Endocrine disruptors and environmental impact in Japan





Various chemical substances are used in business, industries, agriculture and our daily life. It is an important subject to prevent hazards to our health and living environment resulted from environmental contamination by toxic substances. In Japan, we have experienced the contamination caused by harmful chemical substances, such as methylmercury cadmium, PCB in the wastewater from chemical plants. The atmospheric pollution by dioxin, which are formed unintentionally as byproducts from waste incineration and diesel exhaust, also affects the people in the area. Recent investigations suggests that a number of man-made chemicals ranging from plastics products to pesticides may interact with the endocrine system of humans and wildlife populations. Many of them are known by their high toxic levels in animal studies, but not scientifically determined the low level toxic effects in the environment on humans and ecological systems.



Research projects


Many relating projects were recently started in government institutes, universities and industries. So serious is the problem that it could well threaten the very survival of mankind itself. Japanese Environmental Agency completed and presented the Strategic Program on Environmental Endocrine Disruptors '98 (SPEED '98) to the public in May 1998. Based on these programs, studies were started throughout Japan to determine the extent of environmental pollution and its effects on wild life. Other Japanese governments, such as Ministry of Health and Welfare (MHW), Ministry of Agriculture, Forestry and Fisheries (MAFF), Ministry of Construction (MOC), and local governments also started studies on this subject. Relating reports are provided via World Wide Web (WWW) (Table 1).



MHW started work on this issue in 1996 and engaged in research on the High Throughput Pre Screening Methods (HTPS), on mechanisms relating to human health and test methods, and on exposure of foods and containers etc. to fetuses and adults. Examples of references are:

Exposure to endocrine disrupting chemicals: Ema et al. (1999a, 1999b, 2000), Hirose et al. (1999), Ikeda et al. (1999), Nagao et al. (1999a, 1999b, 2000a, 2000b), Ohta et al. (2000), Shirai et al. (1999), Tamura et al. (1999).

Test methods: Akimoto et al. (2000), Minegishi et al. (1997, 1998), Nagasaki et al. (1999a, 1999b), Nakagomi et al. (1999a, 1999b), Ohkura et al. (1999a, 1999b), Ohno et al. (1999), Suzuki et al. (1999a, 1999b).

Rough outlines of these works were shown in Table 2.

The Advisory Committee on Health Influence of Endocrine Disrupting Chemicals was established in April of 1998, in order to examine problems of endocrine disrupting chemicals. An interim report was published in November of the same year and provided via WWW (Table 1).

The Japan Society of Endocrine Disruptor Research, comprised of scientists and engineers from various different fields, was established in June 1998 to shed light on the scientific aspects of the problem. The background of the member scientists is spreading in a wide variety including medical, biological, fisheries, environmental sciences and engineering as well. The International Symposiums on Environmental Endocrine Disruptors were held at Kyoto in December 1998 and in Kobe in December 1999. The main topics in the symposiums and related meetings were: (a) Screening Methods and Cellular Mechanisms; (b) Testing and Hormone Responses; (c) Mechanism of Action; (d) Fish Testing; (e) The Effect of Endocrine Disruptors (ED) on Humans; (f) The Effect of EDs on Wildlife; (g) Dose-response; (h) Potential Effects on Human Health; (i) Basic Biology; (j) Pesticide; (k) Activities in Japan.

Rough outlines of these works were shown in Table 2.





One of the best ways to provide information widely is the use of Internet, especially WWW. Although English is necessary for international information exchange, Japanese is easier for domestic people. In the Home Page of National Institute of Health Sciences (NIHS), the information on Drugs, Food, Chemicals and Environment are represented. Recently, Endocrine Disruptor Information Guide for Researchers was included in the Topics of NIHS Home Page (Table 1). Although it is not yet completed, the Search Engine: (Web search for Endocrine Disruptors) and Lists of Paradigmatic Chemicals were available.

In NIHS, we have been developing related databases, such as Endocrine Disruptor Structure Database (EDSD) (Nakano et al., 1998), Binding Affinity Database for Endocrine Disruptor (BADB) (Kaminuma et al., 2000) and Receptor Database (RDB) (Nakata et al., 1999). In EDSD, chemical names, CAS registry numbers, synonyms, physicochemical properties and two-dimensional and three-dimensional structural data of 149 substances were included. With appropriate viewing software, users can generate three-dimensional images of chemicals. In BADB, experimental data for interaction of exogenous chemicals and bio-molecules were stored. At present, 742 Competition Binding Experimental results and 376 Enzyme Induction Experimental results were included in BADB. Figure 1 shows the relative binding affinity between human estrogen receptor a/b and ligands, which were picked up from BADB.

RDB included various receptor information, for instance structural information (amino acid sequences, secondary structure prediction, three dimensional structure image), functional information (DNA binding sites, ligand binding sites, etc.), genetic information (DNA sequences, gene information), the cell signaling networks information and transcription information. At present (June 2000), RDB contains 1,576 receptor proteins. The three-dimensional structure information was included in 121 receptor proteins. The DNA binding site and ligand binding site information were included in 206 and 130 receptor proteins, respectively. The data for transmembrane region was included in 1,070 receptor proteins. Recent versions of RDB included both information of EDSD and BADB, and much more. Using the table Steroid Hormone Receptor and Possible Endocrine Disruptor in the top page of RDB <http://>, users can refer to a steroid hormone receptor and possible endocrine disrupters (Figure 2).



Global Information Network on Chemicals


In June 1992, the United Nations Conference on Environment and Development Collection (UNCED) was held in Rio de Janeiro, Brazil. Chapter 19 of Agenda 21 of UNCED (UN, 1992) calls on communities "to promote intensified exchanges of information on chemical safety, use and emissions among all involved parties". It recommends that governments and relevant international organizations with the co-operation of industry should strengthen national institutions responsible for information exchange on toxic chemicals and strengthen international institutions and networks responsible for information exchange on toxic chemicals. Within the frame of the Inter-Organization Program for the Sound Management of Chemicals (IOMC), World Health Organization (WHO), International Labour Organization (ILO), United Nations Environmental Programs (UNEP) and Organization for Economic Cooperation and Development (OECD), with the support of the NIHS Japan initiated a project to establish a Global Information Network on Chemicals (GINC). The aim of GINC project is to provide networking arrangements for linking and better access by various users to existing international databases and documents on chemicals, as well as databases and chemical information systems available, or to be developed in each country.

Since the Internet has been launched and the WWW has become popular, the idea spread widely. GINC Home Page < index.html> was designed for the navigation to a wide range of WWW pages and databases in order to find useful information related to chemical safety: regulations, chemical names and synonyms, physico-chemical properties, toxicological data, protection for workers, environmental exposures, assessments and regulations. The United Nations organizations, the OECD, Environmental Protection Agency (EPA, United States), National Institute of Environmental Health Science (NIEHS, United States), and other Center of Excellences (COEs) have provided this information.

For the first GINC meeting at Tokyo 1994, only Korea and Japan were represented from Asia. Since then, attendance from China, Indonesia, Korea, Philippine, Singapore, Sri Lanka, Thailand and Vietnam gathered with the members from International Program on Chemical Safety/WHO (IPCS/WHO), International Register of Potentially Toxic Chemicals (IRPTC/UNEP), International Occupational Safety and Health Information Centre (CIS/ILO), OECD, EPA, NIEHS and Japan in GINC Asia Meeting. The corresponding organizations opened their Web Page for information exchange. These Home Pages and the GINC Asia page <> were connected reciprocally.





EPA formed the Endocrine Disruptor Screening and Testing Committee (EDSTAC) in 1996, and provided the reports EDSTAC via WWW. Under the legislation, some 80,000 existing chemicals and new chemicals were undergoing various screens and/or tests for their potential estrogenicity, as well as other hormonal activities. The integrated computational approach was reported to set priorities for these chemicals for experimental screening and testing (Tong et al., 1999). The OECD endocrine disruptor activity was initiated in 1996. The focus of the activity was to provide information on national and regional activities concerning endocrine disruptors, develop appropriate Test Guidelines and harmonize risk assessment approaches (Huet, 1999).

In the International Symposium on Environmental Endocrine Disruptors at Kobe 1999, many speakers from Denmark, Finland, Germany, Italy, Japan, Netherlands, OECD, Sweden, United Kingdom and United Station reported their works, and had heated discussion with the participants (Table 2). We expect these activities will lead to solutions for the endocrine disruptor problems.


Kotoko Nakata
Division of Chem-Bio Informatics, National Institute of Health Sciences, Tokyo, Japan.





AKIMOTO, T.; SUZUKI, H.; ARAI, Y.; NAKAMA, K. & SUZUKI, K., 2000. A locus responsible for dominant hairless gene (Ht) is located on rat chromosome 10. Experimental Animal, 49:137-140.

CHANG, X. T.; KOBAYASHI, T.; KAJIURA, H.; NAKAMURA, M. & NAGAHAMA, Y., 1997. Isolation and characterization of the cDNA encoding the tilapia (Oreochromis niloticus) cytochrome P450 aromatase (P450arom): Changes in P450arom mRNA, protein and enzyme activity in ovarian follicles during oogenesis. Journal of Molecular Endocrinology, 18:57-66.

EMA, M.; MIYAWAKI, E. & KAWASHIMA, K., 1999a. Suppression of uterine decidualization as a cause of implantation failure induced by triphenyltin chloride in rats. Archives of Toxicology, 73:175-179.

EMA, M.; MIYAWAKI, E. & KAWASHIMA, K., 1999b. Adverse effects of diphenyltin dichloride on initiation and maintenance of pregnancy in rats. Toxicology Letters, 108:17-25.

EMA, M.; MIYAWAKI, E. & KAWASHIMA, K., 2000. Effects of dibutylphtalate on reproductive function in pregnant and pseudopregnant rats. Reproductive Toxicology, 14:13-19.

HIROSE, M.; FUKUSHIMA, S.; IMAIDA, K.; ITO, N. & SHIRAI, T., 1999. Modifying effects of phytic acid and gamma-oryzanol on the promotion stage of rat carcinogenesis. Anticancer Research, 19:3665-3670.

HUET, M.-C., 1999. OECD activity on endocrine disruptors test guideline development. In: International Symposium on Environmental Endocrine Disruptors (ISEED) '99, Program & Abstracts, pp. 74-76. Tokyo: Environmental Health and Safety Division, Environmental Health Department, Environmental Agency.

IGUCHI, T., 1998. Environmental endocrine disruptors. Nippon Rinsho, Japanese Journal of Clinical Medicine, 56:2953-2962.

IKEDA, Y.; TAKAHASHI, S.; KIMURA, J.; CHO, Y.-M.; IMAIDA, K.; SHIRAI, S. & SHIRAI, T., 1999. Anophtalmia in litters of female rats treated with the food-derived carcinogen, 2-amino-1-methy-6-phenylimidazo [4,5-b] pyride. Toxicologic Pathology, 27:628-631.

ISEED '98, 1998. International Symposium on Environmental Endocrine Disruptors '98, Programs & Abstracts. Tokyo: Environmental Health and Safety Division, Environmental Health Department, Environmental Agency.

ISEED '99, 1999. International Symposium on Environmental Endocrine Disruptors '99, Programs & Abstracts. Tokyo: Environmental Health and Safety Division, Environmental Health Department, Environmental Agency.

JIANG, J. Q.; YOUNG, G.; KOBAYASHI, T. & NAGAHAMA, Y., 1998. Eel (Anguilla japonica) testis 11beta-hydroxylase gene is expressed in interrenal tissue and its product lacks aldosterone synthesizing activity. Molecular and Cellular Endocrinology, 146:207-211.

KAMINUMA, T.; TAKAI-IGARASHI, T.; NAKANO, T. & NAKATA, K., 2000. Modeling of signaling pathways for endocrine disruptors. Bio Systems, 55:23-31.

KAWAMURA, Y., 1999. Endocrine disruptor chemicals in food container and utensils. Pharmacia, 35:229-233.

KOBAYASHI, A.; SOGAWA, K. & FUJII-KURIYAMA, Y., 1996. Cooperative interaction between AhR.Arnt and Sp1 for the drug-inducible expression of CYP1A1 gene. Journal of Biological Chemistry, 271:12310-12316.

KUROKI, Y.; IWAMOTO, T.; LEE, J.; YOSHIIKE, M.; NOZAWA, S.; NISHIDA, T.; EWIS, A. A.; NAKAMURA, H.; TODA, T.; TOKUNAGA, K.; KOTLIAROVA, S. E.; KONDOH, N.; KOH, E.; NAMIKI, M.; SHINKA, T. & NAKAHORI, Y., 1999. Spermatogenic ability is different among males in different Y chromosome lineage. Journal of Human Genetics, 44:289-292.

MINEGISHI, K.; SUZUKI, S.; KANEKO, T.; INOUE, T. & TAKAHASHI, A., 1997. Distribution, accumulation and excretion of N,N'-Dimonomethylphenyl-p-phenylenediamine in the 2 year feeding test in rats. Japanese Journal of Toxicology and Environmental Health, 43:336-346.

MINEGISHI, K.; SUZUKI, S.; KANEKO, T.; INOUE, T. & TAKAHASHI, A., 1998. Metabolic fate of N, N'-Dimonomethylphenyl-p-phenylenediamine in the 2 year feeding test. Japanese Journal of Toxicology and Environmental Health, 44:37.

NAGAO, T.; SAITO, Y.; USUMI, K.; NAKAGOMI, M.; YOSHIMURA, S. & ONO, H., 2000a. Disruption of the reproductive system and reproductive performance by administration of nonylphenol to nreborn rats. Human and Experimental Toxicology, 19:284-296.

NAGAO, T.; SAITO, Y.; USUMI, K.; KUWAGATA, M. & IMAI, K., 1999b. Reproductive function in rats exposed neonatally to bisphenol A and estradiol benzoate. Reproductive Toxicology, 13:303-311.

NAGAO, T.; SAITO, Y. & YOSHIMURA, S., 2000b. Possible mechanism of congenital malformations induced by exposure of mouse preimplantaition embryos to mitomycin C. Teratology, 61:248-161.

NAGAO, T.; YOSHIMURA, S.; SAITO, Y. & IMAI, K., 1999a. Developmental toxicity of the topoisomerase inhibitor, etoposide, in rabbits after intravenous administration. Teratogenesis, Carcinogenesis and Mutagenesis, 19:233-241.

NAGASAKI, K.; MAASS, N.; MANABE, T.; HANZAWA, H.; TSUKADA, T.; KIKUCHI, K. & YAMAGUCHI, K., 1999a. Identification of a novel gene, DAM1, amplified at chromosome lp13.3-21 region in human breast cancer cell lines. Cancer Letters, 140: 219-226.

NAGASAKI, K.; SASAKI, K.; MAASS, N.; TSUKADA, T.; HANZAWA, H. & YAMAGUCHI, K., 1999. Staurosporine enhances camp-induced expression of neural-specific gene VGF and tyrosine hydroxylase. Neuroscience Letters, 267:177-180.

NAKAGOMI, M.; IIDA, S.; HARA, Y.; MATSUKI, Y.; NAMBARA, T. & SUZUKI, E., 1999a. Preparation of Specific Antiserum to 15 alpha-Hydroxyestrogens 15-N-acetylglucosaminides. Steroids, 64:491-496.

NAKAGOMI, M.; YAMADA, K.; MATSUKI, Y.; KURIHARA, H. & SUZUKI, E., 1999b. Enzyme immunoassay for the measurement of 17alpha-Estradiol 17-N-acetylglucosaminide in Rabbit Urine. Steroids, 64:301-307.

NAKANO, T.; KOYANO, K.; OHTAKE, C.; YAMAMOTO, M.; HASEGAWA, S.; TAKI, A.; YAMAMOTO, M. & KAMINUMA, T., 1998. Development of endocrine disruptors structure database. In: 21st Symposium on Chemical Information and Computer Science, Abstracts, pp. 94-97. Tokyo: Tatekawa Planning.

NAKATA, K.; TAKAI, T. & KAMINUMA, T., 1999. Development of a receptor database: Application to the endocrine disruptor problem. Bioinformatics, 15:544-552.

NISHIKAWA, J.; SAITO, K.; GOTO, J.; DAKEYAMA, F.; MATSUNO, M. & NISHIHARA, T., 1999. New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator. Toxicology and Applied Pharmacology, 154:76-83.

OBANA, H.; KIKUCHI, M.; OKIHASHI, M. & HORI, S., 1997. Determination of organophosphorus pesticides in foods using an accelerated solvent extraction system. Analyst, 122:217-220.

OHKURA, N.; HOSONO, T.; MARUYAMA, K.; TSUKADA, T. & YAMAGUCHI, K., 1999. The human NGFI-B gene gives rise to two isoforms with different expression profiles. Biomedical Research, 29:213-218.

OHKURA, N.; HOSONO, T.; MARUYAMA, K.; TSUKADA, T. & YAMAGUCHI, K., 1999. An isoform of Nurr1 functions as a negative inhibitors or the NGFI-B family signaling. Biochimica et Biophysica Acta, 1444:69-79.

OHNO, Y.; KANEKO, T.; INOUE, T.; MORIKAWA, Y.; YOSHIDA, T.; FUJII, A.; MATSUDA, M.; OHONO, T.; HAYASHI, M.; MOOMA, J.; UCHIYAMA, T.; CHIBA, K.; IKEDA, N.; IMANISHI, Y.; ITAGAKI, H.; KAKISHIMA, H.; KASAI, Y.; KURISHITA, A.; KOJIMA, H., MATSUKAWA, K.; NAKAMURA, T.; OHKOSHI, K.; OKUMURA, H.; SAIJO, K.; SAKAMOTO, K.; SUZUKI, T.; TAKANO, K.; TANI, N.; USAMI, M. & WATANABE, R., 1999. Inter-laboratory validation of the in vitro eye irritation tests for cosmetic ingredients. 1) Overview of the validation study and Draize scores for evaluation of the tests. Toxicology in Vitro, 13:73-98.

OHTA, R.; MATSUMOTO, A.; SATO, M.; SHIROTA, M.; NAGAO, T.; TOHEI, A. & TAYA, K., 2000. Postnatal behavior in hatano high- and low-avoidance rats following prenatal exposure to low dose of methylazoxymethanol. Neurotoxicology and Teratology, 22:405-413.

OKIHASHI, M.; OBANA, H. & HORI, S., 1998. Determination of N-methylcarbamate pesticides in foods using an accelerated solvent extraction with a mini-column cleanup. Analyst, 123:711-714.

SHIRAI, T.; HIROSE, M. & ITO, N., 1999. Medium-term bioassays in rats for rapid detection of the carcinogenic potential of chemicals. In: The Use of Short- and Medium-Term Test for Carcinogens and Data on Genetic Effects in Carcinogenic Hazard Evaluation (D. B. McGregor, J. M. Rice & S. Venitt, ed.), IARC Scientific Publications 146, pp. 251-273. Lyon: International Agency for Research on Cancer.

SUZUKI, H.; KOKADO, M.; SAITO, K.; KUNIEDA, T. & SUZUKI, K., 1999a. A locus responsible for hypogonadism (hgn). Mammalian Genome, 10: 1106-1107.

SUZUKI, E.; NAKAGOMI, M.; HASHIMOTO, M.; AGUI, M.; IIDA, S.; KONNO, K.; HARA, Y.; KURIHARA, H.; MATSUKI, Y.; IMAI, K. & ONO, H., 1999b. Preparation of Specific Antisera to 15alpha-Hydroxyestrogens. Steroids, 64:551-557.

TAKEYOSHI, M.; ANAI, S. & SHINODA, K., 2000. Changes in serum *2u-globulin levels in male rats given diethylstilbestrol (DES) and their applicability to a screening test for endocrine-disrupting chemicals. Archives of Toxicology, 74:48-53.

TAMURA, T.; MITSUMORI, K.; ONODERA, H.; TAKAHASHI, M.; FUNAKOSHI, T.; YAUHARA, K.; TAKEGAWA, K.; TAKAGI, H. & HIROSE, M., 1999. Time course observation of thyroid proliferative lesions and serum levels of related hormones in rats treated with kojic acid after DHPN initiation. Journal of Toxicological Sciences, 24: 145-155.

TOKUCHI, H.; HIGASHITSUJI, H.; NISHIYAMA, H.; NONOGUCHI, K.; NAGAO, T.; XUE, J. H.; ITOH, K.; OGAWA, O. & FUJITA, J., 1999. Expression of protein tyrosine phosphatase PTP-RL10 and its isoform in the mouse testis. International Journal of Urology, 6:572-577.

TONG, W.; PERKINS, R.; WU, J.; SHI, L.; TU, M.; FANG, H.; BLAIR, R.; BRANHAM, W. & SHEEHAN, D. M., 1999. An integrated computational approach for prioritizing potential estrogenic endocrine disruptors. In: International Symposium on Environmental Endocrine Disruptors (ISEED) '99, Program & Abstracts, pp. 50-51. Tokyo: Environmental Health and Safety Division, Environmental Health Department, Environmental Agency.

UN (United Nations), 1992. Report of the United Nations Conference on Environment and Development. Agenda 21. <>.

YOSHIDA, S.; SAGAI, M.; OSHIO, S.; UMEDA, T.; IHARA, T.; SUGAMATA, M.; SUGAWARA, I. & TAKEDA, K., 1999. Exposure to diesel exhaust affects the male reproductive system of mice. International Journal of Andrology, 22:307-315.

Escola Nacional de Saúde Pública Sergio Arouca, Fundação Oswaldo Cruz Rio de Janeiro - RJ - Brazil