Size-dependent cytotoxic effects of amorphous silica nanoparticles on Langerhans cells.

Amorphous silica nanoparticles (nSPs), are widely used in medicines, cosmetics and food. However, due to their reduced particle size they are suspected to pose new risks induced by changes in biological reactivity and kinetics, which differ from those of bulk materials. In a previous study, we showed that silica particles with a diameter of 70 nm penetrated the stratum corneum (SC) of mouse skin and were taken up by living cells such as keratinocytes and Langerhans cells. To clarify the relationship between particle size, distribution and cellular response, we have evaluated size-dependent intracellular localization and cytotoxicity of silica particles, using the mouse epidermal Langerhans cell line XS52. On treatment with silica particles of diameters 70, 300, and 1000 nm, cellular uptake and cytotoxicity increased with reduction in particle size. These results suggest that smaller sized silica particles induced greater cytotoxicity against Langerhans cells, which was correlated with the quantity of particle uptake into the cells.

Species-specific differences in coumarin-induced hepatotoxicity as an example toxicogenomics-based approach to assessing risk of toxicity to humans.

One expected result from toxicogenomics technology is to overcome the barrier because of species-specific differences in prediction of clinical toxicity using animals. The present study serves as a model case to test if the well-known species-specific difference in the toxicity of coumarin could be elucidated using comprehensive gene expression data from rat in-vivo, rat in-vitro, and human in-vitro systems. Coumarin 150 mg/kg produced obvious pathological changes in the liver of rats after repeated administration for 7 days or more. Moreover, 24 h after a single dose, we observed minor and transient morphological changes, suggesting that some early events leading to hepatic injury occur soon after coumarin is administered to rats. Comprehensive gene expression changes were analyzed using an Affymetrix GeneChip approach, and differentially expressed probe sets were statistically extracted. The changes in expression of the selected probe sets were further examined in primary cultured rat hepatocytes exposed to coumarin, and differentially expressed probe sets common to the in-vivo and in-vitro datasets were selected for further study. These contained many genes related to glutathione metabolism and the oxidative stress response. To incorporate human data, human hepatocyte cultured cells were exposed to coumarin and changes in expression of the bridging gene set were examined. In total, we identified 14 up-regulated and 11 down-regulated probe sets representing rat-human bridging genes. The overall responsiveness of these genes to coumarin was much higher in rats than humans, consistent with the reported species difference in coumarin toxicity. Next, we examined changes in expression of the rat-human bridging genes in cultured rat and human hepatocytes treated with another hepatotoxicant, diclofenac sodium, for which hepatotoxicity does not differ between the species. Both rat and human hepatocytes responded to the marker genes to the same extent when the same concentrations of diclofenac sodium were exposed. We conclude that toxicogenomics-based approaches show promise for overcoming species-specific differences that create a bottleneck in analysis of the toxicity of potential therapeutic treatments.

Lack of chemopreventive effects of alpha-eleostearic acid on 7,12-dimethylbenz[a]anthracene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary and colon carcinogenesis in female Sprague-Dawley rats.

alpha-Eleostearic acid is one of the conjugated linolenic acids from tung oil, which is obtained from the seeds of Aleurites fordii. The effects of dietary alpha-eleostearic acid (18:3, n-5) on the post-initiation period of 7,12-dimethylbenz[a]anthracene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary and colon carcinogenesis were examined using female Sprague-Dawley (SD) rats. For initiation, rats were given subcutaneous injections of 40mg/kg body weight (5 times) and 20mg/kg body weight (3 times) of DMH during the age of 6-8 weeks and a single intragastric administration of 50mg/kg body weight of DMBA at 9 weeks. Then, the animals were treated with 0%, 0.01%, 0.1% or 1.0% alpha-eleostearic acid for 34 weeks. Control rats received the basal diet alone or 1.0% alpha-eleostearic acid without prior initiation treatment. All surviving animals were killed at week 37 of the experiment. There were no statistically significant alterations in any of the parameters for either mammary or colon tumors. These results thus indicate that alpha-eleostearic acid does not exert clear modification effects on DMBA and DMH-induced mammary and colon carcinogenesis, at least under the present experimental conditions.

A subchronic toxicity study of dunaliella carotene in F344 rats.

Dunaliella carotene, extracted from dunaliella alga (Dunaliella bardawil or Dunaliella salina), for use as a food-coloring agent, has beta-carotene as its mainly constituent. As there have been no reports of toxicological evaluation, a 90-day subchronic toxicity study was here performed in F344 rats at dose levels of 0 (control), 0.63%, 1.25%, 2.5% and 5% in powdered basal diet. The average daily intakes of dunaliella carotene were 352, 696, 1420 and 2750 mg/kg/day, respectively, for males, and 370, 748, 1444 and 2879 mg/kg/day for females. No mortality or treatment-related clinical signs were observed throughout the experimental period in any of the groups. Body weight gain was slightly but significantly (p < 0.05) reduced from week 5 to the end of the experiment in 2.5% and 5% males. Increased PLT were observed in 1.25% and 5% males, and 2.5% and 5% females. Significant elevations or tendencies for increase in serum T. Cho and Ca were observed in all treated males and females, with clear dose-dependence in males. Organ weight measurement and histopathological observation revealed no toxicological changes. Based on growth suppression, no-observed-adverse-effect-levels (NOAELs) were estimated to be 1.25% (696 mg/kg/day) for males and 5% (2879 mg/kg/day) for females. As increases in serum Ca were observed in the lowest group in both sexes, a no-observed-effect level (NOEL) could not be determined in this study.

Dose-response studies on the carcinogenicity of potassium bromate in F344 rats after long-term oral administration.

Dose-response studies on the carcinogenicity of potassium bromate (KBrO3), a food additive, were undertaken to examine its effects at low doses. A total of 148 6-week-old male inbred F344 rats were divided into 7 groups. They were given KBrO3 orally in their drinking water at doses of 500, 250, 125, 60, 30, 15, and 0 ppm for 104 weeks, at the end of which time all the surviving animals were autopsied and then examined histopathologically. Shortening of the survival times and marked inhibition of body weight increase were observed in a group given 500 ppm KBrO3. The combined incidences of renal adenocarcinomas and adenomas were significantly increased in rats treated with KBrO3 at doses of 500, 250, and 125 ppm in a dose-related manner. The dose-response curve showed a sigmoid appearance. The value for the virtually safe dose (VSD), calculated by the probit model, was 0.950 ppm KBrO3 at a risk level of 10(-6). However, significant increases in the occurrence of dysplastic foci of the kidney were found in groups at doses higher than 30 ppm KBrO3. The VSD value for the dysplastic foci estimated by the gamma-multi-hit model was 0.148 X 10(-3) ppm KBrO3 at a risk level of 10(-6). In a group tested with 500 ppm KBrO3, the combined incidences for follicular adenocarcinomas and adenomas of the thyroid and for mesotheliomas of the peritoneum were shown to be significantly increased.

Dose-dependent promotion effects of potassium chloride on glandular stomach carcinogenesis in rats after initiation with N-methyl-N'-nitro-N-nitrosoguanidine and the synergistic influence with sodium chloride.

The modifying effects of potassium chloride (KCl) ingestion on glandular stomach carcinogenesis were investigated in male Wistar rats induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and were compared with those of sodium chloride (NaCl). A total of 120 male 6-week-old Wistar rats were divided into six groups, each consisting of 20 animals. After initiation of treatment with a MNNG solution (100 parts/million) as their drinking water for 10 weeks, rats were fed a diet supplemented with 5% NaCl, 2.5% NaCl, 2.5% NaCl plus 2.5% KCl, 5% KCl, 2.5% KCl, or a basal diet alone for the following 62 weeks. Under this experimental condition, there were no statistical differences in the final body weights between groups. The incidences of adenocarcinomas in the glandular stomachs were significantly higher in the 5% NaCl and combined 2.5% NaCl-plus-2.5% KCl groups (P < 0.05 and 0.01) than in the MNNG alone (control) group. The incidences of atypical or precancerous hyperplasias in the glandular stomachs were increased significantly by the 5% NaCl, 2.5% NaCl-plus-2.5% KCl, and 5% KCl treatments (P < 0.05 or 0.01). The multiplicities of adenocarcinomas were significantly greater in the 5% NaCl, 2.5% NaCl, and combined NaCl-plus-KCl groups (P < 0.05 or 0.01) compared with the control value. The multiplicity data for atypical hyperplasias were most striking; namely, their multiplicities were increased significantly by the treatments of NaCl or KCl (P , 0.01) in a clear dose-dependent manner and enhanced synergistically by the combined treatment of NaCl and KCl. Because the concentrations of KCl used in this study were about 1.3 times lower than those of NaCl on a molar basis, although the doses of each chemical were exactly the same on a weight-percent basis, it is suggested that the enhancing effects of KCl might not be much different from those of NaCl. The results in the present study thus indicate that, similarly to NaCl, KCl ingestion exerts dose-dependent promoting effects and a synergistic influence with NaCl when given during the postinitiation phase of two-stage glandular stomach carcinogenesis in rats.

Serum concentrations of hepatocyte growth factor in breast cancer patients.

Hepatocyte growth factor (HGF) was first identified as a potent stimulator of hepatocyte growth and DNA synthesis. Later, it was shown that HGF can promote cell motility and cell proliferation in various types of cells, including tumor cells and endothelial cells. We have examined serum concentrations of HGF in breast cancer patients using an ELISA. Of 134 primary breast cancer patients, 49 (36.6%) showed a significant increase in the circulating level of HGF as compared to healthy controls. The increase in the HGF level was significantly associated with axillary lymph node metastases and histological evidence of venous invasion. No significant correlation between serum HGF concentrations and intratumoral HGF concentrations was found; however, the removal of the primary tumor clearly decreased the serum HGF level, suggesting that the elevation of HGF in the serum was tumor related. Twenty-nine (82.9%) of 35 patients with recurrent breast cancer had an increase in the serum HGF level. The HGF level was significantly higher in patients with liver metastases compared to those with other sites of metastases. Postoperative sequential examinations confirmed that the increase in the serum HGF level was associated with the appearance of relapse. In conclusion, the serum HGF level was significantly increased in breast cancer patients. Circulating HGF might play important roles in tumor progression in this malignancy.

Lack of subchronic toxicity of an aqueous extract of Agaricus blazei Murrill in F344 rats.

Agaricus blazei Murrill, an edible mushroom, is widely used as a functional food due to its possible medicinal effects. Aqueous extracts are also used as food additive to provide an agreeable bitter taste. As a part of its safety assessment, the present 90-day subchronic toxicity study was performed in F344 rats. To establish a no-observed-adverse-effect level (NOAEL), rats were fed powder diet containing A. blazei Murrill aqueous extract at dose levels of 0 (basal diet), 0.63, 1.25, 2.5 and 5% (maximum) for 90 days. During the experiment, there were no remarkable changes in general appearance and no deaths occurred in any experimental group. Although serum blood urea nitrogen was slightly but significantly increased in males of the 2.5 and 5% groups, no related histopathological changes were observed in the kidney, and serum creatinine levels were rather reduced, suggesting the increase of blood urea nitrogen to be of little toxicological significance. Hematology, organ weight measurement and histopathological observation revealed no test compound-related toxicological changes. In conclusion, A. blazei Murrill extract even at 5% in the diet (2654 mg/kgb.w./day for male rats and 2965 mg/kgb.w./day for female rats) did not cause remarkable adverse effects in F344 rats. Thus, the NOAEL was concluded to be 5% in the diet.

Lack of carcinogenicity of D-xylose given in the diet to F344 rats for two years.

The two year carcinogenicity of D-xylose was examined in groups of 50 male and 50 female F344 rats at dietary doses of 0% (control), 2.5% and 5%. The doses were selected on the basis of results from a 13-week subchronic toxicity study. Growth suppression and soft feces were observed in male and female rats of the 5% group. However, no significant differences from the controls were noted with regard to clinical signs, mortality and hematological findings. Decrease in absolute weight and increase in relative weight of the brain in males, and decrease of absolute kidney weight in females were observed in the 5% group, but there were no remarkable histopathological changes. A variety of tumors developed in all groups, including the controls, but all were histologically similar to those known to occur spontaneously in F344 rats, and no statistically significant increase in the incidence of any type of neoplastic lesion was found for either sex in the treated groups. Thus, it was concluded that, under the present experimental conditions, D-xylose is not carcinogenic to F344 rats.

Promoting effect of peroxisome proliferators in two-stage rat renal tumorigenesis.

A two-stage rat renal tumorigenesis model was employed to examine the promoting effects of peroxisome proliferators in the kidney. Groups of 20 male F344 rats were given 0.05% N-ethyl-N-hydroxyethylnitrosamine (EHEN) orally for the first 2 weeks as the initiator. Subsequently they were treated with clofibrate, simfibrate or di(2-ethylhexyl)phthalate (DEHP), respectively, at dietary concentrations of 0.35%, 0.35% and 1.2% for 24 weeks and killed for microscopical examination of the kidney. The incidences of renal cell tumors (RCT) and the numbers of RCT/kidney in rats given DEHP after initiation were significantly increased as compared to the controls. On the other hand, renal promoting effects were not evident in groups given clofibrate or simfibrate. Under the condition of this study, DEHP was found to act as a renal promoter.

Studies on the promoting and complete carcinogenic activities of some oxidizing chemicals in skin carcinogenesis.

Six oxidizing chemicals were tested for promoting and complete carcinogenic activities in skin carcinogenesis using female Sencar mice. In the promotion tests, the chemicals were applied twice a week for 51 weeks after initiation with dimethylbenzanthracene (DMBA). In the tests for complete carcinogenic activities, the chemicals alone were applied for 51 weeks. Benzoyl peroxide was found to be a potent promoter as reported previously. Moreover, possible complete carcinogenic action of this chemical was found in this study. Potential promoting effect was suspected in sodium chlorite. Potassium bromate, ammonium persulphate, hydrogen peroxide and sodium hypochlorite were inactive either as a promoter or a complete carcinogen.

Effects of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on N-nitrosobis(2-oxopropyl)amine (BOP)-initiated carcinogenesis in hamsters.

The effects of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) administration during the post-initiation phase of carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Female Syrian golden hamsters were given a single s.c. injection of BOP at a dose of 10 mg/kg and then administered 3 ppm (H) or 1 ppm (L) NNK in their drinking water for the following 87 weeks. Additional groups of animals received the BOP injection alone, or only the 3 or 1 ppm NNK treatments as BOP-negative controls. At week 88 of the experiment, all surviving animals were sacrificed and development of proliferative lesions was assessed histopathologically. The results showed no statistically significant influence on pancreatic adenocarcinomas or dysplastic lesions, although the incidence and the number of atypical hyperplasias in the pancreas head in the BOP/NNK (L) group was significantly increased as compared to BOP alone group values (P < 0.05). Similarly, the NNK treatments did not affect the incidences or multiplicities of neoplastic or hyperplastic lesions in the endocrine pancreas, lung, liver or kidney. Thus, the present experiment demonstrates that the tobacco-specific carcinogen NNK does not enhance BOP-induced hamster tumorigenesis when given in the promotion phase.

Analysis of proliferative activity in renal lesions induced by N-nitrosobis(2-oxopropyl)amine (BOP) in male Syrian golden hamsters.

Binding of a specific antibody to proliferating cell nuclear antigen (PCNA) and staining of argyrophilic proteins associated with nucleolar organizer regions (AgNORs) were investigated in proliferative lesions induced by N-nitrosobis(2-oxopropyl)amine (BOP) in the hamster kidney. Thirty male Syrian golden hamsters were given three weekly s.c. injections of BOP (10 mg/kg body wt.) and sacrificed for characterization of proliferative changes 30 weeks after the first BOP treatment. Morphologically, lesions of the tubular epithelia were classified either as tubular adenoma or dysplasia, the latter being further classified into small cluster, acidophilic cell, clear cell and cystic types. Immunohistochemistry for PCNA revealed significant increases of cell proliferation activity in adenomas and acidophilic cell types of dysplasia, along with significantly elevated mean numbers of AgNORs per nucleus. The results thus indicate that the acidophilic cell type of dysplasia may be of prime significance as the preneoplastic renal lesion induced by BOP.

Failure of phenethyl isothiocyanate to inhibit hamster tumorigenesis induced by N-nitrosobis(2-oxopropyl)amine when given during the post-initiation phase.

The chemopreventive influence of phenethyl isothiocyanate (PEITC) during the post-initiation stage was investigated in the N-nitrosobis(2-oxopropyl)amine (BOP)-initiated hamster tumorigenesis model. A total of 120 female 5-week-old hamsters were divided into six groups. Animals in groups 1-3, each consisting of 30 hamsters, were injected twice, subcutaneously, with BOP 7 days apart to effect initiation. Starting 1 week after the second BOP injection, hamsters in groups 1 and 2 were fed diets supplemented with 6 micromol/g and 3 micromol/g of PEITC, respectively, for 51 weeks. Animals in group 3 received a basal diet as an initiation positive control. Animals in groups 4-6, each consisting of ten hamsters, were given 6 micromol/g or 3 micromol/g of PEITC alone, or were non-treated, matched negative controls for groups 1-3. At the termination of experimental week 52, the incidences and multiplicities of neoplastic lesions in the target organs including the pancreas, lung, liver and kidney were found to be comparable among the BOP-treated groups. The values for pancreatic adenocarcinomas as well as dysplastic lesions tended to increase although without statistical significance. Taken together with our previous finding that PEITC dramatically inhibited the initiation phase of BOP-induced pancreatic and lung tumorigenesis in hamsters, it can be concluded that PEITC specifically exerts chemopreventive effects only when given concomitantly with the carcinogen.

Failure of dietary alpha-difluoromethylornithine to inhibit gastric carcinogenesis in rats after 8 weeks of treatment with N-methyl-N'-nitro-N-nitrosoguanidine and sodium chloride.

The modifying effects of alpha-difluoromethylomithine (DFMO) on glandular stomach carcinogenesis after initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and sodium chloride were investigated in male outbred Wistar rats. Animals were simultaneously given MNNG solution (100 ppm) as their drinking water and diet supplemented with 10% sodium chloride for 8 weeks, and administered DFMO (dietary levels of 2000 ppm or 500 ppm) and tap water for the following 70 weeks. The DFMO treatment did not show any tendency to inhibit the development of gastric adenocarcinomas. The incidences and multiplicities of atypical hyperplasias in the glandular stomachs were also comparable in all groups of rats given MNNG/sodium chloride. Neither gastric carcinomas nor atypical hyperplasias were observed without the carcinogen treatment. Thus, DFMO did not exert any inhibitory effects when given during the post-initiation phase of two-stage glandular stomach carcinogenesis in rats initiated with MNNG and sodium chloride for 8 weeks.

Antitumor effect of the angiogenesis inhibitor agm-1470 and its combination effect with tamoxifen in dmba induced mammary-tumors in rats.

AGM-1470 is known to inhibit angiogenesis. It inhibited the mitogen induced cell growth in microvascular endothelial cells in vitro. Ten ng/ml AGM-1470 completely inhibited both basic fibroblast growth factor induced cell growth, and interleukin-4 induced cell growth. In an in vivo study, we invesigated the antitumor effect of AGM-1470 in dimethylbenz[a]anthracene (DMBA) induced rat mammary tumors. A dose dependent growth inhibition was seen between administered doses 10 mg/kg and 40 mg/kg. Forty mg/kg 2-week administration of AGM-1470 totally suppressed the tumor growth. In addition, an additive inhibitory effect of AGM-1470 with an antiestrogen tamoxifen was found (p<0.01). It is suggested that the combination of these two drugs might be effective for the treatment of breast cancer.

Promoting effects of ethinyl estradiol on development of renal proliferative lesions induced by N-nitrosobis (2-oxopropyl)amine in female Syrian golden hamsters.

The modulating effects of female sex hormones, ethinylestradiol and levonorgestrel, on the development of renal proliferative lesions after initiation with N-nitrosobis(2-oxopropyl)amine (BOP) were investigated. Three groups of female Syrian golden hamsters, each comprising 30 animals, were given four weekly s.c. injections of BOP (10 mg/kg body weight) and then fed diet containing 1 ppm ethinylestradiol (group 1: BOP/EE), diet containing 10 ppm levonorgestrel (group 2) or basal diet (group 3) for the next 27 weeks. As hormone controls, two groups of 20 female hamsters each received diet containing 1 ppm ethynylestradiol (group 4) and 10 ppm levonorgestrel (group 5) from week 3 for 27 weeks without the prior initiation treatment. The severity of diffuse anisokarya, characterized by varied nuclear size and the incidence of dysplasias of the proximal tubular epithelia induced by BOP, were significantly increased in the BOP/EE group, indicating the promoting effects of the hormone. In the renal dysplastic lesions (small-cluster, cystic, clear-cell and acidophilic cell types), adenomas and nephroblastomas, increases in the numbers of argyrophilic proteins associated with nucleolar organizer regions (NOR) in the nucleus, suggesting a high proliferative activity, were seen in dysplasia of acidophilic cell types and adenomas. In addition, the number of bizarre NOR per nucleus was significantly higher in adenomas than in dysplasias and highest in nephroblastomas. This morphological change in NOR should therefore be a useful parameter for the diagnosis of malignancy of renal tumors.

Nucleolar segregation as an early marker for DNA damage; an experimental study in rats treated with 4-hydroxyaminoquinoline 1-oxide.

Male 6-week-old Sprague Dawley rats were given a single intravenous injection of 4-hydroxyaminoquinoline 1-oxide (4HAQO) at a dose of 20 mg/kg in order to produce ultrastructural changes as possible morphological biomarkers for toxicity. Immunohistochemically demonstrated formation of 4HAQO-DNA adduct was correlated with the changes found. Nucleolar alteration, demonstrable by electron microscopy as segregation of nucleolar components into granular and fibrillar compartments, was evident in cells of the target organs, exocrine pancreas and adrenocortex, but not of the non-target liver parenchyma. Sequential observation clarified that such alteration was highest in frequency 6 h and 4 h after 4HAQO administration in pancreatic acinar cells and adrenocortical cells respectively. Electron microscopically, apoptotic changes of acinar cells were evident 2 h after injection of 4HAQO. DNA adduct formation was consistently demonstrated in the same target organs showing nucleolar segregation, the highest frequency being noted 4 h after 4HAQO treatment in both pancreatic acinar cells and adrenocortical cells. Our results thus indicate an identity of the target cells for nucleolar segregation and 4HAQO-DNA adduct formation which correlates with 4HAQO-toxicity. We suggest that nucleolar segregation occurs subsequent to the generation of DNA damage.

Blocking effects of synthetic trypsin inhibitor (camostat) on pancreatic carcinogenesis in hamsters initiated with N-nitrosobis(2-oxopropyl)amine.

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.

Induction of pancreatic tumors in male Syrian golden hamsters by intraperitoneal N-methyl-N-nitrosourea injection.

The carcinogenic effects of N-methyl-N-nitrosourea (MNU) in male Syrian golden hamsters were investigated. After single i.p. administration of MNU at doses of 50 mg/kg or 10 mg/kg, or after five fractionated i.p. injections to make a total dose of 50 mg/kg body weight (10 mg x 5), histopathological examinations were performed at the end of 40th week of the experiment. Neoplastic changes were observed in various organs, and lesions in the pancreas, forestomach, and adrenal gland were predominant. In the pancreas, three tumor types were observed: ductal adenocarcinomas, acinar cell carcinomas, and islet cell carcinomas. The incidences of pancreatic ductal carcinomas were 56, 27, and 0% in the single 50-mg, fractionated 50-mg, and single 10-mg groups, respectively. Two islet carcinomas were observed in the single 50-mg group, and an islet carcinoma and an acinar cell carcinoma were also observed in the fractionated 50-mg group. Several miscellaneous neoplastic lesions, including squamous cell papillomas/carcinomas in the forestomach, cortical adenomas in the adrenal glands, and a seminoma in the testis were also observed. These results indicate MNU to be a multipotent carcinogen with the pancreas as a target organ in the Syrian golden hamster under this experimental condition. The observed high induction rate for pancreatic ductal carcinoma suggests that this MNU protocol is a useful candidate model for experimental pancreatic ductal carcinogenesis.