title: Hypermethylation of estrogen receptor promoter region in adult testis of rats exposed neonatally to bisphenol A.
abstract: BACKGROUND
  Bisphenol A (BPA) is an estrogenic endocrine disruptor commonly used in manufacture of polycarbonate plastics and epoxy resins. Due to its ubiquitous presence in the environment, health concerns are increasing. Earlier studies from our group have shown that neonatal exposure of male rats to BPA affected spermatogenesis leading to impairment in fertility during adulthood. Further we also observed an altered gene expression of ERα and ERβ in adult testis upon BPA exposure. Based on these results, we hypothesized that apart from endocrine action, BPA might mediate perturbations in expression of ERs via epigenetic mechanism.
  
  
  OBJECTIVE
  The present study was undertaken to determine the effect of exposure of neonatal male rats to BPA on DNA methylation profile of estrogen receptor promoter region and on DNA methylation machinery.
  
  
  METHODS
  In order to test this hypothesis, neonatal male rats were subcutaneously injected with 2.4μg of BPA/day for the first five days of life, i.e., on postnatal days (PND) 1-5, while control group received vehicle (sesame oil). Animals were sacrificed during adulthood (PND-125) and testes were dissected out for analysis. Methylation pattern of promoter region of ERα and ERβ was analyzed in the testis by bisulfite sequencing and expression levels of DNA methyltransferases by quantitative RT-PCR and Western blotting respectively.
  
  
  RESULTS
  Bisulfite sequencing revealed significant hypermethylation of ERα promoter to varying extents from 40% to 60%, and ERβ promoter region with varying extent from 20% to 65%. Approximately 2-fold increase in Dnmt3a and Dnmt3b expression at transcript and protein level was also observed.
  
  
  CONCLUSIONS
  The experimental evidence demonstrated that the neonatal exposure of rats to BPA led to aberrant DNA methylation in testis, indicating methylation mediated epigenetic changes as one of the possible mechanisms of BPA induced adverse effects on spermatogenesis and fertility.
authors.affiliation: National Center for Preclinical Reproductive and Genetic Toxicology, National Institute for Research in Reproductive Health (ICMR), J M Street, Parel, Mumbai 400012, Maharashtra, India.
authors.forename: Tanvi
authors.initials: T
authors.lastname: Doshi
authors.forename: Smita Salian
authors.initials: SS
authors.lastname: Mehta
authors.forename: Vikas
authors.initials: V
authors.lastname: Dighe
authors.forename: Nafisa
authors.initials: N
authors.lastname: Balasinor
authors.forename: Geeta
authors.initials: G
authors.lastname: Vanage
mesh_terms: D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D001559:Benzhydryl Compounds; D019175:DNA Methylation / Q000187:drug effects* / Q000502:physiology; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism*; D004968:Estrogens, Non-Steroidal / Q000008:administration & dosage; D005260:Female; D008297:Male; D010636:Phenols / Q000008:administration & dosage*; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000235:genetics; D013737:Testis / Q000187:drug effects / Q000378:metabolism*; D000098826:Bisphenol A Compounds
chemical_list: D001559:Benzhydryl Compounds; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D004968:Estrogens, Non-Steroidal; D010636:Phenols; D011960:Receptors, Estrogen; C006780:bisphenol A; D000098826:Bisphenol A Compounds