Naoko Mitsui

Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis

There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.

Sandwich ELISAs for quantification of Xenopus laevis vitellogenin and albumin and their application to measurement of estradiol-17β effects on whole animals and primary-cultured hepatocytes

Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for quantification of vitellogenin (VTG) and albumin (ALB) in Xenopus laevis. Working ranges of the ELISAs were 2-1000 ng/ml for VTG and 1-300 ng/ml for ALB. Recoveries of plasma VTG by ELISA were over 90% in dilutions of more than 200 times. The VTG-inducing activity of estradiol-17beta (E2) was measured in whole animals and primary cultured hepatocytes. Immersion of mature male animals in more than 1 nM E2 induced a detectable amount of plasma VTG. VTG induction in younger animals was less potent than in the mature animals but the youngest animals (1.5-3 g body mass) was applicable to the exposure test, irrespective of sex. In vitro exposure of hepatocytes to more than 0.1 nM E2 dose-dependently induced secretion of VTG into the culture medium, while ALB secretion was not significantly affected by E2 treatment. When the VTG-induction levels were normalized by use of a concentration ratio of VTG to ALB, the values obtained from three independent experiments were mutually comparable irrespective of differences in cell density and hepatocyte preparation. Thus, this ratio is thought to be useful for large-scale in vitro screening of estrogenic activities of chemical substances.

Application of metamorphosis assay to a native Japanese amphibian species, Rana rugosa, for assessing effects of thyroid system affecting chemicals

The aims of this study were to assess the utility of a metamorphosis assay for detecting thyroid hormone-disrupting chemicals using Rana rugosa, a domestic frog species in Japan, and to compare species differences in sensitivity to thyroxine (T(4)) and propylthiouracil (PTU) among R. rugosa, Xenopus laevis and Xenopus (Silurana) tropicalis. Tadpoles of R. rugosa (TK stages III/IV) were exposed to standard test chemicals for acceleration (T(4)) and inhibition (PTU) of metamorphosis for 28 days in semi-static condition and total body length and developmental stage (TK stage) were recorded every week. T(4) (0.61 and 2.24 microg/L in actual concentrations) and PTU (19.73, 76.83, and 155.67 mg/L in actual concentrations) induced significant acceleration and inhibition of metamorphosis, respectively. The present results indicate that the metamorphosis assay is successfully applied to the domestic frog species, R. rugosa, suggesting this assay can be used for the assessment of chemicals on ecological impacts in wild frog species.

Developmental disorders and altered gene expression in the tropical clawed frog (Silurana tropicalis) exposed to 17α-ethinylestradiol

Several endocrine‐disrupting chemicals with estrogenic activity can affect sexual development and reproduction in aquatic wildlife. The occurrence of oocytes in the testis (testis‐ova) is one reproductive disorder and can be used as a valid endpoint when studying disruptive effects of estrogenic chemicals. To elucidate the molecular basis of testis‐ova induction, we conducted gene expression analysis in the gonads of Silurana tropicalis exposed to 0, 3, 10 and 30 ng l−1 17α‐ethinylestradiol (EE2) from 2 days after fertilization to the juvenile stage (14 weeks after fertilization). The frequencies of testis‐ova induction or male to female sex‐reversal of the gonads increased in an EE2 dose‐dependent manner. Microarray analysis showed that expressions of a large number of genes were significantly changed by EE2 exposure. Genes including egg envelope composition (zp4, zpax, zpc, zp3.2 and egg cortical granule lectin), 42S particle genes (42Sp50, 42Sp43 and 42Sp48) and regulation of female germ cells (figla) are associated with the testis‐ova and sex‐reversal situation in the gonads. Of those, expression of zpc and 42Sp50 genes is associated with testis‐ova. Thus, we propose that these genes are useful biomarkers for toxicological research in amphibians developmentally exposed to estrogenic chemicals. Copyright

Screening breeding sites of the common toad (Bufo bufo) in England and Wales for evidence of endocrine disrupting activity.

Anuran amphibians are often present in agricultural landscapes and may therefore be exposed to chemicals in surface waters used for breeding. We used passive accumulation devices (SPMD and POCIS) to sample contaminants from nine breeding sites of the Common toad (Bufo bufo) across England and Wales, measuring endocrine activity of the extracts in a recombinant yeast androgen screen (YAS) and yeast estrogen screen (YES) and an in vitro vitellogenin induction screen in primary culture of Xenopus laevis hepatocytes. We also assessed hatching, growth, survival, and development in caged larvae in situ, and sampled metamorphs for gonadal histopathology. None of the SPMD extracts exhibited estrogen receptor or androgen receptor agonist activity, while POCIS extracts from two sites in west-central England exhibited concentration-dependent androgenic activity in the YAS. Three sites exhibited significant estrogenic activity in both the YES and the Xenopus hepatocyte. Hatching rates varied widely among sites, but there was no consistent correlation between hatching rate and intensity of agricultural activity, predicted concentrations of agrochemicals, or endocrine activity measured in YES/YAS assays. While a small number of intersex individuals were observed, their incidence could not be associated with predicted pesticide exposure or endocrine activitity measured in the in vitro screens. There were no significant differences in sex ratio, as determined by gonadal histomorphology among the study sites, and no significant correlation was observed between proportion of males and predicted exposure to agrochemicals. However, a negative correlation did become apparent in later sampling periods between proportion of males and estrogenic activity of the POCIS sample, as measured in the YES. Our results suggest that larval and adult amphibians may be exposed to endocrine disrupting chemicals in breeding ponds, albeit at low concentrations, and that chemical contaminants other than plant protection products may contribute to endocrine activity of surface waters in the agricultural landscape.