Carcinogenicity In Female Rats Research Articles

Carcinogenicity and chronic toxicity of butyl methacrylate in rats and mice by a two-year inhalation study.

We conducted a two-year inhalation study of butyl methacrylate using F344/DuCrlCrlj rats and B6D2F1/Crl mice. Rats were exposed to 0, 30, 125 and 500 ppm (v/v) and mice were exposed to 0, 8, 30 and 125 ppm (v/v) using whole-body inhalation chambers. Non-neoplastic lesions developed in the nasal cavities of both rats and mice, but neoplastic lesions were not found. There was also a positive trend in the incidence of large granular lymphocytic (LGL) leukemia in the spleen of male rats. No changes were observed in female rats. Overall, there is some evidence of carcinogenicity in male rats, but there is no evidence of carcinogenicity in female rats. In male mice, there was a positive trend by Peto's test in the incidence of hepatocellular adenomas, and the incidence of hepatocellular adenomas and hepatocellular carcinomas combined was significantly increased compared to the controls by Fisher's exact test in the 30 ppm exposed male group. In female mice, the incidence of hemangiosarcoma in all organs combined showed a positive trend by Peto's test. Therefore, there is some evidence of carcinogenicity in male mice, and there is equivocal evidence of carcinogenicity in female mice.

Chemical structure-related mechanisms underlying in vivo genotoxicity induced by nitrofurantoin and its constituent moieties in gpt delta rats.

Nitrofurans are antimicrobial compounds containing a nitro group at the 5-position of the furan ring and an amine or hydrazide side chain derivative. One member of the nitrofurans, nitrofurantoin (NFT), is a renal carcinogen in male rats despite its still controversial genotoxicity. We investigated chemical structure-related modes of action of NFT, and reporter gene mutation assays for NFT and its constituent moieties were performed. NFT, 5-nitro-2-furaldehyde (NFA), or 1-aminohydantoin (AHD) was administered to male F344 gpt delta rats by gavage for 4 or 13 weeks at a carcinogenic or the maximum tolerated dose. NFT caused a significant increase in gpt mutant frequency (MF) at 13 weeks with G-base substitution mutations. An increase in gpt MF was also observed in the NFA-treated group at 13 weeks, but not in the AHD-treated group. 8-Hydroxydeoxyguanosine (8-OHdG) levels in the kidney DNA of NFT-treated rats were significantly increased after 4 weeks. NFT caused accumulation of hyaline droplets indicated by positive immunostaining and western blot analysis for α2u-globulin in the proximal tubules. An additional study, in which female gpt delta rats were given NFT at the same dose used for males, was performed to mitigate the effect of α2u-globulin. NFT exerted the same effects on female rat kidneys to the same extent as males in terms of gpt MF and 8-OHdG level. Thus, it is highly probable that the structure of the nitro furan plays a key role in NFT-induced genotoxicity and genotoxic mechanisms including oxidative DNA damage are involved in NFT-induced renal carcinogenesis. α2u-globulin-mediated nephropathy may be a prerequisite for NFT-induced renal carcinogenesis in male rats, and additionally NFT could be a latent carcinogen in female rats and other animal species.

Development of α2u-Globulin Nephropathy and Adrenal Medullary Pheochromocytomas in Male Rats Following Exposure to Stoddard Solvent IIC

Stoddard solvent IIC is widely used as a solvent in paints and varnishes, and for dry cleaning and other grease removal applications. Because concern exists regarding the long-term effects of occupational exposure in industrial settings, the toxicity and carcinogenicity of Stoddard solvent IIC were evaluated in male and female F344/N rats and B6C3F1 mice. Rats and mice were exposed to 0, 138, 275, 550, 1100, or 2200 mg/m3 Stoddard solvent IIC by whole-body inhalation for 3 mo, and to 0, 138 (male rats), 550, 1100, or 2200 (female rats and male and female mice) mg/m3 for 2 yr. The kidney, liver, and adrenal medulla were targets of Stoddard solvent IIC toxicity in rats. After 3 mo of exposure, male rats developed lesions characteristic of α2u-globulin nephropathy. Male and female rats displayed increased liver weights and/or clinical pathology changes suggestive of hepatic injury, although no accompanying histopathologic changes were observed. After 2 yr, increased incidences of adrenal medullary pheochromocytomas provided some evidence of carcinogenicity in male rats. Renal tubule adenomas were slightly increased in male rats after 2 yr, and may have been related to exposure. In mice, there was no chemical-related toxicity after 3 mo, with the exception of increased liver weights in male mice exposed to 2200 mg/m3. After 2 yr, the incidences of hepatocellular adenomas were increased in female mice exposed to 2200 mg/m3; however, these increases were marginal and associated with increases in body weight. There was no evidence of Stoddard solvent IIC carcinogenicity in female rats or male mice. In summary, inhalation exposures of Stoddard solvent IIC resulted in renal toxicity and adrenal medullary pheochromocytomas in male rats. The liver also appeared to be a site of toxicity in male and female rats and mice.

Mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the mammary gland of Big Blue rats on high-and low-fat diets.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a food-borne mutagen and mammary gland carcinogen in female rats. A high-fat diet has been shown to increase the incidence of PhIP-induced mammary gland tumors. The current study used Big Blue rats harboring the lambda lacI mutational reporter transgene, to address whether the promotional effect of a high-fat diet is mediated via modulation in mammary gland mutagenesis. Big Blue rats were given 10 doses of PhIP (75 mg/kg, p.o.) and placed on defined low-fat (5% corn oil) or high-fat (23.5% corn oil) diet for 6 weeks prior to collecting mammary glands. The lacI mutant frequency (mean +/- standard error, n = 3 rats) was 231 +/- 15 (x10(-6)) and 193 +/- 12 (x10(-6)) in the low-and high-fat group, respectively. Values were increased 12-fold over control but were not significantly different between the two diets. In a parallel study, diet did not alter the mutant frequency induced by 7,12-dimethylbenz[a]anthracene (DMBA) (125 mg/kg, p.o.) in the mammary gland. The findings suggest that the promotion by the high-fat diet is not mediated via an increase in mutations. Consistent with the high potency of DMBA as a mammary carcinogen, the mutant frequency was 20-30% higher with DMBA than with PhIP. Sixty-nine and 56 PhIP-induced lacI mutants were sequenced from the low-and high-fat diet groups, respectively. While the percentage of various types of mutations was identical between the diet groups, some difference in the distribution of mutations along the lacI gene was observed. The mutation spectrum in the mammary gland from rats on both diets was consistent with the formation of PhIP-guanine adducts which were detected by a (32)P-post-labeling assay. Guanine base substitutions accounted for approximately 85% of all mutations irrespective of diet. Single base pair deletions at guanine occurred in 11-17% of mutants. G:C to T:A transversions were the predominant base substitution mutation accounting for 35-43% of all mutations. The majority of all guanine mutations (74%) occurred at guanine bases adjacent to another G:C pair. Five out of 125 (4%) mutations involved a guanine deletion in the 5'-GGGA-3' sequence, a PhIP signature mutation reported previously. Twelve out of 125 (10%) mutations involved the guanine base in the sequence 5'-CAG(Purine)-3' (Pu). The findings from these studies suggest that 5'-CAG(Pu)-3' is an additional characteristic target site for PhIP-guanine adduct-induced mutations in vivo in the mammary gland.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) retards mammary gland involution in lactating Sprague-Dawley rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a compound from cooked meat, is an established mammary gland carcinogen in female rats. Four doses of PhIP (150 mg/kg, p.o., once per day) were given to lactating Sprague-Dawley rats separated from their 10-day-old pups to initiate involution of the gland. Twenty-four hours after the last dose, apoptotic index in the mammary gland, as measured by the TUNEL assay, was significantly higher in the gland from control rats than in the PhIP-treated rats (4.757 +/- 1.066 versus 1.905 +/- 0.248%; P < 0.05). In comparison with controls, alveoli in the mammary gland of PhIP-treated rats were also visibly larger and contained more secretory epithelial cells. The expression of Bax, a stimulator of apoptosis, and Bcl-2, an inhibitor of apoptosis, were quantitated by western blotting. Accordingly, Bax expression was 2.7-fold higher in control rats, whereas Bcl-2 expression was 3.1-fold higher in PhIP-treated rats, both changes being statistically different (Student's t-test, P < 0.05). Immunohistochemistry further confirmed a lower expression of Bax and higher expression of Bcl-2 in secretory alveolar epithelial cells of the PhIP-treated mammary gland. The findings are consistent with the notion that exposure to PhIP retarded involution via partial inhibition of programmed cell death. To investigate possible mechanisms for the inhibitory effects of PhIP on mammary gland involution, serum levels of prolactin, an important hormone for the maintenance of lactation, were measured in virgin rats with regular estrous cycles given PhIP (150 mg/kg, p.o.) on the morning of diestrous. After one estrous cycle, on proestrous morning, serum prolactin levels were 1.3-fold higher after PhIP than after control vehicle (one-way ANOVA, Fisher LSD multiple comparison test, P < 0. 05). PhIP exposure during involution was associated with the induction of benign mammary tumors. Seven out of 12 rats developed fibroadenomas, and one developed a tubulopapillary carcinoma within 1 year of receiving PhIP administration during involution (150 mg/kg, p.o., once per day for 5 days), and a high-fat diet (23.5% corn oil). An increase in serum prolactin level and the effects on mammary gland apoptosis seen with PhIP may have implications for the mechanisms of carcinogenic targeting of PhIP to the mammary gland.

Detection of monocyclic aromatic amines, possible mammary carcinogens, in human milk.

Environmental chemicals may play a role in the etiology of human breast cancer. Aromatic amines, industrial chemicals found as environmental pollutants, have been identified as rat mammary carcinogens. We have detected the monocyclic aromatic amines, aniline, o-toluidine, and N-methylaniline at parts per billion levels in human milk samples, by applying solid-phase microextraction coupled with gas chromatography/mass spectrometry. Our findings indicate that human breast ductal epithelial cells are directly exposed to aromatic amines, including o-toluidine, which is a mammary carcinogen in female rats.

Distribution and metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in female rats and their pups at dietary doses.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary carcinogen in female rats and is present in a wide variety of cooked meats. We address here the excretion of PhIP and its metabolites into the breast-milk of lactating rats and the ability of chlorophyllin, a food product derivative with chemopreventive properties, to affect these levels at low PhIP doses. Lactating female F344 rats with suckling pups were orally administered 50, 500 and 1000 ng [14C]PhIP/kg body weight. The excretion of the [14C]PhIP into milk and its distribution among the mammary tissue, liver and blood of the dam, as well as among stomach contents and liver of their suckling pups was measured using accelerator mass spectrometry (AMS). PhIP, PhIP-4'-sulfate, 4'-hydroxy-PhIP, and N2-hydroxy-PhIP-N3-glucuronide were found in the milk at all doses. The chlorophyllin (500 microg/kg) co-administration with PhIP (500 ng/kg) caused increased levels of [14C]PhIP in the milk (32%) and stomach contents (35%) of the pups relative to the animals not receiving chlorophyllin at these low PhIP doses. In contrast, lower [14C]PhIP levels in the chlorophyllin treated animals were observed in the blood (47%) and mammary tissue (68%) of the dam, as well as the pup's liver tissue (37%) compared to the animals receiving only PhIP. Chlorophyllin co-administration resulted in an increased amount of N2-hydroxy-PhIP-N3-glucuronide (42%), increased PhIP (79%) and decreased levels of PhIP-4'-sulphate (77%) relative to the animals not receiving chlorophyllin. These results suggest that PhIP and PhIP metabolites are present in the breast-milk of lactating rats at human dietary PhIP exposures and that PhIP is absorbed by the newborn. Furthermore, these results suggest that other dietary components can affect the dosimetry of PhIP in breast-feeding offspring.

Assessment of the Carcinogenic Potential of Chlorinated Water: Experimental Studies of Chlorine, Chloramine, and Trihalomethanes

Water chlorination has been one of the major disease prevention treatments of this century. While epidemiologic studies suggest an association between cancer in humans and consumption of chlorination byproducts in drinking water, these studies have not been adequate to draw definite conclusions about the carcinogenic potential of the individual byproducts. The purpose of this study was to investigate the carcinogenic potential of chlorinated or chloraminated drinking water and of four organic trihalomethane byproducts of chlorination (chloroform, bromodichloromethane, chlorodibromomethane, and bromoform) in rats and mice. Bromodichloromethane, chlorodibromomethane, bromoform, chlorine, or chloramine was administered to both sexes of F344/N rats and (C57BL/6 x C3H)F1 mice (hereafter called B6C3F1 mice). Chloroform was given to both sexes of Osborne-Mendel rats and B6C3F1 mice. Chlorine or chloramine was administered daily in the drinking water for 2 years at doses ranging from 0.05 to 0.3 mmol/kg per day. The trihalomethanes were administered by gavage in corn oil at doses ranging from 0.15 to 4.0 mmol/kg per day for 2 years, with the exception of chloroform, which was given for 78 weeks. The trihalomethanes were carcinogenic in the liver, kidney, and/or intestine of rodents. There was equivocal evidence for carcinogenicity in female rats that received chlorinated or chloraminated drinking water; this evidence was based on a marginal increase in the incidence of mononuclear cell leukemia. Rodents were generally exposed to lower doses of chlorine and chloramine than to the trihalomethanes, but the doses in these studies were the maximum that the animals would consume in the drinking water. The highest doses used in the chlorine and chloramine studies were equivalent to a daily gavage dose of bromodichloromethane that induced neoplasms of the large intestine in rats. In contrast to the results with the trihalomethanes, administration of chlorine or chloramine did not cause a clear carcinogenic response in rats or mice after long-term exposure. These results suggest that organic byproducts of chlorination are the chemicals of greatest concern in assessment of the carcinogenic potential of chlorinated drinking water.

Mechanisms of dioxin tumor promotion: Implications for risk assessment

TCDD is a liver carcinogen in female rats but not male rats. In a two stage model for hepatocarcinogenesis, using a single initiating dose of diethylnitrosamine (DEN) and chronic exposure to TCDD (25 ng/kg/day for 30 weeks), ovariectomy prevented the tumor promoting actions of TCDD. This finding was consistent with data on cell proliferation which demonstrated that TCDD did not induce cell proliferation in the absence of the ovaries. Dose-response data were obtained for a number of responses to TCDD within the framework of a tumor promotion model. These included cell proliferation, number and size of hepatic preneoplastic foci, induction of cytochrome P-450 isozymes (CYP 1A1 and 1A2), TCDD liver and blood concentrations, epidermal growth factor (EGF) receptor, estrogen receptor, histological changes and clinical chemistries. Dose response relationships were markedly different for the different parameters. For example, CYP 1A1 and 1A2 induction occurred at lower doses than effects of cell proliferation and preneoplastic lesions. The relevance of these data to low dose human risks and rodent to human extrapolations of risks are discussed.

The human relevance of the renal tumor-inducing potential of d-limonene in male rats: Implications for risk assessment

The monoterpene d-limonene is a naturally occurring chemical which is the major component in oil of orange. Currently, d-limonene is widely used as a flavor and fragrance and is listed to be generally recognized as safe (GRAS) in food by the Food and Drug Administration (21 CFR 182.60 in the Code of Federal Regulations). Recently, however, d-limonene has been shown to cause a male rat-specific kidney toxicity referred to as hyaline droplet nephropathy. Furthermore, chronic exposure to d-limonene causes a significant incidence of renal tubular tumors exclusively in male rats. Although d-limonene is not carcinogenic in female rats or male and female mice given much higher dosages, the male rat-specific nephrocarcinogenicity of d-limonene may raise some concern regarding the safety of d-limonene for human consumption. A considerable body of scientific data has indicated that the renal toxicity of d-limonene results from the accumulation of a protein, α2u-globulin, in male rat kidney proximal tuble lysosomes. This protein is synthesized exclusively by adult male rats. Other species, including humans, synthesize proteins that share significant homology with α2u-globulin. However, none of these proteins, including the mouse equivalent of α2u-globulin, can produce this toxicity, indicating a unique specificity for α2u-globulin. With chronic exposure to d-limonene, the hyaline droplet nephropathy progresses and the kidney shows tubular cell necrosis, granular cast formation at the corticomedullary junction, and compensattor cell proliferation. Both d-limonene and cis-d-limonene-1,2-oxide (the major metabolite involved in this toxicity) are negative in in vitro mutagenicity screens. Therefore, the toxicity-related renal cell proliferation is believed to be integrally involved in the carcinogenicity of d-limonene as persistent elevations in renal cell proliferation may increase fixation of spontaneously altered DNA or serve to promote spontaneously initiated cells. The scientific data base demonstrates that the tumorigenic activity of d-limonene in male rats is not relevant to humans. The three major lines of evidence supporting the human safety of d-limonene are (1) the male rat specificity of the nephrotoxicity and carcinogenicity; (2) the pivotal role that α2u-globulin plays in the toxicity, as evidenced by the complete lack of toxicity in other species despite the presence of structurally similar proteins; and (3) the lack of genotoxicity of both d-limonene and d-limonene-1,2-oxide, supporting the concept of a nongenotoxic mechanism, namely, sustained renal cell proliferation. Collectively, the evidence that the renal effects of d-limonene are confined to male rats because of the unique presence of α2u-globulin is quite compelling. In this regard, d-limonene is readily distinguished from classical renal carcinogens and should, therefore, not be subjected to traditional interspecies extrapolation and quantitative risk assessment. As d-limonene shows no toxicity or carcinogenicity in female rats or male and female mice when administered over a lifetime, it is considered safe for human consumption.

Lung neoplasms in rodents after chronic administration of dimethyl hydrogen phosphite

Dimethyl hydrogen phosphite (DMHP), an intermediate in the production of insecticides or herbicides, was administered by p.o. gavage for 2 yr to male Fischer 344/N rats and male and female B6C3F1 mice at doses of 0, 100, or 200 mg/kg and to female Fischer 344/N rats at doses of 0, 50 or 100 mg/kg. Dose related toxicity was seen in the lungs of treated male and female rats. The lung lesions were most prevalent in the high dose male rat group which received a dose twice that given to the high dose female rats. Lung lesions included alveolar epithelial hyperplasia, chemically related pneumonia, alveolar-bronchiolar adenoma, alveolar-bronchiolar carcinoma, and squamous cell carcinoma. DMHP also caused neoplastic and nonneoplastic lesions of the forestomach in male rats; a similar but less pronounced effect was observed in female rats. Nonneoplastic lesions associated with administration of DMHP included mineralization of the cerebellum in male rat and focal calcification of the testis in male mice. Under the conditions of this study, there was clear evidence for carcinogenicity for male rats, equivocal evidence for carcinogenicity in female rats, and no evidence for carcinogenicity in either male or female mice. DMHP caused the highest incidence of lung tumors in the male rat of all chemicals studied to date in the National Cancer Institute-National Toxicology Program Carcinogenesis Testing Program.

High incidence of rat mammary carcinoma by oral administration of ethyl methanesulphonate

Ethyl methanesulphonate (EMS), one of the alkylating agents, is known to be a potent mutagen. We tested EMS for its carcinogenicity in female rats by oral administration. EMS was dissolved in drinking tap water at a concentration of 10(-3) M and was given for the first 12 weeks. The subcutaneous tumors were noticed as early as 16 weeks after initiating the experiment. At the 32nd week, all the surviving rats produced tumors; the majority were multiple tumors in the neck, axillar and inguinal areas corresponding to bilateral mammary glands. Histologically, the prevailing feature of the tumors was infiltrating medullary adenocarcinoma consistent with carcinoma of mammary duct origin. Neither regional lymph node involvement nor distant metastases were shown, but intraductal spread of carcinoma was a marked finding during the 32-week period.