Estrogen-like effects of ultraviolet screen 3-(4-methylbenzylidene)-camphor (Eusolex 6300) on cell proliferation and gene induction in mammalian and amphibian cells.

We tested the ultraviolet screen 3-(4-methylbenzylidene)-camphor (4-MBC; Eusolex 6300), which has been implicated as a potential endocrine disruptor, for its potential to bind to and activate endogenous estrogen receptors (ER) and to mediate ER-dependent changes in gene transcription, in hepatocytes of the water-dwelling South African clawed frog Xenopus laevis. We were able to confirm previous findings that 4-MBC accelerates cell proliferation in estrogen-dependent human breast cancer cells (MCF-7). Results of competitive binding assays of [3H]17beta-estradiol and 4-MBC using cytosolic protein preparations from Xenopus hepatocytes indicated that 4-MBC weakly binds to the ER. 4-MBC at a concentration of 100 micromol/L is not able to completely replace estradiol from the receptor. However, when 4-MBC was tested in a gene induction assay using the relative amount of ER transcript as a marker for ER-dependent transcriptional activation, we found that micromolar concentrations of this substance produced an increase in the amount of ER mRNA that was not different from the amount of mRNA that was observed upon activation of cells with 17beta-estradiol in concentrations above 1 nmol/L. The results indicate that 4-MBC has the potential to change physiological and developmental processes mediated by ER signaling mechanisms. It may therefore be a potentially harmful substance for water-dwelling animals when present in the environment at micromolar concentrations.

Retinol-binding protein as a biomarker to assess endocrine-disrupting compounds in the environment.

Endocrine-disrupting compounds (EDC) are predominantly investigated with respect to their ability to mimic or block estrogenic actions. However, it is well-known that EDC can act as agonists or antagonists of androgen- and estrogen-response systems. For that reason, there is an obvious need for bioassays providing the possibility of detecting (anti-)estrogenic and (anti-)androgenic effects. The retinol-binding protein (RBP) seems to be a useful molecular biomarker for assessing all modes of action of EDC, because it is regulated by sex steroid hormones. This study was conducted to establish RBP as a biomarker for determination of (anti-)estrogenic and (anti-)androgenic effects of EDC using a Xenopus laevis primary hepatocyte culture system. It could be shown that RBP mRNA expression in X. laevis hepatocytes was stimulated by estrogens in a dose-dependent manner whereas a combination of estrogen and androgen or estrogen and anti-estrogen treatment suppressed estrogenic stimulating effects. Androgens testosterone and dihydrotestosterone were able to reduce RBP mRNA expression and the anti-androgen vinclozolin could abolish the mRNA synthesis-suppressing activity of the androgen dihydrotestosterone. These results clearly demonstrated that RBP mRNA expression patterns in Xenopus laevis hepatocytes have different modes of (anti-)estrogenic and (anti-)androgenic action and can be used for examination of suspected EDC. Moreover, water samples from sewage-treatment plant effluents were applied to liver cells and expression levels of RBP and estrogen receptor mRNA (a known estrogenic biomarker) were detected. These samples had high estrogenicity but caused low to moderate induction of RBP mRNA synthesis, leading to the conclusion that RBP levels represent the sum of all possible effects (estrogenic and other effects) of EDC in environmental samples.

Bisphenol A induces feminization in Xenopus laevis tadpoles.

To evaluate possible estrogenic effects of bisphenol A (BPA) in an amphibian model, Xenopus laevis tadpoles were exposed to BPA and 17beta-estradiol (E2) during larval development. After metamorphosis, the gonadal phenotype was determined by gross morphology, and testes were further examined histologically to validate the results. BPA treatment altered the normal sex ratio toward females depending on the BPA concentrations added. Chemical analysis showed a time-dependent decline of BPA during semistatic exposure, indicating that BPA is taken up and metabolized to some extent by tadpoles. In addition, tadpoles were exposed to BPA and E2 for 2 weeks during sensitive stages of sexual differentiation. Afterward, the expression of an estrogenic biomarker, estrogen receptor (ER) mRNA, was assessed by semiquantitative RT-PCR. Both BPA and E2 up-regulated ER mRNA significantly. In conclusion, these results show clear evidence that BPA induces feminization in X. laevis tadpoles, revealing an estrogenic potency of BPA that influences sexual development in amphibians.

Functional genomics and sexual differentiation in amphibians.

In Xenopus laevis the basic mechanisms underlying sexual differentiation were investigated by determining time courses of sexual steroids and their corresponding receptors during complete larval development from egg to juveniles. Androgens as well as estradiol (E2) are derived from maternal origin and accumulate in hatching tadpoles. Sexual steroid contents decreased rapidly after hatching and rose again at the end of metamorphosis indicating endogenous production. In parallel the mRNA expression for corresponding androgen (AR) and estrogen receptors (ER) was measured by means of semiquantitative RT-PCR. Both receptor mRNAs increased dramatically just after hatching and decreased only moderately until end of metamorphosis. In female juveniles E2 and ER-mRNA levels were higher compared with males. Treatment by exogenous E2 elevated both, ER- and AR-mRNA, indicating stimulatory functions of E2 for gene expression of both receptors. Effects on sexual differentiation during larval development were achieved by treatment with E2 and the antiandrogen cyproterone acetate both causing feminization, the antiestrogen tamoxifen resulting in neutralization, and the androgens, methyltestosterone and dihydrotestosterone, but not testosterone, leading to masculinization. The data presented are in accordance with further recent findings and suggest a new hypothesis for functional genomics in sexual differentiation of amphibians.