Gene expression and mutation assessment provide clues of genetic and epigenetic mechanisms in liver tumors of oxazepam-exposed mice.

Liver tumors from a previous National Toxicology Program study were examined using global gene expression and mutation analysis to define the mechanisms of carcinogenesis in mice exposed to oxazepam. Five hepatocellular adenomas and 5 hepatocellular carcinomas from male B6C3F1 mice exposed to 5000 ppm oxazepam and 6 histologically normal liver samples from control animals were examined. One of the major findings in the study was upregulation of the Wnt/ß-catenin signaling pathway. Genes that activate ß-catenin, such as Sox4, were upregulated, whereas genes that inhibit Wnt signaling, such as APC and Crebbp, were downregulated. In addition, liver tumors from oxazepam-exposed mice displayed ß-catenin mutations and increased protein expression of glutamine synthetase, a downstream target in the Wnt signaling pathway. Another important finding in this study was the altered expression of oxidative stress-related genes, specifically increased expression of cytochrome p450 genes, including Cyp1a2 and Cyp2b10, and decreased expression of genes that protect against oxidative stress, such as Sod2 and Cat. Increased oxidative stress was confirmed by measuring isoprostane expression using mass spectrometry. Furthermore, global gene expression identified altered expression of genes that are associated with epigenetic mechanisms of cancer. There was decreased expression of genes that are hypermethylated in human liver cancer, including tumor suppressors APC and Pten. Oxazepam-induced tumors also exhibited decreased expression of genes involved in DNA methylation (Crebbp, Dnmt3b) and histone modification (Sirt1). These data suggest that formation of hepatocellular adenomas and carcinomas in oxazepam-exposed mice involves alteration of the Wnt signaling pathway, oxidative stress, and potential epigenetic alterations.

Lack of modifying effects of eugenol on development of lung proliferative lesions induced by urethane in transgenic mice carrying the human prototype c-Ha-ras gene.

To investigate the modifying effects of eugenol (EUG), a component of cigarette smoke, on lung carcinogenesis, male and female transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) were given a single intraperitoneal injection of 250 mg/kg urethane (UR) or saline, followed by a diet containing 6,000 ppm EUG or basal diet for 26 weeks. Their non-transgenic CB6F1 littermates (non-Tg mice) were also treated in the same manner. In both male and female rasH2 mice, alveolar/bronchiolar hyperplasias, adenomas and carcinomas were observed in all UR-treated groups. However, there were no significant intergroup differences in the incidences and multiplicities of these lesions between the UR alone and UR + EUG groups. In non-Tg mice, alveolar/bronchiolar hyperplasias, adenomas or carcinomas were sporadically observed in UR-treated groups of both sexes, with no significant differences in the incidences and multiplicities between the UR alone and UR + EUG groups. There were no intergroup differences between them in the PCNA-positive ratios of adenomas or carcinomas and the areas of adenomas or carcinomas to the whole lung area examined. The present results suggest that the EUG treatment does not exert modifying effects on lung carcinogenesis induced by UR in both male and female rasH2 mice and non-Tg mice.

Di(2-ethylhexyl)phthalate induces hepatocellular adenoma in transgenic mice carrying a human prototype c-Ha-ras gene in a 26-week carcinogenicity study.

To evaluate the transgenic mouse carrying a human prototype c-Ha-ras gene (rasH2 mouse) as a model for 26-week carcinogenicity tests, Di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, was administered to 15 rasH2 mice/sex/group at concentrations of 1,500, 3,000 or 6,000 ppm, and to 15 wild-type (non-Tg) mice/sex/group at a concentration of 6,000 ppm in their diets for 26 weeks. Survival rates and food consumption in the groups treated with DEHP and in the control group were similar. Body weight gain in rasH2 and non-Tg mice at 6,000 ppm in the terminal week decreased about 10% as compared to the control group. Common findings related to treatment with DEHP in rasH2 and non-Tg mice included hypertrophy with coarse granules and deposit of pigment in the liver, hydronephrosis and tubular regeneration in the kidney, focal atrophy in the testis, and increased eosinophilic body in the nasal cavity. Hepatocellular adenoma was induced by treatment with DEHP, and was confined to male rasH2; mice the incidence being 7%(1/15), 13%(2/15), and 27%(4/15) in the 1,500-, 3,000-, and 6,000-ppm group, respectively. Point mutation was not detected in codon 12 and 61 of human c-Ha-ras transgene upon DNA analyses on frozen samples taken from these hepatocellular adenomas. From the results obtained in this 26-week carcinogenicity study, it is concluded that DEHP is a hepato-carcinogen for transgenic mouse carrying a human prototype c-Ha-ras gene.

Immunohistochemical and ultrastructural studies of 2,6-dimethylaniline-induced nasal proliferative lesions in a rat two-stage nasal carcinogenesis model initiated with N-bis(2-hydroxypropyl)nitrosamine.

Proliferative lesions induced by 2,6-dimethylaniline (DMA) in a two-stage rat nasal carcinogenesis model were immunohistochemically and ultrastructurally investigated. Male F344 rats received diet containing 3,000 ppm DMA for 52 weeks after initiation with a single subcutaneous injection of 2400 mg/kg of N-bis(2-hydroxypropyl)nitrosamine (DHPN). Histopathologically, proliferation of Bowman's glands, glandular hyperplasias, dysplastic foci, adenomas, and carcinomas were observed in treated rats. These nasal lesions mostly arose in the olfactory mucosa of the nasal cavity. Immunohistochemically, they were positive for cytokeratin and/or collagen type IV antibodies. Ultrastructurally, intracytoplasmic dense secretory granules (200-850 nm in diameter), identical to those in normal Bowman's glands, were observed in all the lesions, providing further support from an origin from these glands. Based on their cellular characterization, growth pattern and/or proliferative activity, two morphological continua were evident, one from dysplastic foci to carcinomas and the other from proliferation of Bowman's glands to glandular hyperplasias and adenomas. These results suggest that dysplastic foci arise from Bowman's glands and progress to carcinomas, while proliferation of Bowman's glands result in glandular hyperplasias and adenomas.

Promoting effects of xylazine on development of thyroid tumors in rats initiated with N-bis(2-hydroxypropyl)nitrosamine and the mechanism of action.

To cast light on whether xylazine hydrochloride (XZ), a veterinary medicine commonly used as a sedative agent for food-producing animals, has any promoting potential for thyroid carcinogenesis, the following studies were performed. In Experiment I, male F344 rats received a diet containing 1000 or 0 p.p.m. XZ for 52 weeks with or without initiation with 2400 mg/kg N:-bis(2-hydroxypropyl)nitrosamine (DHPN). Focal follicular cell hyperplasias, adenomas and/or carcinomas were induced in the DHPN alone, XZ alone and DHPN+XZ groups, and the incidences and multiplicities of these lesions in the DHPN+XZ group were significantly increased as compared with the DHPN alone case. In Experiment II, male F344 rats received a diet containing 1000 or 0 p.p.m. XZ and were examined for serum levels of triiodothyronine (T(3)), thyroxine (T(4)) and thyroid-stimulating hormone (TSH) at weeks 1, 2 and 4. In the XZ group, significant increase in thyroid weight and decrease in serum T(4) levels were observed at all time points. Serum T(3) and TSH levels were significantly decreased and increased, respectively, at week 1, but returned to within the control range thereafter. In Experiment III, male F344 rats received a diet containing 1000 or 0 p.p.m. XZ, they were examined for thyroid iodine uptake and organification of XZ after 1 and 2 weeks. The thyroidal iodine uptake per milligram of thyroid and the amount of iodine bound to 1 mg protein showed a tendency for decrease at week 1 and significant decrease at week 2. These results indicate that XZ has tumor-promoting effects on thyroid follicular cells, and suggest an involvement of alterations in thyroid-related hormone levels due to inhibition of thyroid iodine uptake and organification, resulting, provably, in serum TSH stimulation depending on continuous reduction of serum T(4) level through the feedback system in the pituitary-thyroid axis.

A novel "Nihon" rat model of a Mendelian dominantly inherited renal cell carcinoma.

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague-Dawley strain in Japan, and named the rising "Nihon" rat. In this strain, RCs develop from early preneoplastic lesions, which begin to appear at 4 weeks of age, forming adenomas by the age of 16 weeks. The RCs are predominantly of clear cell type. Southern blot, northern blot and SSCP analyses revealed no change in the Tsc1, Tsc2, VHL, and c-Met genes. Thus, the Nihon rat should be a valuable experimental model for understanding renal carcinogenesis, especially clear cell type, which is common among human RCs.

Sequential observation of 2,6-dimethylaniline-induced nasal lesions in a rat two-stage nasal carcinogenesis model after initiation with N-bis(2-hydroxypropyl) nitrosamine.

Male F344 rats received diet containing 3,000 ppm 2,6-dimethylaniline (DMA) after initiation with a single subcutaneous injection of 2,400 mg/kg of N-bis(2-hydroxypropyl)nitrosamine (DHPN), and histological and electron microscopic examinations of the nasal cavity were performed at 4, 13, 26 and 52 weeks to examine sequential changes induced by DMA. Severe atrophy of Bowman's glands and epithelial disarrangement were apparent from week 4, followed by dilatation and/or proliferation of Bowman's glands, degeneration of epithelial cells, and proliferation of undifferentiated epithelial cells from week 13. Focal glandular hyperplasias, dysplastic foci, and adenomas were observed from week 26, and carcinomas at 52 week. These nasal lesions were mostly evident in the olfactory mucosa in the nasal cavity, and their severity and/or incidences, other than atrophy of Bowman's glands, increased with the treatment period. Electron microscopically, carcinoma cells demonstrated desmosomes, dense secretory granules identical to those in normal Bowman's glands, a basement membrane, and microvilli. These results suggest that Bowman's glands are the target of DMA, giving rise to nasal carcinomas after DHPN-initiation.

Repeated dose (28 days) oral toxicity study of flutamide in rats, based on the draft protocol for the 'Enhanced OECD Test Guideline 407' for screening for endocrine-disrupting chemicals.

In association with the international validation project to establish a test protocol for the 'Enhanced OECD Test Guideline 407', we performed a preliminary 28-day, repeated-dose toxicity study of flutamide, a non-steroidal androgen antagonist, and assessed the sensitivity of a list of parameters for detecting endocrine-related effects of endocrine-disrupting chemicals (EDCs). Seven-week-old CD(SD)IGS rats were divided into four groups, each consisting of 10 males and 10 females, and administered flutamide once daily by oral gavage at doses of 0 (control), 0.25, 1 and 4 mg/kg body weight/day. Male rats were killed 1 day after the 28th administration. Female rats were killed on the day they entered the diestrus stage in the estrous cycle following the last treatment. Male rats receiving flutamide at dose levels of 1 and 4 mg/kg showed lobular atrophy of the mammary gland and a decrease in epididymal weight. In addition, 4 mg/kg flutamide-treated males exhibited raised serum testosterone and estradiol levels and decreased weight of the accessory sex glands. In females, a slight prolongation of the estrous cycle was also observed in the 4 mg/kg flutamide-treated group. No dose-related changes could be detected by haematology, serum biochemistry and sperm analysis. Thus, among the parameters tested in the present experimental system, the weight of endocrine-linked organs and their histopathological assessment, serum hormone levels, and estrous cycle stage allowed the detection of endocrine-related effects of flutamide.

Toxicity and blood concentrations of xylazine and its metabolite, 2,6-dimethylaniline, in rats after single or continuous oral administrations.

To cast light on whether the carcinogenic risk of 2,6-dimethylaniline (DMA), a metabolite of xylazine, may increase by ingestion of edible tissues from domestic animals treated with xylazine, the following studies of xylazine and DMA were performed. In Experiment I, male F344 rats received a single oral administration of 150 mg/kg of xylazine hydrochloride. Rats showed symptoms suggesting loss of sensation and pain immediately after the treatment. These signs had disappeared after 3 hr, but the animals died of hydrothorax and pulmonary edema by 9 hr. The plasma concentration of xylazine was 2.88 +/- 0.95 micrograms/ml at 15 min, and then decreased to 0.10 +/- 0.01 microgram/ml at 6 hr. The plasma level of DMA remained at 0.03 to 0.04 microgram/ml during the measurement period. In Experiment II, male F344 rats were fed a diet containing 1000 ppm of xylazine hydrochloride, regarded as the maximum tolerated dose, for 4 weeks. No clear clinical signs were evident and the plasma levels of xylazine and DMA were at the detection limit (0.02 microgram/ml) or less, although follicular cell hypertrophy of the thyroid was observed in all the treated animals. In Experiment III, male F344 rats were fed a diet containing 3000 ppm or 300 ppm of DMA for 4 weeks. Histological changes, such as atrophy of Bowman's gland and irregular arrangement of olfactory epithelial cells, were only observed in the olfactory epithelium of the 3000 ppm group. The plasma levels of DMA were 0.20 to 0.36 microgram/ml in the 3000 ppm group, but under the detection limit in the 300 ppm group. These results suggest that the probability of nasal carcinogenic effects of DNA on consumers via ingestion of edible tissues from food-producing animals treated with xylazine is extremely low, since DMA levels in the blood of rats subjected to continuous administration of high doses of xylazine remained under the detection limit.

Rapid induction of uterine tumors with p53 point mutations in heterozygous p53-deficient CBA mice given a single intraperitoneal administration of N-ethyl-N-nitrosourea.

To investigate the sensitivity of heterozygous p53-deficient CBA mice to carcinogens, 20 female mice [p53(+/-)] and 20 wild-type littermates [p53(+/+)] were given an intraperitoneal injection of 120 mg/kg body wt of N-ethyl-N-nitrosourea (ENU) and were maintained without any other treatment for a further 26 weeks. Histopathology showed that uterine tumors (endometrial polyps and stromal sarcomas) and lung adenomas were induced in both p53(+/-) and p53(+/+) mice. The incidence of uterine tumors and lung adenomas (94% and 81%, respectively) in p53(+/-) mice was significantly greater than that in p53 (+/+) mice (37% and 42%, respectively). Malignant lymphomas were only induced in p53(+/-) mice, at an incidence of 31%. Concerning uterine tumors and preneoplastic lesions, there were endometrial stromal sarcomas and atypical hyperplasias of the endometrial gland in 90% and 63%, respectively, of p53(+/-) mice, with significantly greater incidences than in p53(+/+) mice. Gene analysis revealed GCG-->GTG point mutations in codon 135 of exon 5 of the p53 allele in all of the uterine endometrial stromal sarcomas examined. Our results suggest that female p53(+/-) CBA mice are very susceptible to uterine carcinogenesis, providing a useful model for ENU-induced uterine tumors.

Lack of susceptibility of transgenic mice carrying the human c-Ha-ras proto-oncogene (rasH2 mice) to phenolphthalein in a 6-month carcinogenicity study.

Phenolphthalein has carcinogenic activity, causing malignant lymphomas in B6C3F1 mice at a dietary dose of 3000 ppm in a 2-year carcinogenicity study and in heterozygous p53-deficient female mice at the same dose in a 6-month study. To examine whether phenolphthalein carcinogenic potential can be detected in male and female transgenic (Tg) mice carrying the human c-Ha-ras gene (rasH2 mice) and their wild-type littermates (non-Tg mice), a diet containing 3000, 6000 or 12000 ppm was given for 6 months. Unequivocal induction of neoplastic lesions was not apparent, suggesting that rasH2 mice are resistant to the induction of malignant lymphomas by the treatment of phenolphthalein.

Choriocarcinoma and teratoma in the ovary of a cynomolgus monkey.

An ovarian choriocarcinoma was found in a 13-year-old cynomolgus monkey (Macaca fascicularis). The tumor was accompanied by a mature teratoma in the contralateral ovary. Histologically, the choriocarcinoma was characterized by nests of cells where cytotrophoblasts occupied the periphery with syncytiotrophoblasts at the center. Immunohistochemical staining for anti-human chorionic gonadotropin was positive in the syncytiotrophoblasts. The teratoma consisted of well-differentiated epidermal cells, sebaceous glands, hair follicles, cartilage, bone, and teeth. Choriocarcinoma metastases were in multiple organs. The concomitant development of choriocarcinoma and teratoma in the ovary is a consistent finding with the human counterparts of these lesions.

[A 90-day repeated dose oral toxicity study of magnesium chloride in F344 rats].

In order to examine the toxicity of magnesium chloride hexahydrate, four groups of 10 male and 10 female F344 rats received the compound by dietary supplementation at 2.5, 0.5, 0.1 or 0% for 90 days. No treatment-related death was observed during the study. Transient soft stool and sustained increase in water consumption were observed both in males and females of the 2.5% group and slight reduction in body weight gain was noted in the high-dose males. There were no toxic changes in food consumption, organ weights, hematology and biochemistry, and histopathological examinations in any treated-groups. Based on these results, the no-observed-adverse-effect-level was estimated to be 0.5%, and 2.5% is considered to be appropriate as highest dose for a 2-year carcinogenicity study.

Tumor-promoting activity of 2,6-dimethylaniline in a two-stage nasal carcinogenesis model in N-bis(2-hydroxypropyl)nitrosamine-treated rats.

The potential promotion activity on nasal carcinogenesis of 2,6-dimethylaniline (DMA), an alpha2-adrenergic agonist metabolite of xylazine which is used for food-producing animals as a sedative agent, was examined. Male F344 rats received diet containing 0 or 3000 ppm DMA for 52 weeks after initiation with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Histopathological assessment showed the incidence of carcinomas in the DHPN+DMA group (33%) to be significantly elevated as compared with that for the DHPN-alone group (5%). Incidences and/or multiplicity of epithelial hyperplasias and dysplastic foci were also increased in the DHPN+DMA group. These lesions were exclusively observed in the olfactory mucosa. The lowest plasma levels of DMA in tumor- and dysplastic foci-bearing rats were 0.05 and 0.20 microg/ml, respectively. These results indicate that DHPN acts as an appropriate initiator for nasal carcinogenesis and that DMA exerts a tumor-promoting effect on the olfactory mucosa in the rat nasal cavity.

[A 13-week subchronic oral toxicity study of Perilla extracts in F344 rats].

A 13-week subchronic oral toxicity study of Perilla extracts in drinking water containing 0%, 2.5%, 5% and 10% extracts was performed in both sexes of F344 rats. Rats were randomly divided into 4 groups each consisting of 10 males and 10 females. No animals died during the period of administration. There were no treatment-related changes in body weight gain or in hematological or blood biochemistry values. Nor were any treatment-related histopathological changes observed in the highest dose group. These findings indicate that ingestion of 10% Perilla extracts in drinking water for 13-week does not cause any toxicological changes in rats.

[13-week subchronic oral toxicity study of ammonium sulfate in rats].

A 13-week subchronic oral toxicity study of ammonium sulfate was performed in both sexes of F344 rats by feeding them a CRF-1 powder diet containing concentrations of 0%, 0.38%, 0.75%, 1.5%, and 3.0% of the substance. Rats were randomly divided into 5 groups each consisting of 10 males and 10 females. Male animals in the 3% group exhibited diarrhea during the administration period. No changes indicating obvious ammonium sulfate toxicity were observed in the body weights, organ weights, hematological, serum biochemical, or histopathological examinations. Based on these results, the NOEL (no-observed-effect level) of ammonium sulfate for F344 rats was judged to be 1.5% in males (886 mg/kg/day) and 3% in females (1975 mg/kg/day), and the MTD (maximally tolerated dose) for 2-year carcinogenicity studies in F344 rats was concluded to be 3.0% or more in the diet.