Toxicologic pathology in Europe, as in the rest of the world, has seen a great deal of change over the past ten years, particularly with the advent of many different computerized systems of recording data. Pharmaceutical or chemical products developed by one particular company are distributed and registered today worldwide, and therefore there is a need for an international harmonization of procedures and methods applied in the safety evaluation process of these new components. In order to reduce the number of animal experiments, to allow a comparison between different studies, and to gain better acceptance by regulatory agencies worldwide, the standardization of procedures, the exchange of data in electronic formats, and a harmonization of terms and criteria for interpretation are necessary. All of these require efficient use of computerized systems.
In Europe, the development of an information system designed for use in routine toxicologic pathology, as well as for educational purposes, named RENI (REgistry Nomenclature Information system) in parallel with the establishment of the pathology data base system RITA (Registry of Industrial Toxicology Animal-data) and the implementation of a systematized classification for toxicological pathology. This system of standardized nomenclature and diagnostic criteria has been adopted by the World Health Organization (WHO)/International Agency for Research on Cancer (IARC) after a scientific review by pathology experts from Europe, the United States and Japan.

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The concept of formal quality assessment (QA) or “peer review” in animal toxicity testing began within the National Cancer Institute in the late 1970s. Although there is not a QA requirement in the U.S. regulatory agencies, QA has become widespread within government and private industry. The importance of QA in assuring the accuracy of pathology data has been well documented. Additional benefits of QA are the promotion of standardized nomenclature, and provision of continuing education for the pathologists involved. QA may be used to review a single target organ and/or lesion, or to review an entire study. If a study is particularly complex, or if discrepancies cannot be resolved, then a panel review or Pathology Working Group (PWG) can be convened. The PWG, under the guidance of a chairperson, examines slides “blindly” and records a consensus opinion. In both the QA review and in the PWG, a written report summarizes the findings of each review. Although there may be many sources of disagreement between pathologists, it should be emphasized that QA is not a pathology audit or a process for determining the competency of a pathologist.

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The distribution of lymphocyte populations in peripheral blood and lymphoid tissue in ruminants was examined using a panel of monoclonal antibodies, flow cytometry, and immunohisto-chemistry. In ruminants, the continuous Peyer's patch was proposed as a primary lymphatic organ, with a function similar to that of the bursa of Fabricius in birds. Our findings suggested that the arthritis caused by caprine arthritis-encephalitis virus is due to a hyperresponsive cellular immunity of the cytotoxic and suppressor T cells in the synovial fluid, and N cells in the peripheral blood lymphocytes are a major component of the immune system in this arthritis. The CD8⁺ T cells may play an effector role in suppressing bovine leukemia virus growth in recombinant vaccinia virus-vaccinated animals. Lymphomas of enzootic bovine leukosis were demonstrated as tumors of mature B cells with common characteristics.

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Interleukin-8 (IL-8) is involved in the pathogenesis of a number of inflammatory diseases, such as rheumatoid arthritis, uveitis, pancreatitis, and ulcerative colitis. Molecular cloning of canine IL-8 was performed to establish a basis for its investigation in the canine immune system. Using an LPS-stimulated canine lymph node cDNA library as a temperate, we performed PCR amplification for the isolation of canine IL-8 cDNA. The 400-bp band amplified with the primers, was extracted from the gel and cloned into a pUC118 plasmid vector. The nucleotide sequences were determined. Escherichia coli DH5α strain was used for the transformation and production of fusion protein containing glutath-ione-S-transferase (GST) and canine IL-8. Produced GST-canine IL-8 fusion protein was treated with Factor Xa and recombinant canine IL-8 was purified. Neutrophil shape change assay showed strong biological activity of purified recombinant canine IL-8. In order to clarify the pathophysiological role of IL-8, it is necessary to have an ELISA systems for the quantitation of IL-8 in order to increase the specificity and sensitivity for the evaluation of canine IL-8.

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Immunotoxicology has become a very important field because of the immunological adverse effects of various cytokines (IL-2 and IFNα, etc.) and biological response modifiers in addition to the immunosuppression caused by steroid hormones and anticancer agents and drug (antibiotic)-induced allergies. The adverse effects include the development of infectious diseases or various cancers due to immunosuppression and the occurrence of allergies or autoimmunity due to immunopotentiation. Studies to establish testing guidelines were done worldwide using immunosuppressants (azathioprine and cyclosporin A), and the most suitable marker or test system to detect immunotoxicity of xenobiotics was explored. Moreover, the necessity of immunotoxicity studies is stressed in “Redbood II” (FDA, draft; 1993). In experiments on thymus toxicity in the rat, the following data were obtained. The weight of the thymus increased from birth until 8 weeks of age and then decreased gradually. The weight was also decreased by stress induced by factors such as fasting, the attachment of an Elizabeth collar, etc. and the administration of cyclophosphamide (immunosuppressant). This drug caused degenerative changes in the thymus, spleen, and bone marrow and inhibited the plaque forming cell response to sheep red blood cells in rats. TGP-3 (human recombinant interleukin 2) caused fever via the production of interleukin 1β and increased the number of eosinophils.
When the immune system was suppressed by exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) or chronic administration of an immunosuppressant (cyclophosphamide), the susceptibility to infectious diseases and tumor formation was increased. On the other hand, when the action of the immune system was potentiated by the repeated administration of an immunomodulator (antirheumatic agent), autoimmune diseases rarely occurred. The importance of immunotoxicology and future directions for immunotoxicity testing are discussed in this paper.

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Immunotoxicity study is not required for development of a new drug in GLP guidelines in Japan. However, the need of such a study is growing with the progress of development of biotechnology products, antiviral agents against AIDS, immunotherapeutics, etc. According to the result of the questionnaire undertaken by JPMA, 18 out of 78 pharmaceutical companies have conducted immunotoxicity study on case-by-case basis. The main reasons why the study was conducted were based on the results of general toxicity studies, the pharmacological effects, and/or the trend of related-compounds, such as anti-cancer drugs. The findings judged to be associated with immunotoxicity consisted of histopathological changes in lymphoid tissues, weight changes in lymphoid organs and changes in bone marrow assay, hematology test, and blood chemistry test. Immunotoxicity of drugs were examined for cell-mediated immunity, humoral immunity, non-specific immunity, host resistance, and lymphocyte surface antigens. This survey showed that some companies have already established the testing capabilities for immunotoxicity study, but setting up a guideline for immunotoxicity study should be done with circumspection because determining immunotoxic effects is complicated.

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Pharmaceuticals, mostly nongenotoxic chemicals, induce neoplastic changes with relatively higher incidence in the rodents during chronic carcinogenicity bioassay. The liver is one of the most common target organs developing neoplasms and the results often give a serious impact on the development of new pharmaceuticals unless the results are appropriately interpreted as of non-relevance to human.
In this review article we have been trying to collect as many informations as possible to understand a complex and multistep process of carcinogenesis particular to hepatocarcinogenesis with interaction of pharmaceutical compounds as well as on the practical evidences helpful for risk assessment of pharmaceuticals for human use.
Carcinogenesis caused by pharmacodynamic and hormonal effects has been comparatively well elucidated with pharmaceuticals and not included in this article.
In this article, the following topics are discussed; predictive value of altered hepatocellular foci in some chronic carcinogenicity bioassay, phenobarbital type enzyme induction and tumor promotion, recently proposed mechanisms of carcinogenesis caused by peroxisome proliferators, role of apoptosis in hepatocarcinogenesis, and oncogenes, growth factors, and gap junctional intercellular communication in hepatocarcinogenesis.

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The characteristics of non-genotoxic phenolic compound-induced rat forestomach carcinogenesis were analysed. The incidence of forestomach lesions did not increase with BHA pretreatment, but was elevated when BHA or caffeic acid was administered after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initiation in the 2-step rat forestomach carcinogenesis model. Induction of forestomach tumors was also strongly enhanced by BHA given together with genotoxic carcinogens such as methylnitrosourea (MNU), 3, 2'-dimethyl-4-aminobiphenyl (DMAB) or 2, 2'-dihydroxy-di-n-propyl-nitrosamine (DHPN) as compared to with these carcinogens alone. DNA synthesis in the forestomach epithelium increased within 12 hr to 3 days after commencement of chemical treatment in all cases. Toxicity (inflammation and erosion or ulceration) and cell proliferation became evident subsequent to increase in DNA synthesis in each case. Elevated expression of c-for and c-myc oncogenes in the forestomach epithelium was demontrated 15 min after beginning treatment with BHA, caffeic acid or 4-methoxyphenol. In a reversibility study, although most of the proliferative lesions in the forestomach epithelium induced by these antioxidants regressed after cessation of chemical treatment, some dysplastic lesions were still observed at week 48. The results indicated that these non-genotoxic phenolic compounds possess strong promoting activity, primarily acting as mitogens in rat forestomach epithelium, with regeneration due to toxicity further enhancing cell proliferation. During this continuous elevation of cell turnover, dysplastic lesions appear which persist and presumably play a role in the eventual development of forestomach carcinomas.

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The endocrine system is one of the major communication apparatus covering the whole body. It mediates not only between neighboring cells or organs, but also on distant organs through specific molecules containing an amino-acid sequence.
In carcinogenesis, it is now well accepted that normal cells can be transformed by a given mutagen and these cells may go on to proliferate if it is promoted by any given substances to which the cell has a receptor. Among the factors influencing the development of cancers, the sex hormone is one of importance. Many of the predominant cancers such as those of lung, stomach, esophagus, and liver occur preferentially in the male. However, endocrine-related tumors occurring in the pituitary and thyroid glands mainly occur in the female. Blood-borne tumors such as lymphoma and leukemia are also predominant in the female, and spontaneous or radiation induced tumors in rodents are similar to those of human tumors.
Estrogen plays a role in inducing endocrine tumors by itself without any mutagenic potency. Its effect may be through the estrogen receptor (ER) within the cellular cytoplasm as well as the nuclear acceptor. It enhances thyroid tumorigenesis by way of acting on malignant progression in the methylnitrosourea-induced rat thyroid tumors. In the pituitary gland, it is a potent tumorinogen by itself and the growth and passage of pituitary tumors both in vivo and in vivo have required the existence of estrogen. By the cloning of an estrogen induced pituitary tumor in a female F344 rat, we established an estrogen dependent clone, MtT/Se. Its growth in vivo essentially necessitates estrogen, and shows dosedependent growth from 10^-12 to 10^-9 of estrogen. It has estrogen receptors and produces growth hormone. Its growth seems to be self-regulated by an insulin-like growth factor-1 (IGF-1).

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In vivo-in vitro hepatocyte replicative DNA synthesis (RDS) tests using male F344 rats and B6C3F1 mice were conducted in the present study applying single treatments of 56 nongenotoxic (Ames-negative) hepatocarcinogens, 7 genotoxic (Ames-positive) hepatocarcinogens, 31 nongenotoxic and genotoxic noncarcinogens, and the correlation between early increased RDS events leading to cell proliferation and hepatocarcinogenesis was then reviewed.
In the RDS test, 51 of the 63 nongenotoxic and genotoxic hepatocarcinogens clearly induced increased RDS events, whereas it was very rare for noncarcinogens to cause such increase. A good correlation between early RDS induction and hepatocarcinogenicity was thus achieved. The results suggest that nongenotoxic hepatocarcinogens always give equivocal DNA modifications which never lead to Ames-positive events, such as extremely low levels of oxidative DNA adducts and/or alteration of DNA methylation levels (hypomethylation). Genotoxic hepatocarcinogens might simultaneously act as tumor promoters in multistage carcinogenesis and genetic instability-inducing agents. That early disruption of cell cycle controls, resulting in induction of RDS events by nongenotoxic and genotoxic hepatocarcinogens, is postulated to contribute to carcinogenesis in line with a process of increasing genetic instability. In the present review, the current status of the correlation between cell proliferation and hepatocarcinogenesis is also discussed.

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Hepatocyte-like cells were observed in the stomach of one female and one male B6C3F₁ mice. Gastric hepatocytes in the glandular stomach of a female mouse were observed in adenocarcinoma as same as reported previously. However, the male mouse showed gastric hepatocytes adjacent to the dysplastic gastric epithelium of the glandular stomach. These cells have the histological, ultrastructural and immunohistochemical characteristics resembling those of normal hepatocytes. Since alpha-fetoprotein was also demonstrated in these cells, a comparison with alpha-fetoprotein-producing gastric adenocar-cinoma which develops in humans was attempted. However, the paucity of neoplastic nature of these hepatocyte-like cells suggested that they were metaplastic in origin similarly to the pancreatic hepatocytes.

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Since a myxomatous white mass (8×5×4mm) was grossly observed postmortem in the right ventriclar epicardium of rat F344 which was 106 weeks old, a histopathological examination was conducted. Histologically, there were two different areas. One consisted of spindle-shaped tumor cells, which were arranged in a herring bone pattern, interlacing bundlle pattern, whorl pattern and nuclear palisading (Antoni type A). The other consisted of small round tumor cells, which showed myxomatous proliferation and cyst formation (Antoni type B). These tumor cells were diagnosed as malignant schwannoma by positive immunoreactivity to S-100 protein antigen in the cytoplasm and nuclei, and ultrastructual features of circumferential basal lamina. This case is interesting in that the large tumor which originated in the right ventricle, proliferated extrusively from ventricle to epicardium, and tested strongly positive for S-100 protein antigen in an immunohistochemical staining procedure.

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