Osamu Tooi

Low-dose dioxins alter gene expression related to cholesterol biosynthesis, lipogenesis, and glucose metabolism through the aryl hydrocarbon receptor-mediated pathway in mouse liver

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental contaminant. TCDD binds and activates the transcription factor aryl hydrocarbon receptor (AHR), leading to adverse biological responses via the alteration of the expression of various AHR target genes. Although small amounts of TCDD are consumed via contaminated daily foodstuffs and environmental exposures, the effects of low-dose TCDD on gene expression in animal tissues have not been clarified, while a number of genes affected by high-dose TCDD were reported. In this study, we comprehensively analyzed gene expression profiles in livers of C57BL/6N mice that were orally administered relatively low doses of TCDD (5, 50, or 500 ng/kg body weight (bw) day(-1)) for 18 days. The hepatic TCDD concentrations, measured by gas chromatography-mass spectrometry, were 1.2, 17, and 1063 pg toxicity equivalent quantity (TEQ)/g, respectively. The mRNA level of the cytochrome P450 CYP1A1 was significantly increased by treatment with only TCDD 500 ng/kg bw day(-1). DNA microarray and quantitative RT-PCR analyses revealed changes in the expression of genes involved in the circadian rhythm, cholesterol biosynthesis, fatty acid synthesis, and glucose metabolism in the liver with at all doses of TCDD employed. However, repression of expression of genes involved in energy metabolism was not observed in the livers of Ahr-null mice that were administered the same dose of TCDD. These results indicate that changes in gene expression by TCDD are mediated by AHR and that exposure to low-dose TCDD could affect energy metabolism via alterations of gene expression.

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.

Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect Thyroid-active Substances

In support of an Organization for Economic Cooperation and Development (OECD) Amphibian Metamorphosis Assay (AMA) Test Guideline for the detection of substances that interact with the hypothalamic-pituitary-thyroid axis, a document was developed that provides a standardized approach for evaluating the histology/histopathology of thyroid glands in metamorphosing Xenopus laevis tadpoles. Here, a consolidated description of histology evaluation practices, core diagnostic criteria and severity grading schemes for the AMA, an atlas of the normal architecture of amphibian thyroid glands over the course of metamorphosis, and the core diagnostic criteria with examples of severity grades is provided. Core diagnostic criteria include thyroid gland hypertrophy/atrophy, follicular cell hypertrophy, and follicular cell hyperplasia. The severity grading scheme is semiquantitative and employs a four-grade approach describing ranges of variation within assigned ordinal classes: not remarkable, mild, moderate, and severe. The purpose of this severity grading approach is to provide an efficient, semi-objective tool for comparing changes (compound-related effects) among animals, treatment groups, and studies. Proposed descriptions of lesions for scoring the four core criteria are also given.

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.

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.

Metal ion-responsive transgenic Xenopus laevis as an environmental monitoring animal

We generated germ line-transgenic Xenopus laevis that monitors environmental heavy metal ions. Sperm nuclei were transduced with cDNA of enhanced green fluorescent protein (EGFP) driven by murine metallothionein-1 gene promoters and were microinjected into unfertilized eggs. The eggs developed to sexually matured adults. The transgenic tadpoles at the premetamorphic stage were reared in water containing Zn(2+) and Cd(2+) separately at the concentrations of 0.38-1.52 and 0.09-0.44 μM, respectively. These animals responded to Zn(2+) at as low as 0.38 μM and Cd(2+) at as low as 0.44 μM. The level of EGFP fluorescence emitted by tadpoles increased as the concentration increased up to 1.52 μM and the exposure time prolonged up to 120 h. The fluorescent response was much more sensitive to Cd(2+) than to Zn(2+). We concluded that these transgenic tadpoles are useful as an animal indicator of environmental heavy metal ions.