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Identification of ovarian toxicity is very important for safety assessment of drugs and other environmental chemicals. The detection of interference with ovarian function is very hard without a thorough understanding of the normal ovarian morphology based on reproductive physiology. The focus of the present study was therefore a practical analysis in each stage of the estrous cycles using ovaries obtained from 143 rats demonstrating normal cycling. Transversely dissected maximum areas in the ovaries were examined microscopically for the two major features, follicles and corpora lutea (CL). Classification of growing follicles was in reference to Pedersen and Peters (1968), and functionally divided into follicular stimulating hormone (FSH)-independent and dependent categories. The former, small and medium-sized follicles, respectively primordial/primary and preantral follicles, could be readily detected by immunohistochemical staining for proliferating cell nuclear antigen (PCNA). The large antral and Graafian follicles and large sized atretic follicles showed sequential changes depending on the estrous cycle stage. CL could be divided into currently and previously formed examples. Currently formed CL underwent remarkable changes in their appearance with the cycle, reflecting ovulation and progesterone production. Thus morphological analysis that is synchronized the large antral follicle changes with recently formed CL ones allows the ovary to be classified into the each estrous cycle stage. Morphological deviation from any synchronized combination provides a first pointer of ovarian toxicity. PCNA immunohistochemical staining is also useful to detect small follicles.

Kojic acid (KA) has been widely used as a quasi-drug ingredient. Possible promotion activity of KA was suggested on livers of mouse and rat by findings obtained in genotoxicity and carcinogenicity studies performed thus far. Therefore, in order to examine safety as a quasi-drug ingredient, we investigated the presence of initiation activity in rat liver and the photo-genotoxicity and carcinogenicity in mouse skin. In medium-term carcinogenesis test in rats, 2.0% KA was orally given to F344/DuCrj rats for 4 weeks of the initiation period, followed by the combination of partial hepatectomy and treatment with a hepatocarcinogenesis promoter, phenobarbital (PB). As a result, glutathione S-transferase placental form (GST-P) positive foci of 0.2 mm or more in diameter in the KA group, which is usually used in determination of pre-cancerous lesions, did not increase significantly in both numbers and areas compared with those of the non-initiated controls. In the concurrent analysis, however, numbers of GST-P-positive foci of two cells or more and 0.1 mm or more in diameter increased slightly, and possible weak initiation activity of KA was equivocal. However, considering the known fact that KA exerts promotion activity in the liver of F344 rats by long-term dietary administration, it was suggested that the observed slight increase of the numbers of GST-P-positive foci in rat liver was the effect of promotion activity of KA rather than the initiation activity. In DNA adducts formation assay in a rat liver, no clear adducts derived from KA were detected in male F344/DuCrj rats administered 0.5% or 2% KA orally, and KA was considered not to form DNA adducts in rat liver. In the in vitro photo-reverse mutation assay with bacteria, KA exerted weak photo-mutagenicity. Furthermore, in chromosome aberration study in Chinese hamster lung cells (CHL/IU cells) with UV irradiation, KA induced chromosome aberration at high-dose (1.4 mg/mL) treatment with UV irradiation, but was negative without UV irradiation. However, in the in vivo photo-micronucleus study in mouse, in which 1.0 or 3.0% KA containing cream was applied twice to the back of the animals with a 24-hr interval, KA did not induce micronuclei in mouse epidermal cells. Based on these results, it is considered that the risk of KA to exert photo-carcinogenicity is quite low in the skin. In skin carcinogenesis bioassay for initiation-promotion potential, 3.0% KA cream formulation was applied to the back of the mouse for 1 week (once a day, total 7 times) and for 19 weeks (5 times a week, total 95 times) during the initiation and the promotion stages, respectively. No skin nodules were observed in any animal skins formed due to KA treatment given in either stage. Therefore, KA is considered not to possess initiation nor promotion activity of skin carcinogenesis. Furthermore, from the above findings, it is suggested that KA is virtually safe as a quasi-drug ingredient.

Two-generation reproductive toxicity studies using rats of benzophenone, n-butylbenzene, butyl benzyl phthalate, 2,4-dichlorophenol, dicychlohexyl phthalate, diethyl phthalate, 4-nitrotoluene, lindane and vinclozolin, were performed to investigate whether these chemicals have endocrine-mediated effects with the support of the Ministry of Economy, Trade and Industry and the Ministry of the Environment. Benzophenone exposure was via the diet at concentrations of 0, 100, 450 or 2000 ppm, n-butylbenzene was administered orally by gavage at dose levels of 0, 30, 100 or 300 mg/kg/day, butyl benzyl phthalate was administered orally by gavage at dose levels of 0, 100, 200, or 400 mg/kg/day, 2,4-dichlorophenol was administered in the diet at concentrations of 0, 500, 2000 or 8000 ppm, dicyclohexyl phthalate was given in the diet at concentrations of 0, 240, 1200 or 6000 ppm, diethyl phthalate was administered in the diet at concentrations of 0, 600, 3000 or 15000 ppm, 4-nitrotoluene was administered orally by gavage at doses of 0, 40, 80, or 160 mg/kg/day, lindane exposure was in the diet at concentrations of 0, 10, 60, or 300 ppm, and vinclozolin treatment was by feeding diet at concentrations of 0, 40, 200 or 1000 ppm.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a heterocyclic amine found in cooked meat, is a strong mutagen in the Salmonella/microsome assay and was proven to be a hepatocarcinogen in rodents. We used the lacI transgenic (Big Blue(R)) mouse to investigate MeIQx genotoxicity in vivo. lacI mutant frequencies were examined in liver and colon after single intragastric administration of MeIQx (males) or 12 weeks of feeding in the diet (males and females). Micronucleus induction was monitored in the peripheral blood and cell proliferating activity was monitored by proliferating cell nuclear antigen (PCNA) immunostaining, but only after the intragastric administration. Intragastric treatment with MeIQx (100 mg/kg) did not increase mutant frequency (MF) in liver or colon but it did induce a slight but statistically significant increase in the incidence of micronucleated reticulocytes 48 h after the treatment. No apparent increase in PCNA-positive foci was observed in any of tissues analyzed 14 days after the treatment. Administration of MeIQx (300 ppm) in diet for 12 weeks, however, caused MF increases in liver and colon in male and female mice, with greater increases in the females. An increase was also obvious after 4 weeks, but only in females. The sex difference in MF is consistent with the fact that female mice are more susceptible to MeIQx carcinogenesis. These results demonstrated that in the transgenic mouse mutation assay, long-term feeding of MeIQx was more effective than single gastric exposures in revealing the compound's mutagenicity in the target organs of carcinogenicity and that sex differences in susceptibility can also be observed.

The modifying effects of dietary L-methionine in the post-initiation phase of pancreatic carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Groups consisting of 20 and 30 animals, respectively, were given BOP subcutaneously, once a week five times at a dose of 10 mg/kg body wt. and then continuously fed diet supplemented with 2% (group 1) or 0% (group 2) methionine (weeks 5-32). After five subcutaneous injections of saline, group 3 animals were similarly fed diet supplemented with 2% methionine for the same period. The incidence of pancreatic ductal adenocarcinomas was significantly lower in group 1 (36.8%, P

The National Institute of Health Sciences and 28 member companies of the Japan Pharmaceutical Manufacturers Association (JPMA) have conducted a validation study intended to examine whether or not lesions in the male reproductive organs noted in 4-week treatment studies can also be detected after 2-week treatment. In this study, lesions in the male reproductive organs after 2-week treatment were, therefore, compared with those after 4-week treatment. A total of 24 test substances was evaluated, these being nucleic acid modulators, cell division inhibitors, central hormonal modulators, hormonal drugs and their antagonists, other drugs and general chemicals. Among these substances, theophylline did not cause any appreciable lesions in the male reproductive organs even after 4-week treatment in the preliminary studies. With busulfan, data reported in the literature was not reproduced in the preliminary study and all animals died. Therefore, detailed examinations were not conducted for busulfan and theophylline. The remaining 22 test substances, when given to animals for 2 weeks at doses equal to or higher than for 4-week treatment, caused lesions similar to those noted after 4 weeks. It is evident from these findings that effects of pharmaceuticals on the male reproductive organs can be detected in most cases with 2-week treatment.

The effects of long-term concurrent administration of powdered fish meal and sodium nitrite were examined in F344 rats. A total of 600, 6-week-old rats were divided into 6 male and 6 female groups, each consisting of 50 animals. Rats in groups 1-3 and 7-9 were respectively fed diets supplemented with 64%, 32% and 8% (basal diet) fish meal, and simultaneously given 0.12% sodium nitrite in their drinking water. Groups 4-6 and 10-12 were respectively given 64%, 32% and 8% fish meal and tap water. At the 104th week, all surviving animals were killed and examined histopathologically. Treatment with fish meal dose-dependently increased the incidences and multiplicities of atypical tubules, adenomas and renal cell carcinomas in sodium nitrite-treated males. Females were less susceptible than males for renal tumor induction. In males given the 64% fish meal diet alone, the incidence and multiplicity of atypical tubules were also significantly increased as compared with the 8% fish meal alone case. Nephropathy was apparent in fish meal-treated groups in a clear dose-dependent manner, irrespective of the sodium nitrite treatment, and was more prominent in males than in females. Dimethylnitrosamine was found in the stomach contents after 4-week treatment with 64% fish meal plus 0.12% sodium nitrite, at a level twice that in the 8% fish meal plus 0.12% sodium nitrite group. The results clearly indicate that concurrent administration of fish meal and sodium nitrite induces renal epithelial tumors. Further studies are required to elucidate how nephropathy and nitrosamines produced in stomach contents may contribute to the observed renal tumor induction.

A newborn mouse two-stage tumorigenesis assay was evaluated as a possible alternative to chronic rodent carcinogenicity bioassays by investigating the carcinogenicity of two major heterocyclic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). One week after birth, Crj:CD-1 mice of both sexes were subcutaneously administered N-nitrosobis(2-oxopropyl)amine (BOP) at a dose of 50 mg/kg as an initiation treatment and starting 2 weeks thereafter they were fed diets supplemented with MeIQx at concentrations of 300, 30, 3 or 0 ppm or PhIP at 200, 50, 10 or 0 ppm for 23 weeks. Animals in all groups predominantly developed tumors of the lung and liver. Pulmonary adenomas and adenocarcinomas were observed in all groups with high incidences, without any significant differences between the groups. MeIQx and PhIP did not influence the multiplicity except in the group given 10 ppm PhIP where it significantly increased the number of pulmonary adenomas (P

At the present time, there are no uniform standards for the duration of non-rodent chronic toxicity studies. The European Union (EU) requires a 6-month non-rodent study. In Japan, a 6-month study is sufficient for most, but not all, compounds. The U.S. Food and Drug Administration (FDA) maintains its standard duration of 12 months for non-rodents, with 6-month studies accepted for some clinical indications on a case-by-case basis. To achieve harmonization on the duration of non-rodent toxicity studies, each member regulatory region (EU, U.S., and Japan) of the International Conference on Harmonization (ICH) collected non-rodent studies with significant new toxicological findings that had occurred after 6 months. An ICH expert working group was organized that included representatives from the regulatory authorities of each ICH region, to jointly review all available case studies for the purpose of arriving at a consensus on the best duration time for non-rodent toxicity studies. Eighteen case studies were identified and evaluated (16 original cases plus 2 additional FDA cases); most of the toxicities identified fell into the following categories: (1) toxicities identified at 6 months; (2) toxicities observed at 12 months, which were absent or considered isolated and not noteworthy findings at 6 months; (3) drug-related deaths or morbidity that occurred between 6 and 12 months, with a pattern of toxicity that permitted the interpolation of findings to an intermediate interval between 6 and 12 months; and (4) a shift in the dose response for toxicity with increasing duration of drug exposure. Of the 18 cases evaluated, 11 supported a study-duration of 9-12 months, 4 supported a duration of 12 months, and the 3 remaining cases indicated that a 6-month study would be adequate. The working group concluded that there was sufficient evidence to support a harmonized 9-month duration for non-rodent toxicity studies, which would be applicable for most categories of pharmaceuticals.

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on cytoplasmic free calcium ion concentration ([Ca2+]i) were investigated in primary cultured rat hepatocytes. A high concentration (4.0 microM) of TBT increased resting levels of [Ca2+]i and then induced cell blebs resulting in cell death within 2 h. The increase in [Ca2+]i, but not the cell death, depended on the presence of extracellular Ca2+, suggesting that the increase in [Ca2+]i is not critical for the cytotoxicity of TBT. A low concentration (0.1 microM) of TBT did not have any toxic effect (decrease in ATP content, decrease in viability, and shape change) on cultured hepatocytes and did not change [Ca2+]i. However, the calcium responses induced by phenylephrine, [Arg8]-vasopressin, and ATP were suppressed in the cells pretreated with 0.1 microM TBT for 30 min. The suppression was not observed in the cells pretreated with 0.1 microM TBT for only 1 min. Pretreatment with 0.1 microM TBT for 30 min had no effect on the inositol 1,4,5-triphosphate content or its increase in response to hormonal stimulation. These results suggest that TBT suppresses hormone-induced calcium responses at nontoxic low concentrations.

In recent intervention studies, beta-carotene failed to reduce or even increased the incidence of lung cancers in smokers. In the present investigation, the modifying effects of beta-carotene at various doses on the development of upper respiratory tract tumors were investigated in Syrian hamsters treated with diethylnitrosamine (DEN) and cigarette smoke. A total of 120 male 5-week-old hamsters were divided into 4 groups, each consisting of 30 animals. After a single subcutaneous (s.c.) injection of 100 mg/kg DEN, hamsters in groups 1-4 were respectively administered diets supplemented with beta-carotene at doses of 0.5%, 0.05%, 0.005% or 0% during experimental weeks 1 to 13, and simultaneously exposed to cigarette smoke. The duration of cigarette smoke exposure was 9 min twice a day, 5 days a week. Because of a marked reduction of body weight in group 1, the highest dose of beta-carotene was changed to 0.25% after 10 days. In all groups, epithelial hyperplasias and/or papillomas were induced in the larynx and trachea. However, the incidence and multiplicity of papillomas in group 1 were significantly (P < 0.05) lower than the group 4 values. Moreover, the beta-carotene treatments significantly (P < 0.05 or 0.01) reduced both the incidence and multiplicity of hyperplasias in a dose-dependent manner. The levels of retinol and beta-carotene in the serum, and the retinol level in the liver, were also elevated with dose dependence. Our results thus indicate that beta-carotene inhibits tumorigenesis, even at the high dose of 0.25%, under the present experimental conditions.

A view that 14 stages of rat spermatogenic cycle could be arranged into 4 groups, viz., conventional stages I-VI, VII-VIII, IX-XI and XII-XIV, according to the features of elongated spermatids was previously presented. A novel morphometry of seminiferous epithelia based on these 4 groups was also proposed. In the present study, utility of the proposed morphometry in the histopathological evaluations of testicular toxicities was monitored in comparison with the conventional one. After administrating adriamycin, ethylene glycol monomethyl ether or 1, 3-dinitrobenzene to rats, the viability of their germ cells was estimated by the proposed morphometry and the conventional one employed stages II-III, V, VII, X and XII. In every case, the evaluating results of the proposed morphometry were similar to those of the conventional one. Thus, it was verified that the proposed morphometry was identical with the conventional one in respect of reliable detection of the testicular toxicities. In addition, in situ terminal dUTP nick end labeling indicated that death of spermatogonia, pachytene spermatocytes or round spermatids induced by the above 3 toxic cornpounds was exclusively apoptotic death. In conclusion, the proposed morphometry would be useful as a practical tool in the evaluation of testicular toxicities.

In order to examine whether kojic acid (KA) exerts a promoting effect on thyroid carcinogenesis, male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine (BHP; 2800 mg/kg body wt, single s.c. injection) and, starting 1 week later, received pulverized basal diet containing 2 or 0% KA for 12 weeks. Untreated control rats were given basal diet for 13 weeks. As an additional experiment, two groups without BHP initiation received basal diet or diet containing 2% KA for 20 weeks. The serum triiodothyronine (T3) and thyroxine (T4) levels were significantly decreased (half to one-third of values of the BHP alone group) and serum thyroid-stimulating hormone (TSH) was markedly increased (13-19 times higher than the values of the BHP-alone group) in the BHP + KA group at weeks 4 and 12. Similar changes in serum thyroid-related hormones were observed in the group with 2% KA alone at week 4, but not at week 20. Thyroid weights were significantly increased in the BHP + KA and KA-alone groups. Focal thyroid follicular hyperplasias and adenomas were observed in 4/5 and 3/ 5 rats in the BHP + KA group at week 4, respectively. At weeks 12, these lesions were observed in all rats in the BHP + KA group. Animals of the KA alone group showed marked diffuse hypertrophy of follicular epithelial cells at weeks 4 and 20. No changes in thyroid-related hormone levels or thyroid histopathological lesions were observed in either the BHP alone or the untreated control groups. Measurement of liver T4-uridine diphosphate glucuronosyltransferase (UDP-GT) activity at week 4 revealed no significant intergroup differences. These results suggest that thyroid proliferative lesions were induced by KA administration due to continuous serum TSH stimulation through the negative feedback mechanism of the pituitary-thyroid axis, with decreases of T3 and T4 caused by a mechanism independent of T4-UDP-GT activity.

Piperonyl butoxide, alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylenedioxy-2-propyltol uene, is a widely used pesticide-synergist. Recently, results were reported indicating that piperonyl butoxide is a hepatocarcinogen in rat. Since the underlying mechanism was not elucidated, we examined the effects on rat liver cells in detail. For this purpose male F344 rats were administered piperonyl butoxide mixed in the diet at concentrations of 0 (negative control), 0.05, 0.2 or 2% for 2 days, 1, 2, and 4 weeks. As a positive control, phenobarbital was administered to rats for up to 4 weeks as a 0.1% solution in the drinking water. Increased liver weight, centrilobular hepatocellular hypertrophy due to increased smooth endoplasmic reticulum, decreased numbers and areas of connexin 32-positive spots per hepatocyte, and increased cell proliferation were observed in rats treated with 0.2 and 2% piperonyl butoxide. Similar results were obtained for 0.1% phenobarbital treated rats. Hepatocellular necrosis suggestive of hepatotoxicity was also observed in the 2% piperonyl butoxide group. These results indicate that the promoting mechanism of piperonyl butoxide in hepatocarcinogenesis is similar to that of phenobarbital, involving an ability to induce CYP isoenzymes and inhibit gap junctional intercellular communication. In addition, increased cell proliferation following hepatocellular necrosis may also play a role at high doses.

This paper is the first version of a Japanese glossary of terms for structural developmental abnormalities in laboratory animals, mainly rats, mice and rabbits. This is a translation of the glossary entitled Terminology of Developmental Abnormalities in Common Laboratory Mammals that was edited by the International Federation of Teratology Societies (IFTS) Committee on International Harmonization of Nomenclature in Developmental Toxicology (IFTS glossary). The purpose of the present Japanese glossary is to provide a common vocabulary, terms and definitions in the description of abnormalities observed in reproductive and developmental toxicity studies. The glossary contains 868 observations, synonyms, related terms, definitions of the abnormalities and non-preferred terms for external, visceral and skeletal abnormalities in fetuses or neonates of laboratory animals. Modifying terms used repeatedly in the glossary (e. g., absent, atresia) are listed separately in Appendix A, and syndrome names that are generally known are listed in Appendix B. Translation was made into Japanese based on the following: 1) Observations are translated in order of organ/finding (e. g., thymus/absent, maxilla/fused) with exception of the terms in common use, 2) Words are appended where appropriate, when literal translation does not provide an adequate description, 3) A supplementary explanation is appended as an “annotation”, where appropriate, 4) Among the synonyms or related terms, the terms listed in Appendix A or the same Japanese translations as the observations are not described repeatedly, and 5) Non-preferred terms are not literally translated, and only those terms which are considered as adequate as Japanese are described. Suggestions, questions, and additions are welcomed on the Japanese terms and translations in the glossary. Revisions of the Japanese glossary are planned based on the comments received.

In a 2-year carcinogenicity study of potassium iodide (KI) in F344/DuCrj rats, squamous cell carcinomas (SCCs) were observed in the salivary glands of 4/40 males and 3/40 females receiving 1000 ppm KI in the drinking water. Ductular proliferation with lobular atrophy was observed at high incidence in the submandibular glands of the high-dose animals, and squamous metaplasia was frequently evident within the proliferative ductules and the larger interlobular ducts. A transition from metaplasia to SCC was apparent. The results suggest that squamous metaplasia in proliferative ductules, occurring secondarily to lobular impairment induced by KI, may develop into SCCs via a non-genotoxic, proliferation-dependent mechanism.

The modifying effects of oltipraz on induction of glandular stomach carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in a total of 120 male 6-week-old Wistar rats, divided into six groups. Groups 1-3 (30 animals each) were given 100 p.p.m. MNNG in their drinking water for 10 weeks as an initiation treatment for gastric cancer induction and respectively fed diets supplemented with 0.04%, 0.02% and 0% oltipraz for 12 weeks, starting 1 week before and finishing 1 week after the carcinogen exposure. Groups 4-6 (10 animals each) were similarly treated without the application of MNNG. At the end of the 80th experimental week, all surviving animals were autopsied and examined histopathologically for the existence of gastric proliferative lesions. The incidence and multiplicity of adenocarcinomas were significantly (P < 0.01) lower in group 1 than in group 3. In addition, the multiplicity of atypical hyperplasias in the pyloric region was significantly (P < 0.05) decreased in group 1 as compared with the group 3 value. No gastric proliferative lesions were found in groups 4-6. In an additional short-term experiment, oltipraz significantly reduced cell proliferative activity (P < 0.01) and elevated glutathione levels (P < 0.05) in the glandular stomach mucosa of rats treated with MNNG. Thus our results clearly indicate that oltipraz can inhibit induction of proliferative glandular stomach lesions by MNNG in the rat.

The modifying effects of quinacrine administration during the post-initiation phase of carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Female Syrian hamsters were given three weekly s.c. injections of BOP at a dose of 10 mg/kg and then 300 or 100 ppm quinacrine in their diet for 37 weeks. Additional groups of animals received the BOP injection alone, or only the 300 ppm quinacrine treatment as BOP-negative controls. At week 40 of the experiment, all surviving animals were killed and development of proliferative lesions was assessed histopathologically. The multiplicity of pancreatic adenocarcinomas and dysplastic lesions per hamster was significantly higher (P

Changes of TSH-producing cells in the pituitary and thyroid expression of the growth factors, transforming growth factor alpha (TGF alpha) and epidermal growth factor receptor (EGFR), as well as cyclin D1, were investigated immunohistochemically in order to clarify their contribution to the enhancing effects of excess vitamin A (VA) on thyroidal carcinogenesis induced by thiourea (TU). Male rats were allocated to 4 groups, control, TU, VA, and TU + VA, respectively, receiving no treatment, water containing 0.2% TU, diet containing 0.1% VA, and both for 10 or 19 weeks after a single s.c. injection of DHPN (2800 mg/kg) for initiation. Immunohistochemistry using antibodies against TSH demonstrated enlargement of TSH-producing cells in the TU + VA group as compared to the TU group, supporting our conclusion that enhanced TSH stimulation is mainly responsible for promoting the effects of excess VA. Since the expression of TGF alpha, EGFR, and cyclin D1 in thyroid proliferative lesions did not exhibit any differences between the TU and TU + VA groups in the present study, these factors are unlikely to participate in VA enhancement of carcinogenesis.

Dimethylnitrosamine (DMN) produces tumors in mice predominantly in the liver, but also in the kidney and lung. It forms O6-methylguanine adducts in DNA, which induce G:C-->A:T transitions. We have analyzed the spectra of spontaneous and DMN-induced mutations in the lacI transgene of the Big Blue mouse (C57BL/6). In both cases, mutations in the liver, kidney and lung were predominantly base substitutions, among which G:C-->A:T transitions were the most frequent. In contrast, a high incidence of short deletions (2-23 bp) was only found in the liver of treated mice. The deletions often occurred at direct repeat sequences. Single-base deletion incidence was also higher in the liver than in the kidney and lung. These results imply that accumulation of DNA lesions or their repair in liver is different from other organs. Spontaneous and induced base substitutions and deletions appeared to be randomly distributed in the lacI gene and an apparent hotspot was not observed, except for a 4 bp deletion of a (TGGC)3 sequence at positions 621-632. The present data demonstrate, for the first time, that DMN induces short deletions especially in the liver, although the mechanism involved needs further investigation.

The influence of phenethyl isothiocyanate (PEITC) on cell kinetics in the target organs of N‐nitroso‐bis(2‐oxopropyl)amine (BOP) tumorigenicity and on xenobiotic‐metabolizing enzymes was investigated in hamsters. Female 5‐week‐old Syrian hamsters were given a single s.c. dose of 0, 20 or 50 mg/ kg of BOP 2 h after receiving PEITC by gavage at a dose of 0, 100 or 250 μmol/animal (0, 16.3 or 40.8 mg/animal). Six and 22 h after the BOP administration, hamsters were killed and tissues were sampled. Proliferating cell nuclear antigen immunohistochemistry demonstrated significant reduction (P< 0.05–0.001) by PEITC of the labeling indices in the pancreatic acini and ducts, bronchioles, and renal tubules of the BOP‐treated animals in a dose‐dependent manner. In the lungs, the PEITC pre treat merit significantly (P< 0.001) reduced the 06‐methyldeoxyguanosine levels as compared to the BOP‐alone value. Immunoblot analysis of liver cytochrome P450 isoenzymes showed CYP 2B1 to be mainly involved in the metabolic activation of BOP. PEITC significantly (P

Recently, we have shown that dimethylnitrosamine (DMN) treatments increase lacI mutant frequency in the liver, kidney and lung but not in other organs, and also enhance cell proliferation only in the bronchial epithelia. In the present study, organ specificity of tumorigenicity induced by DMN was compared to those of lacI mutation and cell proliferation in Big Blue ® mice. Male 8-week-old Big Blue ® mice were treated with daily i.p. injections of 1 or 10 mg/kg DMN for 5 days, or a single i.p. injection of 5 or 10 mg/kg DMN. Except for the 10 mg/kg×5 DMN group, all animals survived until 78 weeks after the first treatment of DMN. In the present study, the induction of cell proliferation in the bronchial epithelia was confirmed in a dose-dependent manner. At the termination of 78 weeks, it was histopathologically shown that the DMN-treated mice developed liver cell tumor in three out of seven (43%) of the 5 mg/kg group, renal tubule dysplasia in three out of seven (43%) of the 1 mg/kg×5 group, and duodenal adenocarcinoma in one of seven (14%) of the 1 mg/kg×5 group, although no neoplastic or preneoplastic lesions were found in the control mice. Because non-transgenic C57BL/6 mice are resistant to developing spontaneous liver cell and duodenal tumors, it was speculated that even these low doses of DMN could be sufficient to initiate target cells. Our results thus suggest that organ specificity of tumorigenicity by DMN is in favorable agreement with that of lacI mutation but not with possibly temporal cell proliferation induced by DMN.

To clarify relationships between serum thyroid-stimulating hormone (TSH) levels and ultrastructural changes in thyrotrophs caused by intermittent or continuous treatments with antithyroid compound, male Fischer-344 rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN) were given water containing 0.1% sulfadimethoxine (SM) for 8 wk and then sacrificed (Group 1). Additional groups were examined 2 wk after withdrawal (Group 2), after 4 wk retreatment following a 2-wk withdrawal period (Group 3), and after 14 wk continuous exposure (Group 4). Control rats (Group 5) remained untreated for 8 wk after the DHPN initiation. Microscopic examination revealed hypertrophy of thyrotrophs and depletion of TSH-positive material in Groups 1, 3, and 4 but a return to normal in Group 2. Electron microscopic examination of thyrotrophs in the anterior pituitary in Groups 1, 3, and 4 revealed dilated rough endoplasmic reticulum (ER) cisternae with intracisternal dense granules as well as diminished numbers of intracytoplasmic secretory granules, these changes being most marked in Group 1 and least pronounced in Group 4. The number of intracytoplasmic secretory granules in Group 3 was much lower than in Group 4, as demonstrated by morphometric analysis. In Group 2, thyrotrophs showed dilated rough ER cisternae without intracisternal dense granules and essentially the same component of intracytoplasmic secretory granules as Group 5. The present study suggests that while prolonged continuous treatment with SM results in gradual acclimation to an increased demand for TSH, intermittent treatment elicits a persistent state of reduced TSH storage in thyrotrophs due to a continued strong feedback through the hypothalamus-pituitary-thyroid axis.

In order to investigate the effects of vitamin A (VA) supplementation on thyroid proliferative lesions associated with goitrogens other than thiourea, F344 rats were given VA in the diet at 0.1% and drinking water containing sulfadimethox-ine, propylthiouracil, potassium thiocyanate or phenobarbital for 19 weeks after initiation with N-bis (2-hydroxypropyl) nitrosamine. At the end of the treatment period, serum T3, T4, and TSH levels, T4-uridinedi phosphate glucuronosyltransferase (UDP-GT) activity in the liver, and thyroid weights were determined. In addition, thyroid proliferative lesions were histologically examined and evaluated for their PCNA labeling indices. Although serum T4 levels were lowered by VA in all goitrogen-treatment cases, serum levels of TSH, which is responsible for control of proliferation of thyroid follicular cells, were not elevated by the VA supplementation. There were also no enhancing effects of VA in terms of the other parameters, suggesting that the enhancement observed ealier with thiourea is a special case.

Female Syrian golden hamsters were given a subcutaneous injection of 0.6 mg of N-methyl-N-nitrosourethane (MNUR) every 2 weeks for 8 weeks (a total of 5 doses) and then maintained without any treatment for the next 26 weeks. The MNUR-induced bronchiolo-alveolar cell tumors were all adenomas composed of basophilic or clear cells in a papillary growth pattern or papillary/solid tumors consisting of pleomorphic cells. Ultrastructurally, most of the tumor cells had the lamellar bodies characteristic of alveolar type II cells in their cytoplasm. Positive cyclin D1 immunostaining was associated with pleomorphic cell type adenomas, whereas no overexpression of p53 protein, hsp70 or mdm2 gene protein was detected in any of the tumors. The lung tumor samples were examined for the presence of mutations in the K-ras gene in codon 12, 13, and 61 by a non-isotopic method for selective oligonucleotide hybridization after PCR amplification of DNA from formalin fixed tissue embedded in paraffin. Lung tumors from 6 of 18 (33%) tumor bearers contained a point mutation in codon 13 or 61, 3 involving GGC→GAC transitions at the second base of codon 13 and 3 affecting the 61st codon, one CAA→CTA, and two CAA→CGA. No mutations were detected in codon 12. These findings indicate that, at least, a point mutation of the K-ras gene was related to the development of bronchiolo-alveolar cell adenomas induced by MNUR and may suggest that cyclin DI overexpression in pleomorphic cell type tumors plays a role in the process of malignant transformation.

The modifying effects of captafol and protective effects of L-cysteine on the development of glutathione S-transferase placental form-positive (GST-P +) foci of the liver and expression of proliferating cell nuclear antigen (PCNA) in the kidney were investigated in a medium-term bioassay using D-galactosamine (DGA) in rats. Male 6-week-old F344 rats were initially given a single i.p. injection (200 mg/kg) of diethylnitrosamine (DEN) and after 2 weeks on basal diet, received two i.p. injections of DGA (300 mg/kg) at the ends of weeks 2 and 5, and were fed a diet supplemented with test chemicals for weeks 3-8. Animals in group 1 were given 1500 ppm captafol in the diet, while group 2 received 1500 ppm captafol in diet as well as 1500 ppm L-cysteine in drinking water, animals in control group being given basal diet alone. Positive results regarding increased numbers and areas of GST-P + liver cell foci were obtained in rats treated with captafol alone. On the other hand, significant reduction by L-cysteine in the areas of GST-P + liver cell foci initiated by DEN and promoted by captafol was observed. In addition, the PCNA-labelling indices of renal tubule cells were elevated in rats treated with captafol alone and significantly reduced in rats treated simultaneously with L-cysteine. The protocol used in the present study therefore allowed the in vivo determination of promoting effects of captafol and inhibitory influence of L-cysteine by analyzing GST-P + foci in the livers as marker lesions, within a relatively short period of 8 weeks. Thus, this bioassay protocol could have applicability as a new in vivo assay system for the screening of hepatic carcinogenic or anti-carcinogenic agents.

Changes observed in lympho-hematopoietic organs in rats given piperonyl butoxide may be attributable either to direct toxic effects or to undernutrition. Male F344 rats were therefore fed diet containing 2.5% piperonyl butoxide or subjected to a 64% restriction of food intake for 2 weeks. Marked inhibition of body weight gain, decreased white blood cell count, depletion of T/B lymphocytes in lymphoid tissues, hypoplasia of the bone marrow, and decreased proliferating cell nuclear antigen (PCNA) labeling indices in these tissues were seen in both dietary restriction and 2.5% piperonyl butoxide groups. The depletion of T lymphocytes in the thymus and spleen was stronger in the 2.5% piperonyl butoxide group, as indicated by PCNA labeling indices and image analysis of T lymphocyte areas of the spleen, however, the toxicological profile observed for the chemically treated group was essentially the same as for animals on the restricted diet. These results suggest that the lympho-hematopoietic findings in rats receiving 2.5% piperonyl butoxide are probably due to undernutrition resulting from a reduced food intake.

In order to examine modifying effects of simultaneous treatment with large amounts of vitamin A (VA) and thiourea (TU) on the thyroid tumorigenesis in rats, male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine (2800 mg/kg body weight, single s.c. injection), and starting 1 week later received diet containing 0.1% VA (VA group), drinking water containing 0.2% TU (TU group), 0.2% TU + 0.1% VA (TU + VA group) or tap water/basal diet (control group) for 19 weeks. Serum T3 and T4 in the TU and TU + VA groups were significantly decreased as compared to the control group, while serum TSH levels were remarkably increased. The ratios of T3 and T4 decrease and TSH increase in the TU + VA group were remarkably more pronounced than in the TU group. Thyroid neoplastic lesions were only induced in the TU and TU + VA groups. The multiplicity of intracapsular follicular cell proliferative foci in the TU + VA group was significantly increased as compared to the TU group value. Cell proliferation of hypertrophic and subcapsular follicular cells, as well as in hyperplasias, and neoplasias with adenomatous growth pattern was significantly higher in the combined treatment case than after TU alone. In the liver, centrilobular hypertrophy of hepatocytes was seen in the TU and TU + VA groups, this being especially marked in the latter group. In the combined group case the affected cells were strongly positive for GST-P antibody binding. The results of the present study suggest that cell proliferation of thyroid follicular cell proliferative lesions in rats is enhanced by strong TSH stimulation with simultaneous treatment of TU and large amounts of VA.

The mechanism of increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) by removal of extracellular Na + , which phenomena were reported previously (Japan.J. Pharmacol. 61 83–91 1993), was investigated in cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent Ca 2+ indicator, fura-2, by digital ratio imaging microscopy. Isotonic substitution of choline chloride for NaCl induced a transient increase in [Ca 2+ ] i . The pretreatment of thapsigargin (0.5 μM), but not nicardipine (10 μM), suppressed the transient increase completely. In solutions containing micromolar concentrations of free Ca 2+ (nominally Ca 2+ -free solution), the Na + -free induced transient increase was observed, but neither the second cell exposure to the Na + -free solution nor the following application of histamine increased [Ca 2+ ] i , indicating that removal of extracellular Na + releases Ca 2+ from intracellular stores including inositol 1,4,5-trisphosphate (IP 3 )-releasable pools. The Na + -free-induced transient increase required the presence of more than micromolar concentrations of extracellular free Ca 2+ and releasable Ca 2+ within the stores, but ryanodine did not affect the transient increase. These results suggest that undetectable influx of Ca 2+ by the reverse-mode action of the Na + /Ca 2+ exchanger can release Ca 2+ from the thapsigargin-sensitive intracellular stores including IP3-releasable pools.

Changes resulting from toxicity to the rat testis and epididymis induced by ethylene-1, 2-dimeth-anesulfonate (EDS) were histomorphometrically studied. In experiment I, ten-week old male rats received a single dose of 100mg/kg body weight EDS, and at 1, 3, 7, 14, and 28 days thereafter were sacrificed for histopathological evaluation of the testis, epididymis, prostate, and seminal vesicle. Lesions involving Leydig cells and seminiferous tubules in the testis wereanalyzed using a quantitative method. In the treated animals, Leydig cells disappeared within 3 days of the EDS treatment, and decreases of pachytene spermatocytes and round spermatids were subsequently observed after 7 days. In contrast, necrosis of epithelia was already observed in the epididymis at 1 day after the EDS treatment, and the severity of the lesion increased up to day 3. At day 7, although loss of Leydig cells was still evident, recovery of epithelia in the epididymis was observed. In the prostate and seminal vesicles, atrophic changes wereseen without necrotic lesions. In experiment II, castrated animals were injected with EDS and sacrificed after 3 days for histophathological observation. Changes similar to those in uncastrated animals treated with EDS were recognized in the epididymis, and with more pronounced severity than those observed in castratedanimals without EDS treatment. These results suggest that toxic lesions of the epididymis induced by EDS are not secondary to those in the testis, but rather are due to direct effects of the compound.

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.

Suitable conditions of bleomycin (BLM)-treatment to effectively induce pulmonary fibrosis without high mortality were investigated in Syrian golden hamsters. Male 6-week-old hamsters were divided into 5 groups, each consisting of 5 animals. Groups 1-4 were intratracheally instilled with 2.5U/kg of BLM on day O and then repeatedly given BLM as follows: group 1, 2.5U/kg intratracheally on day 14; group 2, 1.0U/kg intratracheally on day 14; group 3, 1.0U/kg intratracheally each on days 7 and 14; group 4, 20U/kg intraperitoneally each on days 10and 14. Group 5 served as a vehicle control. The survival rate was 4/5 (80%) in groups l and 3, and 5/5 (100%) in other groups. One animal each in groups I and 3 was found dead on day 22, of which lungs histopathologically showed evident fibrosis accompanied with congestion, edema, inflammatory cell infiltration, and hemorrhage. The lung weight at the termination of the experiment on day 28 was the highest in group 1, and significantly higher in groups 1 (p

The relationship of pulmonary fibrosis to development of lung tumors was investigated in Syrian golden hamsters treated with N-methyl-N-nitrosourethane (MNUR). Female animals in groups 1 and 2 received five subcutaneous (sc) injections of 0.6 and 0.2mg, respectively, of the carcinogen, whereas males in group 3 were given a single 0.3mg sc dose. After initiation the animals in each group were maintained, together with controls, without any further treatment for 26 weeks, and then examined for whether proliferative lesions in the lung are secondary to pulmonary inflammatory lesions induced by MNUR. Lung tumors were induced in all hamsters of group 1, but only 56% and 36%, respectively, were accompanied by inflammatory lesions and pulmonary fibrosis. Similar results were also obtained for tumors in group 2 and papillary hyperplasias in the female treated groups, many being observed without any involvement of fibrotic lesions. Glandular metaplasia was observed at high frequency in groups 1-3, but no evidence of a histogenetic link with development of lung tumors was found. A close relation between development of lung tumors and papillary hyperplasias was noted, however. The present study thus suggested that a considerable proportion of lung tumors induced in hamsters by MNUR are independent of toxicity-related changes and presumably due to direct DNA initiating action of the carcinogen.

To clarify the biological behavior of karyomegalic alveolar epithelial cells induced by N-methyl-N-nitrosourethane (MNUR) and whether these cells progress to lung tumors, female Syrian golden hamsters, 6 weeks old, were given five subcutaneous injections of 0.6 mg/animal of MNUR at two week intervals and their lungs were examined at weeks 1, 4, 8 and 12 after the termination of treatment. At week 1, in severely affected areas where marked multifocal thickening of alveolar walls due to interstitial edema and cellular infiltration was observed, some regenerative alveolar epithelial cells had abundant eosinophilic cytoplasm and gigantic bizarre nuclei. The cells were confirmed ultrastructurally to be derived from alveolar type II cells. The number of these karyomegalic epithelial cells became significantly decreased thereafter, together with the reduction of inflammatory changes. On AgNOR staining, normal alveolar epithelial cells had 1.8 +/- 0.03 black dots within their nuclei while the karyomegalic epithelial cells had 4 black dots or more, from 1 week. The PCNA labeling index of the karyomegalic epithelial cells at week 1 was 14.6 +/- 2.4, and was significantly decreased from 4 week. This epithelial cell population also displayed a wider range of DNA contents (2.1-5.5C) than normal epithelial cells (1.6-2.3C). These results suggest that karyomegalic alveolar epithelial cells may be mutant cells which occur after initiation with MNUR, but the possibility that they can act as progenitors of alveolar epithelial cell tumors was considered to be extremely low.

This study was performed for developing a new medium-term carcinogenicity bioassay treated with d-galactosamine (DGA) as a non-surgical method without partial hepatectomy (PH). In male F344 rats initiated with diethylnitrosamine (DEN, 200 mg/kg i.p.), enhancing effects of DGA (300 mg/kg i.p.) given twice 3 weeks apart during the promotion procedure with 2-acetylaminofluorene (2-AAF, 0.01% in diet) were compared along with those of PH by analyzing preneoplastic glutathione S-transferase placental form positive (GST-P+) hepatocyte foci as endpoint marker lesions. The DGA treatment did not affect the body weight gain whereas the PH treatment caused a transient body weight loss. Although both bioassay protocols were effective to detect the potential hepatocarcinogenicity of 2-AAF, the number and area of GST-P positive foci per cm2 were larger in the bioassay using DGA than in that using PH, the number being statistically significant (P

To cast light on the relationship between the development of pulmonary fibrosis and lung cancer, female Syrian golden hamsters were given 5 sc injections of 0.6 mg/animal of N-methyl- N-nitrosourethane (MNUR) at 2-wk intervals and then maintained without any treatment for 26 wk. Bronchiolo-alveolar cell tumors and hyperplasias, which were recognized from week 4 after termination of treatment, were each subdivided morphologically into 3 types. Bronchiolo-alveolar cell tumors included papillary tumors consisting of basophilic cells, papillary tumors consisting of clear cells, and papillary/solid tumors consisting of pleomorphic cells, with papillary tumors consisting of basophilic cells predominating. Bronchiolo-alveolar cell hyperplasias encountered were glandular metaplasia, simple hyperplasia, and papillary hyperplasia. Glandular metaplasia was the most common of the hyperplastic lesions. To some extent, all the proliferative lesions were associated with inflammatory cell infiltration, but this varied greatly. The rate for papillary tumors consisting of basophilic cells with connective tissue proliferation was 35%. Among the hyperplastic lesions, the cell prolif erative activity of papillary hyperplasia (12.5%) was significantly higher than in other hyperplastic lesions, suggesting that this lesion might be a preneoplastic change. None of the lung proliferative lesions showed any unequivocal immunoreactivity for the p53 protein. The present study suggests that most lung tumors may develop from pulmonary inflammatory lesions induced by MNUR, but the possibility that DNA injuries to the respiratory epithelial cells by MNUR may cause lung tumors cannot be precluded.

To determine whether production of thyroid proliferative lesions would be enhanced by intermittent rather than continuous treatment with a goitrogen, male F344 rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN, 2800 mg/kg body weight, single s.c. injection) were given water containing 0.1% sulfadimethoxine (SM) for 20 weeks (group 1) or 0.1% SM for the first 8 weeks followed by 2 cycles consisting of 2 weeks withdrawal and 4 weeks retreatment with 0.1% SM (group 2). Control rats (group 3) were untreated for 20 weeks after the DHPN initiation. Serum T3 and T4 levels were significantly decreased in groups 1 and 2 compared to group 3. Serum thyroid stimulating hormone level was significantly increased in all treated groups compared to group 3. The numbers of follicular cell hyperplasias were significantly increased in group 2 compared to group 1. BrdU labeling indices for follicular cells and hyperplasias were also significantly elevated in group 2 compared to group 1. Electron microscopic examination of thyrotrophs in the anterior pituitary in groups 1 and 2 revealed dilated rough ER cisternae with intracisternal dense granules. The number of intracytoplasmic secretory granules in group 2 was moderately decreased compared to group 1. Therefore, the results of the present study suggest that it may be possible to enhance production of thyroid neoplastic lesions by intermittent treatment.

The tripartite-harmonized International Conference on Harmonization reproductive guideline (1993) recommends administration of test substances for 4 weeks to male rats before mating. However, scientific or experimental rationale for this recommendation is not firmly based, and the most appropriate parameters have not been established in experimental models. Therefore, a team consisting of 16 Japanese pharmaceutical companies and the National Institute of Health Sciences performed a collaborative study to determine the optimal period and parameters for detection of male fertility disorders in rats. Sixteen compounds, including four anticancer drugs, two psychotropic drugs, two nootropic drugs, two vitamins, two hormones, one antihypertensive agent, one diuretic drug, and two general chemicals were administered to male rats for 4 or 9 weeks before mating. Parameters used to examine effects on the male reproductive system were organ weights, sper-matogenic endpoints, mating behavior, cesarean section findings, and histopathology. From the results, treatment for 4 weeks before mating was concluded to be sufficient to detect adverse effects on male fertility, with the histopathology of the testis being the most sensitive index for the drugs used. Sperm parameters, especially number, and genital organ weight determination provided information confirming toxicity. Tests of reproductive activity were generally found to be insensitive, except where the drugs affected sperm maturation. Based on this study, it is concluded that a 4-week treatment period is appropriate for detection of drug effects on male fertility, and that histopath-ological examination of the testis is the most sensitive approach.

The effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), on bleomycin-induced diffuse alveolar damage (DAD) were investigated in male Syrian golden hamsters. Six-week-old animals were treated with a single intratracheal administration of bleomycin (BLM; 5mg/5ml/kg) or an equivalent volume of saline. Starting 1 day after the treatment, animals were given 1% BHA or 1% BHT in the diet or basal diet alone for 31 days. The survival rate of the BLM/BHA group (55%) was significantly higher than that of the BLM alone (15%) or BLM/BHT (15%) groups (p

A thirteen week feeding study was conducted by feeding young adult male and female Sprague Dawley [Crl:CD®(SD)] rats diets containing grain from genetically modified (GM) DP-ØØ4114-3 maize that was either untreated (4114) or treated in the field with glufosinate ammonium (4114GLU). Control rats were fed diets containing the same concentration of near isogenic, non-GM maize grain (091) or one of three types of commercially available non-GM maize grain. At the end of the in-life phase, renal tubule tumors were reported in two male rats consuming diets containing 4114 maize grain. An expert panel of pathologists was convened as a Pathology Working Group (PWG) to review coded kidney histology sections from control (091) and treated (4114 and 4114GLU) male rats. The objectives were for the panel to characterize the histopathologic findings and to interpret their relationship to consumption of the indicated diet. The PWG concluded unanimously that the kidney tumors were characteristic of amphophilic-vacuolar (AV) tumors and AV atypical tubular hyperplasia which represent a distinctive phenotype that has been reported to occur sporadically in young Sprague Dawley Rats. The PWG determined that the neoplasms and atypical tubular hyperplasias were multicentric and bilateral which typifies tumors of familial origin. Degenerative/regenerative or cytotoxic changes consistent with nephrotoxicity leading to tumor induction were not observed in these rats and thus supports the conclusion that tumors were unrelated to consumption of the test diet. It was the unanimous opinion of the PWG that the proliferative renal tubule cell lesions were spontaneous and not related to consumption of diets containing 4114 maize grain.

Time course changes in serum TSH and quantitative data for thyroid proliferative lesions in male F344 rats administered N-bis(2-hydroxypropyl)nitrosamine (DHPN: 2000 mg/kg body weight, single s.c. injection) followed by 0.1% thiourea (TU), were assessed at weeks 1, 2, 4, 8, 12 and 16 of treatment. The serum T4 level in the TU group was markedly decreased at week 1 and remained significantly lowered throughout the experiment. Serum TSH levels, in contrast, were elevated up to a peak at around week 4 with a return to the normal range at week 12. Thyroid weights in the TU group were increased significantly in a treatment period-dependent manner. Histopathologically, marked hypertrophy of thyroid follicular cells occurred at the early stage of TU treatment. Proliferative lesions, such as hyperplasia and adenomas, occurred from weeks 2 and 4, respectively, and increased with the later treatment period. The cell proliferative activity of follicular cells, assessed by BrdU incorporation, was high until week 2, but then returned to normal. The initially appearing hyperplasias and adenomas were characterized by marked proliferation but this also greatly decreased at later stages when TSH was no longer elevated. The results of our study thus suggest that a high serum TSH level plays an important role in the early phase of thyroid tumorigenesis and 8 weeks treatment with test substances is sufficient for detection of thyroid tumor promoter potential in two-stage thyroid carcinogenesis models.

Influences of cigarette smoke on N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic duct and respiratory tract tumorigenesis were investigated using a hamster two-stage carcinogenesis model. Male 5-week-old hamsters were divided into 5 groups. Group 1 was s.c. injected with BOP at a dose of 10 mg/kg once a week for 3 weeks as an initiation treatment together with cigarette smoke exposure over the same 4-week period. Group 2 was exposed to cigarette smoke for 26 weeks after the BOP-initiation. Groups 3 and 4 were respectively given the BOP-initiation alone and the 26-week cigarette smoke exposure without initiation. Group 5 served as a sham-smoked negative control. The experiment was terminated 30 weeks after the first BOP injection. The incidence of pancreatic adenocarcinomas was significantly decreased in Group 1 as compared to the Group 3 value (P < 0.01) while the Group 2 value did not show any change. In contrast, the incidence of laryngeal and tracheal proliferative lesions (hyperplasias and papillomas) was significantly increased in Group 2 over Group 3 (P < 0.01). The incidence of pulmonary hyperplasias was also increased in Group 2 over Group 3 (P < 0.05), although that of pulmonary adenomas or adenocarcinomas was decreased in Group 2 as compared to the Group 3 value (P < 0.01). Cigarette smoke exposure in the BOP-initiation phase (Group 1) did not affect the development of respiratory proliferative lesions. No animals in Groups 4 and 5 developed any tumors in the pancreas or respiratory tract. Our results thus indicate that cigarette smoke exposure inhibits pancreatic carcinogenesis when given in the initiation phase, whereas it modulates (enhances or suppresses) the development of proliferative lesions in the respiratory tract if applied during the promotion stage to hamsters pretreated with BOP.

Nucleolar organizer regions, stainable with the one-step silver colloid method (AgNORs), were evaluated for estimation of proliferative activity of pancreatic lesions, including ductal hyperplasias, dysplasias, adenomas, and adenocarcinomas induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) in dogs and compared with values for proliferating cell nuclear antigen (PCNA) positive cells. The mean numbers of AgNORs per nucleus of cells in adenocarcinoma, dysplasia and hyperplasia cases were 2.37 (p

The effects of concomitant administration of a synthetic trypsin inhibitor (camostat) on pancreatic carcinogenesis in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP) were investigated. Thirty-two female Syrian golden hamsters were given weekly 10 mg/kg s.c. injections of BOP for 5 weeks while simultaneously receiving a 500 ppm camostat diet (BOP + camostat group). Additional groups of 30 animals received either the s.c. injections of BOP (BOP group), or the 500 ppm camostat diet (camostat group) during the same 5-week period. Thirty weeks after the first BOP administration, the incidence of pancreatic adenocarcinomas in the BOP + camostat group was significantly lower than in the group administered BOP only (p < 0.05). Similarly, the total numbers of pancreatic adenocarcinomas or dysplastic lesions were significantly decreased in the BOP + camostat group as compared with the BOP group (p < 0.01). None of the animals receiving camostat alone developed any adenocarcinomas or dysplastic lesions of the pancreas. The results of the present experiments clearly show that camostat can inhibit induction of hamster pancreatic ductal neoplasms when administered simultaneously with BOP.

In order to examine the carcinogenicity of 3‐amino‐l‐methyl‐5H‐pyrido[4,3‐β]indole acetate (Trp‐P‐2), 30 male and 30 female F344 rats were maintained on diet containing 0, 30, or 100 ppm Trp‐P‐2 for 112 weeks. The overall mean chemical intakes in the 100 ppm and 30 ppm groups were 3.84 and 1.14 mg/kg/day in males, and 4.57 and 1.34 ing/kg/day in females, respectively. Females of the 100 ppm group showed increased mortality in the late period of the study. In the 100 ppm group, significant increases in the incidences of neoplastic lesions were found in the liver, urinary bladder and mammary gland in males, and in the mammary gland, hematopoietic system and clitoral gland in females. Histologically, tumors induced by Trp‐P‐2 were hepatocellular adenomas, transitional cell tumors (papillomas and carcinomas) of the urinary bladder, fibroadenomas/fibromas of the mammary gland, malignant lymphomas and clitoral gland tumors (adenomas and adenocarcinomas). These results indicate multi‐target carcinogenicity of Trp‐P‐2 in F344 rats and provide evidence that the urinary bladder is also a target for heterocyclic amine action.