title: Mixture effects of levonorgestrel and ethinylestradiol: estrogenic biomarkers and hormone receptor mRNA expression during sexual programming.
abstract: Synthetic progesterone (progestins) and estrogens are widely used pharmaceuticals. Given that their simultaneous unintentional exposure occurs in wildlife and also in human infants, data on mixture effects of combined exposures to these hormones during development is needed. Using the Xenopus (Silurana) tropicalis test system we investigated mixture effects of levonorgestrel (LNG) and ethinylestradiol (EE2) on hormone sensitive endpoints. After larval exposure to LNG (0.1nM), or EE2 (0.1nM) singly, or in combination with LNG (0.01, 0.1, 1.0nM), the gonadal sex ratio was determined histologically and hepatic mRNA levels of genes encoding vitellogenin (vtg beta1) and the estrogen (esr1, esr2), progesterone (ipgr) and androgen (ar) receptors were quantified using quantitative PCR. All EE2-exposed groups showed female-biased sex ratios and increased vtg beta1 mRNA levels compared with the controls. Compared with the EE2-alone group (positive control) there were no significant alterations in vtg beta1 levels or in sex ratios in the co-exposure groups. Exposure to LNG-alone caused an increase in ar mRNA levels in females, but not in males, compared to the controls and the co-exposed groups, indicating that co-exposure to EE2 counteracted the LNG-induced ar levels. No treatment related impacts on the mRNA expression of esr1, esr2, and ipgr in female tadpoles were found, suggesting that these endpoints are insensitive to long-term exposure to estrogen or progestin. Due to the EE2-induced female-biased sex ratios, the mRNA expression data for the low number of males in the EE2-exposed groups were not statistically analyzed. In conclusion, our results suggest that induced vtg expression is a robust biomarker for estrogenic activity in exposure scenarios involving both estrogens and progestins. Developmental exposure to LNG caused an induction of hepatic ar mRNA expression that was antagonized by combined exposure to EE2 and LNG. To our knowledge this is the first study to report effects of combined exposures to EE2 and LNG during the period of sexual programming.
authors.affiliation: Uppsala University, Department of Environmental Toxicology, Centre for Reproductive Biology in Uppsala, Norbyvägen 18A, 752 36 Uppsala, Sweden. Electronic address: moa.kvarnryd@ebc.uu.se.
authors.forename: Moa
authors.initials: M
authors.lastname: Säfholm
authors.affiliation: Uppsala University, Department of Environmental Toxicology, Centre for Reproductive Biology in Uppsala, Norbyvägen 18A, 752 36 Uppsala, Sweden. Electronic address: erika.jansson@ebc.uu.se.
authors.forename: Erika
authors.initials: E
authors.lastname: Jansson
authors.affiliation: Umeå University, Department of Chemistry, KBC 6A, Linnaeus väg 6, 901 87 Umeå, Sweden. Electronic address: jerker.fick@umu.se.
authors.forename: Jerker
authors.initials: J
authors.lastname: Fick
authors.affiliation: Uppsala University, Department of Environmental Toxicology, Centre for Reproductive Biology in Uppsala, Norbyvägen 18A, 752 36 Uppsala, Sweden. Electronic address: cecilia.berg@ebc.uu.se.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Berg
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D004357:Drug Synergism; D004625:Embryo, Nonmammalian / Q000187:drug effects; D004967:Estrogens / Q000235:genetics; D004997:Ethinyl Estradiol / Q000633:toxicity*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D006066:Gonads / Q000187:drug effects; D016912:Levonorgestrel / Q000633:toxicity*; D008099:Liver / Q000187:drug effects; D008297:Male; D011944:Receptors, Androgen / Q000235:genetics*; D011980:Receptors, Progesterone / Q000235:genetics*; D012744:Sex Ratio; D014819:Vitellogenins / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000633:toxicity; D014981:Xenopus / Q000196:embryology
chemical_list: D015415:Biomarkers; D004967:Estrogens; D011944:Receptors, Androgen; D011980:Receptors, Progesterone; D014819:Vitellogenins; D014874:Water Pollutants, Chemical; D004997:Ethinyl Estradiol; D016912:Levonorgestrel
keywords: Amphibians; Androgen receptor; Endocrine disruption; Pharmaceuticals; Sex differentiation; Vitellogenin