Development of biomarkers of endocrine disrupting activity in emerging amphibian model, Silurana (Xenopus) tropicalis.

Because amphibians show peculiar ecological features and interesting responses to some hormones, it is conceivable that amphibians are very useful animals for assessing the toxic effects of environmental contaminants, including endocrine disrupters. To develop methods of detecting endocrine toxicity of environmental chemicals in amphibians, we have started to assemble a biomarker tool kit for an emerging amphibian model, Silurana (Xenopus) tropicalis. We isolated full-length cDNAs encoding estrogen receptor alpha (ERalpha), ERbeta, thyroid hormone receptor alpha (TRalpha), and TRbeta of S. (X.) tropicalis to develop a reporter gene assay system, as an estimation tool for environmental chemicals. The amino acid sequences inferred from the four full-length cDNAs were highly homologous to those of ERalpha, TRalpha and TRbeta of X. laevis, and ERbeta of the Japanese quail. In particular, the S. (X.) tropicalis ERalpha shared a higher similarity of amino acid sequence with X. laevis ERalpha than the previously reported S. (X.) tropicalis ERalpha, as determined by Wu et al. RT-PCR analysis showed that the two ERalpha and ERbeta transcripts were expressed relatively abundantly in the brain, liver, and gonad/kidney complex of the S. (X.) tropicalis tadpole after gonadal sex differentiation occurring at developmental stages 54-59, suggesting that they are susceptible to estrogenic substances. A similar result was obtained in the two TR transcripts, although their expression levels were lower in the gonad/kidney complex than in the other tissues. Moreover, we identified vitellogenin A (Vtg A) and Vtg B as estrogen-responsive genes expressed in the female S. (X.) tropicalis liver using macroarray analysis and RT-PCR. In addition, Rana japonica Vtg was purified from serum using anion-exchange chromatography to produce anti-Vtg antibody as a protein marker. In the future, we are going to construct reporter gene assay systems using the full-length ER and TR cDNAs, analyze histologically the differentiation of gonads and thyroid glands in the S. (X.) tropicalis tadpole exposed to estrogenic chemicals, and produce sex-reversed male S. (X.) tropicalis to obtain all-male tadpoles. Using these tools, we hope to be able to identify endocrine disrupting compounds in laboratory experiments for hazard assessment purposes, and also detect endocrine toxicity in environmental samples as part of an integrated approach to assessing the impact of environmental contaminants on wild amphibian populations in Japan and the UK.