan endemic nephropathy, in a cohort of Romanian patients


A severe, chronic and irreversible kidney disease affecting discrete rural populations in the Balkan Peninsula countries, Balkan endemic nephropathy (BEN) has been a scientific puzzle for more than half a century. Many environmental and other factors have been suggested as the primary cause and recent significant findings have linked BEN to aristolochic acids, phytotoxins derived from the plant Aristolochia clematitis, found in high density in the endemic areas. However, given that the incidence of BEN is less than 10% in affected villages, and it tends to have a family aggregation, as yet unidentified genetic factors may also play a role. To further explore this possibility, a pilot study was initiated to investigate the DNA methylation of CYP1A1, CYP1A2, NAT1, NQO1 and GSTT1 in blood samples from a group of Romanian BEN patients, compared to healthy controls and non-BEN chronic kidney disease (CKD) subjects. Our study revealed a more pronounced hypomethylation pattern in BEN and non-BEN CKD groups, compared to the healthy control group at specific CpGs across all five genes interrogated. Average methylation across the five regions investigated indicated significant differences only at GSTT1, in both BEN patients (p = 0.028) and non-BEN disease subjects (p = 0.015), relative to healthy individuals. Since GSTT1 active genotype appears to be a common feature of Serbian and Romanian BEN patients, GSTT1 epigenetic variation and increased gene activity could act as a predisposing (co)factor in BEN populations from the affected countries. BEN and non-BEN CKD groups show similar methylation patterns with exception of GSTT1 CpG8 (p = 0.046).

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Pavlović NM (2013) Balkan endemic nephropathy—current status and future perspectives. Clin Kidney J 6(3):257–265.

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Andonova IE, Sarueva RB, Horvath AD, Simenov VA, Dimitrov PS, Petropoulos EA et al (2004) Balkan endemic nephropathy and genetic variants of glutathione S transferases. J Nephrol 17(3):390–398

    CAS  PubMed  Google Scholar 

  3. 3.

    Staneva R, Rukova B, Hadjidekova S, Nesheva D, Antonova O, Dimitrov P et al (2013) Whole genome methylation array analysis reveals new aspects in Balkan endemic nephropathy etiology. BMC Nephrol 14:225.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Gruia AT, Oprean C, Ivan A, Cean A, Cristea M, Draghia L et al (2018) Balkan endemic nephropathy and aristolochic acid I: an investigation into the role of soil and soil organic matter contamination, as a potential natural exposure pathway. Environ Geochem Health 40:1437–1448.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Karimi R (2015) Biomedical & biopharmaceutical sciences with patient care correlations. Jhons Bartlett Learning, Burlington

    Google Scholar 

  6. 6.

    Knights KP (2013) Renal drug metabolism in humans: the potential for drug–endobiotic interactions involving cytochrome P450 (CYP) and UDPglucuronosyltransferase (UGT). Brit J Clin Pharmacol 76(4):587–602.

    CAS  Article  Google Scholar 

  7. 7.

    Chen B, Bai Y, Sun M, Ni X, Yang Y et al (2012) Glutathione S-transferases T1 null genotype is associated with susceptibility to aristolochic acid nephropathy. Int Urol Nephrol 44:301–307.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Grant DM (1991) Detoxification pathways in the liver. J Inherit Metab Dis 14(4):421–430

    CAS  Article  Google Scholar 

  9. 9.

    Gu X, Manautou J (2012) Molecular mechanisms underlying chemical liver injury. Expert Rev Mol Med 14:E4.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Toncheva DI, Von Ahsen N, Atanasova SY, Dimitrov TG, Armstrong VW, Oellerich M (2004) Identification of NQO1 and GSTs genotype frequencies in Bulgarian patients with Balkan endemic nephropathy. J Nephrol 7(3):384–389

    Google Scholar 

  11. 11.

    Ghotbi R, Gomez A, Milani L, Tybring G, Syvänen AC, Bertilsson L et al (2009) Allele-specific expression and gene methylation in the control of CYP1A2 mRNA level in human livers. Pharmacogenom J 9:208–217

    CAS  Article  Google Scholar 

  12. 12.

    Coolen MW, Statham AL, Gardiner-Garden M, Clark SJ (2007) Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: critical evaluation and improvements. Nucleic Acids Res 35:e119.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Martino D, Loke YJ, Gordon L, Ollikainen M, Cruickshank MN, Saffery R et al (2013) Longitudinal, genome-scale analysis of DNA methylation in twins from birth to 18 months of age reveals rapid epigenetic change in early life and pair-specific effects of discordance. Genome Biol 14:R42.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Chan W, Luo HB, Zheng Y, Cheng YK, Cai Z (2007) Investigation of the metabolism and reproductive activation of carcinogenic aristolochic acids in rats. Drug Metab Dispos 35(6):866–874.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Stiborová M, Arlt VM, Schmeiser HH (2017) DNA adducts formed by aristolochic acid are unique biomarkers of exposure and explain the initiation phase of upper urothelial cancer. Int J Mol Sci 18(10):2144.

    CAS  Article  PubMed Central  Google Scholar 

  16. 16.

    Atanasova S, von Ahsen N, Dimitrov T, Oellerich M, Toncheva D (2004) First study of NATl and NAT2 polymorphisms in bulgarian patients with Balkan endemic nephropathy (BEN) and healthy controls. Biotechnol Biotechnol Equip 18(1):95–103.

    CAS  Article  Google Scholar 

  17. 17.

    Arlt VM, Ferluga D, Stiborova M, Pfohl-Leszkowicz A, Vukelic M, Vdovic S et al (2002) Is aristolochic acid a risk factor for Balkan endemic nephropathy-associated urothelial cancer? Int J Cancer 101:500–502.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Stiborová M, Arlt VM, Henderson CJ, Wolf CR, Kotrbová V, Moserová M et al (2008) Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: studies with the hepatic NADPH:cytochrome P450 reductase null mouse. Toxicol Appl Pharmacol 226:318–327.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Stiborová M, Arlt VM, Schmeiser HH (2016) Balkan endemic nephropathy: an update on its aetiology. Arch Toxicol 90(11):2595–2615.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Stiborova M, Frei E, Sopko B, Wiessler M, Schmeiser HH (2002) Carcinogenic aristolochic acids upon activation by DT-diaphorase form adducts found in DNA of patients with Chinese herbs nephropathy. Carcinogenesis 23:617–625.

    Article  PubMed  Google Scholar 

  21. 21.

    Stiborova M, Frei E, Sopko B, Sopkova K, Markova V, Lankova M et al (2003) Human cytosolic enzymes involved in the metabolic activation of carcinogenic aristolochic acid: evidence for reductive activation by human NAD(P)H: quinone oxidoreductase. Carcinogenesis 24:1695–1703.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Stiborová M, Mareîs J, Frei E, Volker M, Arlt VM, Heinz HS (2011) The human carcinogen aristolochic acid I is activated to form DNA adducts by human NAD(P)H:quinone oxidoreductase without the contribution of acetyltransferases or sulfotransferases. Environ Mol Mutagen 52(6):448–459.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Lee SB, Kim CY, Lee HJ, Yun JH, Nho CW (2009) Induction of the phase II detoxification enzyme NQO1 in hepatocarcinoma cells by lignans from the fruit of Schisandra chinensis through nuclear accumulation of Nrf2. Planta Med 75(12):1314–1318.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Stiborová M, Volker M, Arlt VM, Heinz H, Schmeiser HH (2017) DNA adducts formed by aristolochic acid are unique biomarkers of exposure and explain the initiation phase of upper urothelial cancer. Int J Mol Sci 18(10):2144.

    CAS  Article  PubMed Central  Google Scholar 

  25. 25.

    Matic MG, Coric VM, Savic-Radojevic AR, Bulat PV, Pljesa-Ercegovac MS et al (2017) Does occupational exposure to solvents and pesticides in association with glutathione S-transferase A1, M1, P1, and T1 polymorphisms increase the risk of bladder cancer? The Belgrade Case–Control Study. PLoS ONE 9(6):e99448.

    CAS  Article  Google Scholar 

  26. 26.

    Bachmann K (2009) Drug metabolism. In: Hacker M, Messer WS II, Bachmann KA (eds) Pharmacology principles and practice. Academic Press, Cambridge, pp 131–173

    Google Scholar 

  27. 27.

    Kellen EM, Zeegers A, Paulussen M, Van Dongen M, Buntinx F et al (2006) Fruit consumption reduces the effect of smoking on bladder cancer risk. The Belgian case control study on bladder cancer. Int J Cancer 118:2572–2578

    CAS  Article  Google Scholar 

  28. 28.

    Raimondi S, Paracchini V, Autrup H, Barros-Dios JM, Benhamou S, Boffetta P et al (2006) Meta- and pooled analysis of GSTT1 and lung cancer: a HuGE-GSEC review. Am J Epidemiol 164:1027–1042.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Saadat M (2006) Genetic polymorphisms of glutathione S-transferase T1 (GSTT1) and susceptibility to gastric cancer: a meta-analysis. Cancer Sci 97:505–509

    CAS  Article  Google Scholar 

  30. 30.

    Ye Z, Song H (2005) Gluthatione S-transferase polymorphism (GSTM1, TESTP1 and GSTT) and risk of acute leukemia: a systemic review of meta-analysis. Eur J Cancer 41:980–989.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Reljic Z, Zlatovic M, Savic-Radojevic A, Pekmezovic T, Djukanovic L, Matic M et al (2013) Is increased susceptibility to balkan endemic nephropathy in carriers of common GSTA1 (*A/*B) polymorphism linked with the catalytic role of GSTA1 in ochratoxin A biotransformation? Serbian case control study and in silico analysis. Toxins (Basel) 6(8):2348–2362.

    CAS  Article  Google Scholar 

  32. 32.

    Matic M, Dragicevic B, Pekmezovic T, Suvakov S, Savic-Radojevic A, Pljesa-Ercegovac M, Dragicevic D, Smiljic J, Simic T (2016) Common polymorphisms in GSTA1, GSTM1 and GSTT1 are associated with susceptibility to urinary bladder cancer in individuals from Balkan endemic nephropathy areas of Serbia, Tohoku. J Exp Med 240:25–30

    CAS  Google Scholar 

  33. 33.

    Amenya HZ, Chiharu T, Seiichiroh O (2016) Dioxin induces Ahr-dependent robust DNA demethylation of the Cyp1a1 promoter via Tdg in the mouse liver. Sci Rep 66:34989.

    CAS  Article  Google Scholar 

  34. 34.

    Dietrich C (2016) Antioxidant functions of the aryl hydrocarbon receptor. Stem Cell Int 16:1–10.

    CAS  Article  Google Scholar 

  35. 35.

    Sunaga N, Kohno T, Yanagitani N, Sugimura H, Kunitoh H, Tamura T et al (2002) Contribution of the NQO1 and GSTT1 polymorphisms to lung adenocarcinoma susceptibility. Cancer Epidemiol Biomark Prev 11(8):730–738

    CAS  Google Scholar 

  36. 36.

    Taningher M, Malacarne D, Izzotti A, Ugolini D, Parodi S (1999) Drug metabolism polymorphisms as modulators of cancer susceptibility. Mutat Res 436:227–261.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Bartsch H, Nair U, Risch A, Rojas M, Wikman H, Alexandrov K (2000) Genetic polymorphism of CYP genes, alone or in combination, as a risk modifier of tobacco-related cancers. Cancer Epidemiol Biomark Prev 9:3–28

    CAS  Google Scholar 

  38. 38.

    Kihara M, Noda K (1994) Lung cancer risk of GSTM1 null genotype is dependent on the extent of tobacco smoke exposure. Carcinogenesis (Lond) 15:415–418

    CAS  Article  Google Scholar 

  39. 39.

    Rebbeck TR (1997) Molecular epidemiology of the human glutathione S-transferase genotypes GSTM1 and GSTT1 in cancer susceptibility. Cancer Epidemiol Biomark Prev 6:733–743

    CAS  Google Scholar 

  40. 40.

    Hayashi S, Watanabe J, Kawajiri K (1992) High susceptibility to lung cancer analyzed in terms of combined genotypes of P450IA1 and Mu-class glutathione S-transferase genes. Jpn J Cancer Res 83:866–870

    CAS  Article  Google Scholar 

  41. 41.

    Le Marchand L, Sivaraman L, Pierce L, Seifried A, Lum A, Wilkens LR et al (1998) Associations of CYP1A1, GSTM1, and CYP2E1 polymorphisms with lung cancer suggest cell type specificities to tobacco carcinogens. Cancer Res 58:4858–4863

    PubMed  Google Scholar 

Download references


This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS-UEFISCDI, Project Number II-RU-TE-2014-4-0151.

Author information




Designed the experiments: AI, CAT, RS, VP, performed the experiments: AI, DL, MIC, AT, ATG, CO, FM, FMB, CAT, analyzed the data: AI, DL, RS, CAT, contributed reagents/materials/analysis tools: AI, DL, RS, VP, CAT, wrote the paper: AI, DL, RS, CAT. All the authors have read and approved the final, submitted version of manuscript.

Corresponding author

Correspondence to Alexandra Ivan.

Ethics declarations

Conflict of interest

Alexandra Ivan, Dilys Lam, Mirabela Iustina Cristea, Ada Telea, Alexandra Teodora Gruia, Camelia Oprean, Florin Margineanu, Florina Maria Bojin, Richard Saffery, Virgil Paunescu, and Calin Adrian Tatu declare that they have no competing interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethical Committee for scientific research approval of the County Hospital “Pius Branzeu’ Timisoara, Romania and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Patient consent for publication

All participants gave their written informed consent for sample collection and publication of the data included in this case report study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 1300 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ivan, A., Lam, D., Cristea, M.I. et al. Differential methylation pattern of xenobiotic metabolizing genes and susceptibility to Balkan endemic nephropathy, in a cohort of Romanian patients. J Nephrol 33, 91–100 (2020).

Download citation


  • Balkan endemic nephropathy
  • Aristolochic acid
  • Gene methylation
  • Epigenetics
  • Xenobiotic metabolism
  • GSTT1