The cytochrome P450 (CYP) catalyzes the oxidation of a variety of endogenous and xenobiotic compounds. The expression levels of CYP isozymes are modulated by endogenous hormones and xenobiotic chemicals. In our previous study, we performed a field survey of the mRNA expression levels of the major CYP isozymes in roof-rats captured in environmentally distinct two areas (Hokkaido, Osaka prefecture). The mRNA expression levels of CYP isozymes were analyzed using competitive RT-PCR and compared them between the two areas. The mRNA expression of the adult male-specific CYP isozymes was detected in many of the adult female roof-rats trapped in Osaka prefecture but not in the adult female roof-rats trapped in Hokkaido at all. The adult male-specific CYP isozymes can be induced in female rat by the administration of various xenobiotics, e.g., lindane (pesticide), DDT (pesticide), dexamethason (synthetic adrenocortical hormone), phenytoin (anticonvulsant hydantoin derivative), and oxandrolone (protein anabolic steroid). The aim of the present study is to investigate the mRNA induction of adult male-specific CYP isozymes and its mechanism in the adult female rat by the administration of the xenobiotic chemical inducers in order to propose that the mRNA expression of adult male-specific CYP isozymes in the female roof-rat liver will be a useful biomarker for assessing the level of exposure to environmental chemicals.

1. A Survey on Heavy Metal Contamination of Wild Animals
   Recently the environmental pollution is in progress in the habitat of wild animals due to the development of mountainous district or illegally dumped industrial wastes. The wild animals may ingest various pollutants including heavy metals. Although, lead poisoning of wild birds occurs frequently by ingesting shotgun pellets, there are few data on the heavy metal contamination of wild animals. Therefore, the liver, kidney and hair samples collected from wild animals such as Japanese black bear and sika deer are analyzed by PIXE (particle induced X-ray emission) to determine the concentration of heavy metals. This study is carried out in cooperation with Iwate Prefecture, Morioka Zoological Park and Nishina Memorial Cyclotron Center, Japan Radioisotope Association.
2. A Survey on Mutagenic Pollutants in Natural Water
   Various chemicals are flowing into the river via the industrial or domestic waste water. Air and soil pollutants also affect the quality of river water. River, lake and other natural water is the habitat of aquatic animals and is also essential for many other wild animals. In this study, polycyclic compounds are selectively adsorbed from natural water to blue rayon and their mutagenicity is examined by the umu test and the comet assay. The adsorption compounds are also analyzed by the GC-MS (Gas Chromatography-Mass Spectrometry) to assess the pollution of natural water.
3. Toxicological Study on Fluorinated Organic Compounds
   Fluorinated organic compounds (FOCs) are widely used for waterproof agents, coating agents, polymers, lubricants, etc. Persistent FOCs such as perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) remain eternally in nature. It has been reported that these compounds are accumulated not only in humans but also in wild animals. However, there are few information on the toxicity of persistent FOCs. In this study, therefore, toxicological examinations are carried out using experimental animals and aquatic protozoa, paramecium, to elucidate the toxicity of FOCs and to assess the effect on wild animals.

It is reported that great cormorant is one of heavily contaminated wild birds by organochlorines (OCs) in Japan. Great cormorants are widely distributing along fresh water, i.e. rivers and lakes, and shores in Japan. They mostly depend on fishes for food. Therefore they are at the top of food-chain in the aquatic ecosystems. It has reported by the Ministry of Environment that the great cormorant in some polluted areas highly accumulated organochlorines including dioxins. In the first part of the COE research, relationship between contamination of OCs and histological changes in thyroid function was investigated. Great cormorants contaminated heavily by OCs showed extensive morphological changes of thyroid glands with low levels of circulating thyroid hormones. The residue levels of dioxins were higher in adult than juvenile. The extent of morphological changes in the thyroid gland was higher in great cormorants captured from Tokyo Bay than those captured from Lake Biwa. There were prominent morphological changes of thyroid glands such as increased density of small follicles, and increased number of follicular epithelial cells in the follicular lumen. The higher degree of thyroid lesions in great cormorants from Tokyo Bay area was associated with significantly higher levels of OCs. Thus, these results clearly demonstrated that morphological changes of thyroid glands have a possible connection with the levels of dioxin contamination in Japan. The aim of the second part of the COE study is to examine relationship among thyroid function, endocrine and immune function in wild great cormorant in Japan and to establish a new animal model ranking high in food-chain for assessment of environmental contamination.

Air pollution has become a very serious international problem for all the flora and fauna that exists in this earth including humankind. Especially in big cities around the world, the major cause for air pollution is thought to be from exhaust particulates emitted by diesel engines. There have been much reports of the possible health impact of DEP, such as to the respiratory and the circulatory system. But Suzuki and his coworkers recently reported that there are compounds from DEP that exert estrogenic effects, and have isolated 5 major nitrophenols as active compounds. These nitrophenols are a known degradation product of the insecticide fenitrothion, which is used widely in our country as well as many countries around the world and is thought to be accumulated in large amounts to the environment and therefore, the effects to the wildlife is much in concern. However, it is difficult to consider the immediate effect upon the wildlife animals because there are no basic data available about these compounds. So the aim of the present study is to clarify the mechanisms of the effects to the reproductive function of the compounds. We used animal models for this study such as mice, rats or quails, comprehending to the mammal or the avian animal models in real life.

1. Steroid receptor binding analysis by using recombinant yeast screen assays: To define the possible receptor or anti- binding ability of the five compounds isolated from DEP such as estrogen, testosterone, progesterone, cortisol receptors.
   
2. Reproductive cell line exposure evaluation: To evaluate the effects of the compounds mentioned above to the reproductive cells in vitro, by using endocrine or molecular techniques.
   
3. Animal exposure evaluation: To evaluate the effects of the compounds using mice, rats and quails as animal models. To examine the effects to the female reproductive system, exposure of the compounds to the pregnant animals to see the effects of the developmental stages of the embryo. As for the male reproductive system, the testis of the animals exposed to the compounds will be analyzed by using computer-assisted sperm motility analysis system and radioimmunoassay methods.

Nowadays, the disease patterns of humans observed in Japan are changing; for example, metabolic diseases (life style-related diseases), allergic diseases, persistent viral infections, neurological diseases and senile disorders predominate. By using nonhuman primates, that are ideally suited because of their evolutional and genetically close alliance to humans, we can assess these combined diseases and affection of environmental pollution, especially the endocrine-disturbing chemicals. We investigate free-living wild monkeys, long-term maintained monkeys, and monkeys in bleeding colonies in this study. Based on collaboration with the Society for Primate Diseases and Pathology (SPDP), the following studies will be promoted;

1. Coordination for an international workshop on Primate Diseases in 2005 and 2006 collaborated with SPDP.
   
2. Publication of a color atlas for primate diseases collaborated with SPDP.
   
3. Morphological analysis of environmental pollution-related changes in nonhuman primates.
   
4. Detection and characterization of Zoonosis between humans and nonhuman primates.
   Important zoonosis including T.B are surveyed in colonies of monkeys that are freely living, zoo-maintained or kept in breeding colony.
   
5. Evaluation of spontaneous lesions including neoplasms and aging-related changes in nonhuman primates.
   
6. Experimental studies on environmental pollutants or infectious agents such as EHV9 in nonhuman primate to evaluate human diseases.

Generally, environmental pollutants have toxicity to nerve, immune and genital systems. Among the toxicity, the genital toxicity has been well documented in several environmental pollutants, and some toxic mechanisms are already elucidated. However, the toxic mechanisms for nerve and immune systems are not necessarily resolved so far. In the present study, we focus on the neurotoxicity of organotin compounds, such as tributyltin (TBT). TBT have been given much attention as environmental endocrine disruptors and considered to be hazardous if exposed even at lower concentrations. It has been reported that TBT induces neuronal cell apoptosis due to cytosolic Ca2+ overload. However, the precise mechanisms for TBT-induced apoptosis remain to be elucidated. Therefore, using cultured cell lines originated from neuronal cell (PC12 cell), we will explore how TBT mobilizes intracellular Ca2+ to induce apoptosis.

In our previous studies, the following findings are obtained in regard to neurotoxicity of TBT. (1) TBT (0.01-10 ?M) decreased the number of viable PC12 cells by promoting apoptosis (Figure 1). The TBT effect was dependent on its concentration and treatment period. (2) When intracellular Ca2+ concentration ([Ca2+]i) was monitored using fura-2 fluorimetry technique, TBT (0.01-10 ?M) concentration-dependently increased [Ca2+]i in fura-2 loaded PC12 cells (Figure 2). The 50 % effective concentration of TBT (0.07 ?M) was similar to a concentration required for inducing 50 % cell death (0.09 ?M). Dibutyltin (DBT) and monobutyltin (MBT) also decreased the number of viable cells and increased [Ca2+]i, and the rank order of potencies were TBT>DBT>MBT and TBT>DBT?MBT, respectively. (3) When Ca2+ was removed from the extracellular solution, or the cell membrane was depolarized using 140 mM K+ extracellular solution, increase in [Ca2+]i by TBT (10 ?M) was markedly diminished. Treatment of thapsigargin, an inhibitor of intracellular store function, had little or no effect on TBT-induced increase in [Ca2+]i. Nicardipine, a L-type voltage dependent Ca2+ channel blocker, but not -conotoxin, an N-type Ca2+ channel blocker, inhibited the TBT-induced increase in [Ca2+]i. These results suggest that TBT induces apoptosis by mobilizing Ca2+ into cytosol through the activation of L-type voltage dependent Ca2+ channels (Figure 3).

In the second term of our project, we will study about signal transduction mechanism for TBT-induced activation of L-type Ca2+ channels. To do this, the following points will be investigated using electrophysiological techniques. (1) To test a possibility that TBT indirectly activates L-type Ca2+ channels by depolarizing the cell membrane, membrane potential responses to TBT are monitored using current-clamp technique. (2) To test a possibility that TBT directly activates L-type Ca2+ channels, effect of TBT on L-type Ca2+ channel current is investigated using voltage-clamp technique. (3) Because TBT has been known to activate protein kinase C (PKC), which modulates activity of some types of ion channels, effects of PKC inhibitors on the TBT action are examined. These investigations may give useful information for TBT-induced neurotoxicity, and thereby for risk assessment of organotin compounds.