Mixture effects of levonorgestrel and ethinylestradiol: Estrogenic biomarkers and hormone receptor mRNA expression during sexual programming

Säfholm, Moa; Jansson, Erika; Fick, Jerker; Berg, Cecilia

doi:10.1016/j.aquatox.2015.02.004

Cited by 19 publications

(7 citation statements)

References 76 publications

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“…In the same way, in the juvenile African clawed frog, no estrogenic effect of LNG alone or in mixture with EE2 is observed on the ER-regulated expression of vtg beta1 gene or on the sex ratio (Säfholm et al 2015). All these studies show that additive mixture responses of EE2 and LNG do not occur on all ER-regulated endpoints in all tissues and, as previously stated by others, at all levels of biological organization (Runnalls et al 2015, Säfholm et al 2015.…”

supporting

confidence: 62%

“…LNG exerts biological activities that differ from the natural progestin (progesterone) since it has progestagenic and androgenic activities (Besse and Garric 2009) and also estrogenic activities both in vitro and in vivo (Jeng et al 1992, Zucchi et al 2012, Creusot et al 2014, Kroupova et al 2014. However, despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined toxicity (Runnalls et al 2015, Säfholm et al 2015). …”

Section: Introductionmentioning

confidence: 99%

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Additive effects of levonorgestrel and ethinylestradiol on brain aromatase ( cyp19a1b ) in zebrafish specific in vitro and in vivo bioassays

Hinfray¹,

Tebby

Garoche³

et al. 2016

Toxicology and Applied Pharmacology

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Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-tranfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentrationresponse relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures.

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supporting

confidence: 62%

Section: Introductionmentioning

confidence: 99%

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase ( cyp19a1b ) in zebrafish specific in vitro and in vivo bioassays

Hinfray¹,

Tebby

Garoche³

et al. 2016

Toxicology and Applied Pharmacology

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“…In adult exposures, levonorgestrel, but not P4, altered the male mating behavior (Hoffmann and Kloas, 2012). When tadpoles were exposed to mixtures of levonorgestrel and EE2, no femalebiased sex ratios occurred as compared to the exposures of tadpoles to EE2 alone (Säfholm et al, 2015). The induction of the AR transcript by levonorgestrel was antagonized when there was combined exposure to levonorgestrel and EE2.…”

Section: Effects In Invertebrates and Amphibiansmentioning

confidence: 96%

Progestins as endocrine disrupters in aquatic ecosystems: Concentrations, effects and risk assessment

Fent

2015

Environment International

203

129

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“…hormono-regulated genes and altered reproductive outputs at environmentally relevant concentrations (Bluthgen et al, 2013;Han et al, 2014;Liang et al, 2015;Overturf et al, 2014;Safholm et al, 2015;Zucchi et al, 2014).…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish

Cano-Nicolau

Garoche²,

Hinfray³

et al. 2016

Toxicology and Applied Pharmacology

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The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. We showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC50 ranging from the low nM range to hundreds nM. The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfERα, zfERβ1 or zfERβ2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity. Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation.

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Mixture effects of levonorgestrel and ethinylestradiol: Estrogenic biomarkers and hormone receptor mRNA expression during sexual programming

Cited by 19 publications

References 76 publications

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase ( cyp19a1b ) in zebrafish specific in vitro and in vivo bioassays

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase ( cyp19a1b ) in zebrafish specific in vitro and in vivo bioassays

Progestins as endocrine disrupters in aquatic ecosystems: Concentrations, effects and risk assessment

Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish

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