Hypocatalasemic mice were treated with N-methyl-N-nitrosourea (MNU) in their drinking water or gastric intubation for 16 weeks or were given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in their drinking water for 6 months. With MNU treatment in the drinking water, well differentiated tumors were observed in the glandular stomachs of one male (4%) and 5 female (15%) animals. Sarcomas of the dermis also appeared in 10 males (40%). Adrenal lesions developed in I I females (35%) along with kidney, ovary, and uterus tumors. In the Group given MNU intubation, squamous cell carcinomas of the forestomach appeared from 122 days after the first treatment, reaching incidences of 81% in males, 94% in females, with invasion of the diaphragm in 3 male cases and the diaphragm, liver, pancreas, or adrenal in I I female cases. Glandular stomach tumors occurred in 12 males (52%), with 3 being signet ring cell carcinomas, and in 12 females (35%). In a sub-Group undergoing forestomach removal after MNU intubation esophagectasis appeared from 80 days after the operation at incidences of 63% in males and 74% in females. The glandular stomach tumors including 3 signet ring cell carcinomas were found in 17 of 46 (40%) males, 2 of 18 (11%) females demonstrated lesion, one being a signet ring cell carcinoma. In the Group receiving MNNG treatment, anemia appeared from the 5.5 month time point and no gastric tumors were observed. The present results do not support the conclusion that MNU induction of glandular stomach lesions in hypocatalasemic mice may provide a useful model of signet ring cell carcinomas

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In 1993, the Society of Toxicologic Pathologists (STP) and the International Life Sciences Institute (ILSI) established a Joint STP-ILSI Committee in aid of international harmonization of nomenclature and diagnostic criteria used in toxicologic pathology. The objective of the Committee's work is to develop standardized nomenclature and diagnostic criteria for proliferative and nonproliferative lesions in laboratory animals used in chemical toxicity/carcinogenicity studies so that they are accepted and used by toxicologic pathologists and regulatory agencies around the world. Since its establishment in 1993, the Joint STP-ILSI Committee has made significant progress in standardization for rat proliferative/nonproliferative lesions and also started the work on those in the mouse. An initial draft outline of hepatic proliferative lesions in mice was presented at the 14th International Symposium of the STP in June 1995, and the development of standardized nomenclature and diagnostic criteria is currently underway. The Committee is planning to finalize this work on mouse proliferative lesions in the liver as well as those in other organ systems by April 1997. After reaching consensus, the final manuscript will be used both for the International Agency for Research on Cancer (IARC) publication, “International Classification of Rodent Tumours, Part II, The Mouse, ” and the STP “Guides on Standardized System of Nomenclature and Diagnostic Criteria” (SSNDC).

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Secondary polycythemia was observed in hepatocellular carcinoma-bearing B6C3F₁ mice which were used as control group animals in 2-year carcinogenicity studies. Statistically significant increases in erythrocyte count, hematocrit value, hemoglobin concentration, and absolute reticulocyte count were noted in the peripheral blood of hepatocellular carcinoma-bearing mice as compared with non-tumor-bearing mice. A decrease in mean corpuscular volume and mean corpuscular hemoglobin value was also noted in hepatocellular carcinoma-bearing mice. Mean corpuscular hemoglobin concentration and relative reticulocyte count in hepatocellular carcinoma-bearing mice were comparable to those in non-tumor-bearing mice. In addition, the plasma erythropoietin level in hepatocellular carcinoma-bearing mice was significantly higher than that in hepatocellular adenoma-bearing mice and that in non-tumor-bearing mice. Therefore, the hematological changes observed in hepatocellular carcinoma-bearing mice were diagnosed as secondary polycythemia. At necropsy, the spleen in hepatocellular carcinoma-bearing mice was dark-red and enlarged 2 to 4 times compared with that in non-tumor-bearing mice. Histopathologic examination revealed proliferation of the erythroid cells and megakaryocytes in the spleen and bone marrow in hepatocellular carcinoma-bearing mice, suggesting an increase in erythropoietic activity. These findings indicate that increased levels of plasma erythropoietin in hepatocellular carcinoma-bearing mice stimulate erythropoiesis in the hematopoietic organs and result in proliferation of microcytic and hypochromic erythrocytes. Possible mechanisms for the induction of secondary polycythemia in hepatocellular carcinoma-bearing mice are discussed.

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Effects of simultaneous administration of 5 or 10 heterocyclic amines at low dose levels on rat liver carcinogenesis were investigated using our medium-term bioassay protocol. Male F344 rats were initially given diethylnitrosamine (DEN, 200mg/kg, i.p.) and beginning 2 weeks later received heterocyclic amines for 6 weeks. All animals were subjected to partial hepatectomy at week 3 and killed at week 8. Five carcinogenic heterocyclic amines in each experiment 1 (Trp-P- 1, Glu-P-2, IQ, MeIQ, MeIQx) and experiment 2 (Trp-P-2, Glu-P- 1, McAαC, AαC, PhIP) were administered together or individually in the diet at levels of 1/1, 1/5, or 1/25 carcinogenic doses, and all 10 chemicals of 1/10 or 1/100 levels in experiment 3. Induction of immunohisto-chemically-demonstrated glutathione S-transferase placental form (GST-P) positive foci, reliable preneoplastic lesions, was generally increased in the combination groups over the sum of individual effects. Thus an apparent synergism was observed based on the heteroadditive model, the most obvious effects being found in the group given all 10 chemicals at the 1/10 dose levels. However, the values in groups given chemical mixtures were generally close to means of 5 or 10 individual data in their corresponding higher dose groups, indicating that isoadditivity might occur in those groups. The theoretical basis underlying the concept of synergism is briefly reviewed and a simple method for analysis of isoadditivity is proposed.

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Short- and mid-term model systems for analyzing the early proliferative responses of hepatocytes following administration of hepatocarcinogens were used to elucidate the biological nature and morphogenesis of preneoplastic and neoplastic lesions involving hepatocytes of B6C3F₁ mice exposed to test chemicals in a long-term carcinogenicity study. Immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression, in addition to immunohistochemical analysis of the induction of cytochrome P-450 isozymes in the livers of these mice were compared one week after receiving a single dose of one of the following chemicals; diethylnitrosamine, safrole, phenobarbital, and non-genotoxic carcinogens (Xa and Xb). Differences in the time or the site of the appearance of PCNA-positive hepatocytes, and the regional expression of P-450 isozymes in the liver, were seen between these chemicals. Each type of preneoplastic foci of cell alteration (FCA) and neoplasia, both hepatocellular adenoma (HCA) and carcinoma (HCC), were analyzed at the 18 and 25 week stages in studies on diethylnitrosamine-initiated two-step, hepatocarcinogenesis in mice promoted by phenobarbital and/or sex hormones (testosterone in the males, and ethinylestradiol in the females). Marked heterogeneity in the staining of each phenotype of basophilic and eosinophilic FCA, or HCA with biomarkers, including PAS, P-450, G-6-Pase, γ-GT, π-GST, and PCNA, was seen. There were no apparent differences between the characteristics of each type of chemically-induced proliferative nodular lesions and the spontaneously occurring ones. Biomarker analysis of the proliferative hepatocytes from short- and mid-term model mice may help to predict the hepatocarcinogenicity and the potency of test chemicals, although further studies with a variety of known hepatocarcinogens will be needed.

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A retrospective evaluation of spontaneous hepatocellular tumors in B6C3F, mice of 9 two-year and 4 seventy eight-week carcinogenicity studies was conducted. The incidences of hepatocellular adenomas and carcinomas (HCCs) in males were significantly higher than those in females in both series, and development of less-well-differentiated carcinomas with metastases was clearly linked to aging.
PCNA positive indices of well-differentiated HCCs were significantly higher than those of hepatocyte foci, hepatocellular adenomas, and moderately differentiated HCCs. This parameter was useful for the definition of foci and adenomas because in these cases the PCNA positive cells demonstrated a uniform distribution pattern. The number of AgNOR dots tended to increase in accordance with the progression of hepatocarcinogenesis, with bizarre-irregularly shaped forms found in the nuclei of moderately differentiated HCC cells. This parameter therefore appears to be useful for evaluation of the degree of malignancy in hepatocellular carcinomas.
While no c-H-ras protein expression could be detected in 9 cases of well-differentiated HCCs examined in the present study, 18 to 50% of preneoplastic lesions (foci) and neoplastic lesions including adenoma, carcinoma within adenoma, and moderately differentiated HCC.
The present study showed that assessment of the atypical grade in HCC is paramount for evaluation of malignancy, requiring appropriate methods for objective diagnosis.

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Serial ultrasonographic examinations of cephaloridine (CER)-induced renal lesions were performed in rabbits. The nephrosonograms in CER-injected rabbits showed enlarged renal echoic area, increased cortical echogenicity, increased medullary hypoechoic area, and enhanced corticomedullary demarcation. The kidneys in CER-injected rabbits showed macroscopically renal enlargement, cortical discoloration, and medullary enlargement and congestion, and showed histopathologically cortical tubular necrosis, urinary casts in the distal nephrons, and congestion in the outer medulla. These results suggest that drug-induced tubular necrosis is indicated by increased renal cortical echogenicity and that ultrasonographic examination is useful for evaluation of drug-induced nephrotoxicity.

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FK506-induced nephrotoxicity has been reported to occur by arteriolar constriction of the renal cortex, which is induced through the production of such vasoactive factors as thromboxane A₂ (TX A₂), renin, etc. In the present study, we investigated whether FK506-induced nephrotoxicity is improved by concomitant antagonists for TX A₂, angiotensin II, endothelin, adenosine, adrenergic α₂ receptor, platelet activating factor, and calcium channel, and found that some showed a potentiality of being antidotes to FK506-induced nephrotoxicity. A calcium antagonist showed the best improving effects and was followed by a TXA₂ dual inhibitor and an angiotensin II antagonist. An α₂ blocked showed a slight improvement, but endothelin, adenosine, and platelet activating factor (PAF) antagonists had no effects on the nephrotoxicity. The results suggested that FK506-induced nephrotoxicity occurs by constriction of the renal artery, and that various vasoactive factors are involved. In the present study, FK506 and antagonists for vasoconstrictive factors were dosed to rats and the parameters of nephrotoxicity were examined. In this kind of study, antidotes need not only improve the toxicity of the main drug; they must not diminish its pharmacological action. Accordingly, both toxic and pharmacological parameters must be investigated in a study aimed at finding an antidote to side effects of medicine. Thus, in the present paper we put forth our opinions about estimation of concomitant dosing.

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In order to examine the usefulness of simplified quantitative morphometry with stage grouping analysis of spermatogenic cycle for assessing testicular toxicity, ten-week-old male rats received a single dose of 100mg/kg body weight ethylene-1, 2-dimethanesulfonate (EDS). At 1, 3, 7, 14, and 28 days after the EDS treatment, animals were sacrificed for histopathological evaluation. The stages of the spermatogenic cycle were classified into four major groups (stages I-VI, VII-VIII, IX-XI, XII-XIV) for simplified screening. The numbers of seminiferous epithelial cells at each designated stage were counted in a total of 5 seminiferous tubules of each group per animal. The results were compared with those obtained from a strict stage analysis of the seminiferous tubules in the individual stages II-III, V, VII, X, and XII. With both analyses, Leydig cells were found to have disappeared in the interstitial tissue within 3 days after the EDS treatment, and decreases of pachytene spermatocytes and round spermatids were observed in the seminiferous tubules thereafter. The results of the present screening morphometry, using classification of stages into four groups, were similar to those from the strict stage analysis. We propose that the simplified approach tested in the present study can be applied to advantage for the evaluation of testicular toxicity.

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Spontaneous gastric carcinoids were observed in a 104-week-old male and a 112-week-old female Crl: CD (SD) Br rat. The tumors showed intramucosal proliferation and gave positive results in argyrophilic reaction. Ultrastructurally, variable numbers of electron-dense or electron-opaque secretory granules were observed in the cytoplasm of the tumor cells. These tumors were considered to be of enterochromaffin- like (ECL) cell origin.

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