There are continued concerns about endocrine-disrupting chemical effects, and appropriate vertebrate models for assessment of risk are a high priority. Frog tadpoles are very sensitive to environmental substances because of their habitat and the complex processes of metamorphosis regulated by the endocrine system, mainly thyroid hormones. During metamorphosis, marked alteration in hormonal factors occurs, as well as dramatic structural and functional changes in larval tissues. There are a variety of mechanisms determining thyroid hormone balance or disruption directly or indirectly. Direct-acting agents can cause changes in thyroxine synthesis and/or secretion in thyroid through effects on peroxidases, thyroidal iodide uptake, deiodinase, and proteolysis. At the same time, indirect action may result from biochemical processes such as sulfation, deiodination and glucuronidation. Because their potential to disrupt thyroid hormones has been identified as an important consideration for the regulation of chemicals, the OECD and the EPA have each established guidelines that make use of larval African clawed frogs (Xenopus laevis) and frog metamorphosis for screening and testing of potential endocrine disrupters. The guidelines are based on evaluation of alteration in the hypothalamic-pituitary-thyroid axis. One of the primary endpoints is thyroid gland histopathology. Others are mortality, developmental stage, hind limb length, snout-vent length and wet body weight. Regarding histopathological features, the guidelines include core criteria and additional qualitative parameters along with grading. Taking into account the difficulties in evaluating amphibian thyroid glands, which change continuously throughout metamorphosis, histopathological examination has been shown to be a very sensitive approach.

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The toxic effects of pesticides on earthworms, one of the most important bioindicators in the terrestrial environment, are closely related to their body burden determined by uptake, metabolism and excretion processes. Not only the passive diffusion via the outer skin from a dissolved fraction of pesticide but also the ingestion of contaminated soil and food governs the uptake process, with each contribution controlled by either the hydrophobicity of the pesticide or the soil organic matter. Although the available information is limited, earthworms are likely to metabolize pesticides via hydrolysis and oxidation (Phase I) followed by conjugation (Phase II), and low bioaccumulation is observed as a result for most pesticides. The acute toxicity in the soil exposure can be partly explained by the dissolved fraction of pesticide in pore water, but the contribution of dietary uptake and metabolism should be further studied to correctly evaluate pesticide toxicity.

Exposure to pesticide residues is claimed to be one of the possible causes of frog decline. Knowledge of basic information on the uptake, metabolism and depuration processes of pesticides in the frog is needed to understand the relationship between exposure and toxic effects from their actual body burden, together with their bioconcentration. The hydrophobicity of pesticides and industrial chemicals was one of the most important factors controlling bioconcentration, similarly to fish, when frogs are exposed to contaminated water. Skin absorption was also a key route in the uptake process especially in the adult frog. The metabolic profiles in the frog, mainly examined by an intraperitoneal injection technique, were common to other aquatic species without any frog-specific transformation reaction. The effects of developmental stage, sex, species and environmental factors such as temperature were observed for bioconcentration and metabolism.

Bioconcentration and metabolism of pyriproxyfen uniformly labeled with C at the phenoxyphenyl ring were studied using tadpoles of African clawed frog, Xenopus laevis, exposed to water at the nominal concentrations of 3 and 300 ppb for 22 days under the flow-through conditions, with a following 3 day depuration phase. Neither meaningful mortality nor abnormal behavior was observed in control and exposure groups throughout the study. After the rapid uptake to tadpoles, pyriproxyfen was extensively metabolized and excreted, and as a result, steady-state bioconcentration factors and depuration half-lives ranged from 550 to 610 and from 0.34 to 0.54 days, respectively. The metabolites were mostly distributed in the liver or gastrointestinal tract. The major metabolic reactions were hydroxylation at the 4' position of the phenoxyphenyl group and cleavage of the ether linkage, followed by sulfate conjugation.

INTRODUCTIONHyperprolactinemia is a common clinical disorder due to long-term administration of chemicals such as dopamine D2 receptor antagonists. The role of toxicological evaluation is to protect humans from toxicity induced by drugs or other chemicals and unwanted side effects. It is necessary to assess and differentiate direct toxicological effects of compounds from their secondary effects.The anterior pituitary-grafted rat is well known as a useful model animal for research on hyperprolactinemia; many of which focused on hormonal changes, body weight or glucose metabolism (Matsuda et al., 1994;Reis et al., 1997;Adler, 1986) by means of this model although they had only patchy information. On the other hand, in females, physiological hyperprolactinemia is observed during pregnancy and the subsequent post partum lactation period. Many studies have revealed that prolactin (PRL) is mainly characterized by increases in insulin secretion and islet cell mass in vivo and in vitro and is closely related to glucose (GLU) metabolism in females et al., 1993;Parsons et al., 1995). However, there is lack of information in males especially regarding the effects of hyperprolactinemia on toxicological parameters.The present study was designed to clarify the effects of hyperprolactinemia on toxicological parameters using anterior pituitary-grafted male rats. The study took into account sex difference of donors since PRL contents and the proportion of lactotrophs in female rat-derived anterior pituitary grafts are greater than those in their counterparts derived from male rats (McArthur et al., 2006). Also, the study included the effects of hyperprolactinemia on islet cell proliferation in male rats because sever- ABSTRACT -Prolactin has a wide variety of biological effects. Consequences of hyperprolactinemia on islet B cell proliferation as well as general toxicological parameters were here examined using anterior pituitary-grafted rats. Three or six anterior pituitary glands were implanted under single renal capsules of F344 male rats and left there for 13 weeks afterward. Clinical observation along with measurement of body weight and food consumption was conducted during the observation period, and subsequently hematology, blood biochemistry, gross pathology, organ weights and histopathology were examined. In addition, the proliferation rate of islet B cells was measured by a 5-bromo-2'-deoxy-uridine (BrdU) labeling technique. Serum prolactin concentrations at week 13 were 36, 70, 75 and 105 ng/ml in the shamoperated, 3-pituitary-grafted groups from male or female donors, and 6-pituitary-grafted group from male donors, respectively. Higher cholinesterase and total cholesterol values, lower trigriceride and leutenizing hormones (LH) values, and higher adrenal weights compared to those in the sham-operated group were apparent in the 3-and/or 6-pituitary-grafted groups. Also, the study revealed that mammary gland structure was transformed with change of differentiation from a male to a female acinar pattern. Furthermore a -...

Tadpoles during metamorphosis are sensitive to chemical exposure as shown in the amphibian metamorphosis assay, which is a method to detect effects of chemicals on the functions of hypothalamus‐pituitary‐thyroid axis. The present study reports existence of different modes of action between pyriproxyfen (PYR) and 6‐propyl‐2‐thiouracil (PTU) under different feeding conditions based on gene expression profiles (transcriptomics) in the thyroid glands of tadpoles of the African clawed frog, Xenopus laevis. PTU and PYR were exposed to the tadpoles during metamorphosis under normal (fed groups, both of PTU and PRY) and restricted feeding (fasted groups, PTU only) conditions; and effects were compared to control groups. Delayed development based on decreased Nieuwkoop and Faber developmental stage number without any histopathological changes was observed in the control of restricted feeding (control‐fasted) group, and the PYR group with reduced food consumption. Clear developmental retardation with typical thyroid histopathological changes was observed in the PTU groups. To find clusters of all samples based on their similarity of expression patterns, hierarchical clustering analysis using selected gene probes was conducted. It revealed gene profiles from samples of the PYR group were quite similar to those of the control‐fasted group, followed by the control group with normal feeding (control‐fed). The results suggest that key events in the thyroid glands of tadpoles induced by PYR should be quite similar to those of control‐fasted, and quite different from those of the PTU groups. Our findings demonstrated the usefulness of transcriptomics, which enabled recognition of the different modes of actions.