Sulfuric acid esters as major ultimate electrophilic and hepatocarcinogenic metabolites of 4-aminoazobenzene and its N-methyl derivatives in infant male C57BL/6J x C3H/HeJ F1 (B6C3F1) mice

Liver cytosols from 12-day-old male C57BL/6 X C3H/HeJ F1 (B6C3F1) mice contain 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase activity for N-hydroxy-4-aminoazobenzene and N-hydroxy-N-methyl-4-aminoazobenzene. No acetyl co-enzyme A-dependent transacetylase activity for these hydroxylamines was detected in the cytosols. Pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol were only moderately active inhibitors of the sulfotransferase activity; at a 100-microM concentration each compound inhibited the activity by only 50-80%. A single dose of 0.04 mumol/g body weight of PCP administered to 12-day-old male B6C3F1 mice 45 min prior to a single dose of 0.1 mumol/g body weight of [3H]4-aminoazobenzene ([3H]AB) or [3H]N,N-dimethyl-4-aminoazobenzene ([3H]DAB) inhibited DNA adduct formation by approximately 50%. Under identical conditions, PCP also reduced the average number of hepatomas induced per mouse at 9 months by AB and N-methyl-4-aminoazobenzene (MAB) by 52 and 36%, respectively. PCP strongly inhibited the hepatocarcinogenicity of DAB or AB when this agent was administered in the diet with either dye to female CD-1 mice over a 10- month period. Single doses of 0.15 mumol/g body weight of [3H]AB and [3H]DAB bound to hepatic DNA of 12-day-old brachymorphic B6C3F2 mice, which are deficient in the synthesis of PAPS, at levels 15 and 20%, respectively, of those found in their phenotypically normal litter mates. Under identical conditions, the incidence of hepatomas in brachymorphic mice at 9 months were 11 and 29%, with averages of 0.2 and 0.8 hepatomas/mouse for AB and MAB, respectively. Incidences of 77 and 86%, with averages of 6.6 and 5.4 hepatomas/mouse, respectively, were found in their phenotypically normal litter mates. These data strongly indicate that N-sulfo?xy-AB is a major ultimate electrophilic and hepatocarcinogenic metabolite of AB in mice. Similarly, this ester and N-sulfo?xy-N-methyl-4-aminoazobenzene appear to be critical metabolites for these activities of DAB and MAB.     

Carcinogenicity of o-ethoxybenzamide in (C57BL/6N X C3H/HeN)F1 mice

The carcinogenicity of o-ethoxybenzamide (CAS: 938-73-8), which is also called ethenzamide and which is widely used as an antipyretic anodyne in Japan, was examined in 298 (C57BL/6N X C3H/HeN)F1 mice. Groups of males and females were fed a diet containing 0 (control), 0.4, or 1.2% o-ethoxybenzamide for 96 weeks and sacrificed at the 100th week. Among the male mice fed the higher dose of the drug, the total incidence of liver cell tumors was 68%, with 18% of the mice developing hepatocellular carcinomas; both yields were significantly higher than those in the controls. In o-ethoxybenzamide-treated male mice the multiplicities of the hepatic cell tumors were also significantly higher than the multiplicity of the hepatic tumor in male control mice. A dose-response relationship with regard to both incidence and multiplicities of hepatic cell tumors in male mice was observed. In female mice fed o-ethoxybenzamide the incidence and multiplicities of the liver cell tumors were increased compared to those of the controls, but statistical significance was observed only in the multiplicity of tumors in mice given the lower dose. In both sexes hepatic cell tumors developed earlier than in the controls. These results show that o-ethoxybenzamide enhances the development of hepatic cell tumors in male (C57BL/6N X C3H/HeN)F1 mice.     

Carcinogenicity of bucetin in (C57BL/6 X C3H)F1 mice

The carcinogenicity of bucetin [(3-hydroxy-p-butyrophenetidide) CAS: 1083-57-4], an antipyretic analgesic drug, was examined in 300 (C57BL/6 X C3H)F1 mice. Groups of 50 mice of each sex were treated with 1.5 or 0.75% bucetin in their basal diet for 76 weeks and then fed a basal diet for 8 weeks. Control groups were given a basal diet for 84 weeks. In 10 of 46 (22%) male mice given the high dose of bucetin and in 6 of 45 (13%) given the low dose, renal cell tumors were induced. Dysplastic lesions of the proximal tubules were frequently seen in the males given bucetin in a dose-related fashion. Neither tumorous nor preneoplastic lesions developed in the kidneys of bucetin-treated female mice and control animals. Papilloma of the urinary bladder in 1 male mouse and papillary or nodular hyperplasia in 9 mice of both sexes were observed in groups given the high dose of bucetin.     

Histopathology of liver carcinomas in (C57BL/6N X C3H/HeN)F1 mice ingesting chlordane.

(C57BL/6N X C3H/HeN)F1 male mice that ingested 30 or 56 ppm chlordane and female mice that ingested 30 or 64 ppm chlordane in the diet had highly significant incidences of carcinomas of the liver. The carcinomas varied from well differentiated to poorly differentiated and undifferentiated and were capable of invasion and metastasis. They were more poorly differentiated in mice receiving chlordane than in controls.     

alpha-Fetoprotein levels and hepatic alterations during chemical carcinogenesis in C57BL/6N mice.

Hepatocellular carcinomas were induced by administration of acetylaminofluorene or chlordane to C57BL/6N mice. Lesions which closely resembled the neoplastic nodule described as a putative premalignant lesion in rats were evident. alpha-Fetoprotein elevations were noted only in the presence of the malignant lesions for both carcinogens. In this regard, the responses of these mice were similar to those seen during spontaneous hepatocarcinogenesis.     

Enzyme and immunohistochemical phenotyping of diethylnitrosamine-induced liver lesions of male C3H/He, B6C3F1 and C57BL/6J mice.

Male C3H/He, B6C3F1 and C57BL/6J mice were given a single injection of diethylnitrosamine (20 micrograms/g body wt) on day 15 after birth and animals were killed 17-29 (C3H/He and B6C3F1) and 29-46 weeks (C57BL/6J) after treatment. Carcinogen-induced liver lesions were identified by a deficiency in the marker enzyme glucose-6-phosphatase and the enzymatic phenotypes of these lesions were studied by enzyme and immunohistochemical methods using serial liver sections stained for seven additional histochemical markers. In all three mouse strains, liver lesions were characterized by an increased basophilia and a decreased expression of UDP-glucuronosyl-transferase, microsomal epoxide hydrolase and NADPH-cytochrome P450 reductase, while the cytochrome P450 isoenzymes 1A2, 2C6 and 2E1 were virtually unexpressed. Quantitative analyses revealed that throughout all periods of investigation, on average greater than 70% of the glucose-6-phosphatase-deficient lesions occupying up to 99% of the total volumetric fraction expressed concomitant alterations in at least one of these additional marker stainings. Upon determination of the phenotypic complexity levels, between 70 and 90% of lesions were found to contain alterations in at least six of the markers analysed, while lesions with alterations in less than three markers were comparatively infrequent. In the light of previous observations in the rat liver system, the relative homogeneity of enzyme phenotypes and the apparent lack of time-dependent changes in enzyme expression suggest that the majority of lesions of all three mouse strains possess an increased neoplastic character already from the very early beginning of the carcinogenic process in liver.     

Circadian variation in the induction of intestinal tumors by N-methyl-N-nitrosourea in male C57BL/6N mice

Male C57BL/6N mice were administered a single ip injection of 30 mg of N-methyl-N-nitrosourea (MNU)/kg of body weight. Additional groups were treated similarly every 3 hours for the next 24 hours. Adenocarcinomas of the small intestine were the major treatment-related tumors, with the total incidence being 38% at 250 days after injection. There was a significant circadian variation for tumor induction; the maximum number of intestinal tumors (approximately equal to 55%) tended to occur when the MNU was administered during the middle of the light period (6:00 to 18:00), while the tumor incidence was at a minimum (approximately equal to 10%) when the MNU was given in the middle of the dark phase (18:00 to 6:00). These data are discussed in relation to DNA synthesis and repair and MNU-induced cellular toxicity.     

Morphology and classification of ovarian neoplasms in F344 rats and (C57BL/6 X C3H)F1 mice

For the updating of the National Toxicology Program (NTP) classification system for rat and mouse ovarian tumors, all the primary ovarian tumors from the archives of the National Cancer Institute and NTP Carcinogenesis Testing Programs were reviewed. The relative frequency and principal diagnostic features of 204 ovarian tumors from 39,851 female F344 rats and of 587 ovarian tumors from 41,102 female (C57BL/6 X C3H)F1 (B6C3F1) mice were described. The most frequently observed neoplasms in F344 rats were malignant granulosa cell tumors (29% of primary rat ovarian neoplasms observed), benign undifferentiated sex cord-stromal tumors (26%), benign granulosa cell tumors (16%), and benign Sertoli cell tumors (7%). The most frequent neoplasms in B6C3F1 mice were cystadenomas (24%), tubulostromal adenomas (24%), benign granulosa cell tumors (21%), and benign teratomas (8%). Ovarian neoplasms were not significantly related to treatment in rats. Tubulostromal adenomas, benign and malignant granulosa cell tumors, and benign teratomas were significantly more frequent in treated mice than in controls.     

Disposition and metabolism of aniline in Fischer 344 rats and C57BL/6 X C3H F1 mice

We examined the metabolism and disposition of aniline, which induces spleen hemangiosarcomas in rats but no tumors in mice, in normal and predosed Fischer 344 rats, and C57BL/6 X C3H F1 mice administered low (50 and 100 mg/kg, respectively) or high (250 and 500 mg/kg, respectively) doses. Of 11 tissues examined, the highest levels of binding of [14C]aniline to DNA were in the kidney, large intestine, and spleen of high-dose rats that had received prior dosing; these tissues had covalent binding indices of 14.2, 4.3, and 3.7 mumol/mol nucleotides/dose, respectively. Protein and RNA were the major macromolecular targets for binding of radioactivity from [14C]aniline. Relative to controls, most tissues from predosed mice (low dose and high dose) showed less binding to protein and RNA; but for most tissues from predosed rats administered 50-mg/kg doses of [14C]aniline, there was more extensive binding. Also relative to controls, binding of radioactivity in the spleen of predosed rats given [14C]aniline (50 mg/kg) was 148% greater for protein and 302% greater for RNA. For rats administered 250 mg of [14C]aniline per kg, however, there were no outstanding differences in binding to RNA and protein between normal and predosed animals. The profiles of urinary metabolites produced by rats and mice were not appreciably different in animals predosed with aniline. For rats, however, the profiles were different for the low and high doses, suggesting that the main metabolic pathway was saturated at the higher dose. p-Acetamidophenyl sulfate represented over 70% of the total radioactivity recovered from the urine of rats dosed with 50 mg of aniline per kg but only 30% in the urine of those dosed with 250 mg/kg. The urine of the high-dose rats contained greater percentages of p-aminophenyl sulfate, p-acetamidophenyl glucuronide, and unconjugated metabolites. In mouse urine, p-acetamidophenyl glucuronide, representing 29 to 32% of the total radioactivity, was the major metabolite. Nevertheless, mice produced more ortho derivatives than did rats, for in acid-treated urine, the ratio of p- to o-aminophenol was 8.1 for rats and 1.6 for mice. Predosing of rats and mice did not change the kinetic values for liver aniline p-hydroxylase or N-hydroxylase but increased the amount of mouse liver cytochrome P-450 from 0.231 to 0.491 nmol/mg protein. For p-hydroxylase of rat liver, the apparent Km value was higher, and the apparent Vmax value lower than in mouse liver. Kinetic values for rat and mouse N-hydroxylase were similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neoplasms observed in untreated and corn oil gavage control groups of F344/N rats and (C57BL/6N X C3H/HeN)F1 (B6C3F1) mice

Control data on F344/N rats and (C57BL/6N X C3H/HeN)F1 (B6C3F1) mammary tumor virus-free mice from the National Toxicology Program (NTP) were examined to determine if animals receiving corn oil by gavage showed tumor incidences that differed from those of untreated control animals. Analyses of these data were adjusted for interlaboratory variability, time-related trends, and supplier effects. Two biologically significant effects were found: Male F344/N control rats receiving corn oil by gavage showed a higher (P less than .05) incidence of pancreatic acinar cell adenoma and a lower (P less than .001) incidence of leukemia (primarily mononuclear cell leukemia) than did the corresponding untreated controls. The increased incidences of pancreatic acinar cell adenoma seen in male rats administered corn oil by gavage were associated with elevated body weights observed in these animals relative to untreated controls. Female F344 rats and male and female B6C3F1 mice showed little or no evidence of a difference in tumor incidence between corn oil gavage-treated and untreated controls. A review of nearly 300 carcinogenesis studies done by the National Cancer Institute (NCI) and the NTP revealed that there were no corn oil gavage studies in which increased incidences of pancreatic acinar cell tumors or leukemia in male F344/N rats were the sole evidence of the carcinogenicity of a test chemical. Thus use of corn oil appears to have little impact on the interpretation of NCI-NTP carcinogenicity studies.     

Forestomach and kidney carcinogenicity of caffeic acid in F344 rats and C57BL/6N x C3H/HeN F1 mice

The carcinogenic potential of caffeic acid was investigated in both sexes of F344 rats and C57BL/6N x C3H/HeN F1 mice. After groups of 30 animals received diet containing 0 and 2.0% caffeic acid for 104 weeks in rats or 96 weeks in mice, detailed histopathological examination revealed induction of forestomach squamous cell papillomas or carcinomas in rats at high incidence (77% for males; 80% for females) and in mice at low incidence (13% for males; 3% for females). Invasion to the abdominal cavity of these squamous cell carcinomas was observed in three rats and two mice. In addition, renal tubular cell hyperplasias and adenomas, clearly related to toxic lesions, were found in treated rats at high incidence for males (73 and 13%) and low incidence for females (20 and 0%). In mice, renal tubular cell hyperplasias and tumors also occurred in treated females (97 and 28%), and at a lower incidence in treated males (27 and 3%). No toxic renal injuries were apparent in mice. Alveolar type II cell tumors also developed in treated male mice (27%) with statistical significance. Thus, the current investigation showed caffeic acid to exert carcinogenic activity for the forestomach squamous cell epithelium in both sexes of F344 rats and C57BL/6N x C3H/HeN F1 mice, for the renal tubular cell in male rats and female mice, and for the alveolar type II cell in male mice.     

Inheritance of susceptibility to phototumorigenesis and persistent hyperplasia in F1 hybrids between SENCAR mice and BALB/c or C57BL/6 mice

SENCAR mice are selectively bred for hypersusceptibility to two-stage chemical skin carcinogenesis. They are also hypersusceptible to UV radiation tumorigenesis with single high-dose, but not chronic low-dose, exposures. In addition, SENCAR mice exhibit an exaggerated and persistent epidermal hyperplasia (due to sustained proliferation of the basal cells) in response to UV-induced tissue damage. In the present study, we have examined the inheritance of susceptibility to both phototumorigenesis and persistent hyperplasia in the F1 offspring of SENCAR mice crossed with either of two inbred strains (BALB/c or C57BL/6) which are relatively resistant to phototumorigenesis. A total of 428 mice from the parental strains and reciprocal F1 crosses were given a single high dose (8.64 x 10(4) J/m2) of UV radiation (FS40 sunlamps) which causes persistent hyperplasia and tumorigenesis in many SENCAR, but no BALB/c or C57BL/6, mice. F1 hybrids between SENCAR and C57BL/6 mice did not develop persistent hyperplasia or skin tumors, which indicates that susceptibility to both traits is completely recessive to the C57BL/6 genotype. In contrast, F1 hybrids between SENCAR and BALB/c mice developed both persistent hyperplasia and skin tumors, although at a much lower incidence than the SENCAR mice, indicating that susceptibility to both traits is only partially (incompletely) recessive to the BALB/c genotype. Thus, in either F1 cross, susceptibility to phototumorigenesis decreased in parallel with persistent hyperplasia. These results are consistent with the hypothesis that the two characteristics are mechanistically related.     

Induction of peroxisome proliferation and hepatic tumours in C57BL/6N mice by ciprofibrate, a hypolipidaemic compound

The hepatic effects of ciprofibrate, a potent peroxisome proliferator, were evaluated in male C57BL/6N mice, a mouse strain with very low incidence of spontaneous liver tumour development. Dietary feeding of ciprofibrate (0.0125% or 0.025% w/w) for 2 weeks resulted in a marked proliferation of peroxisomes (9-fold increase) and several-fold increase (8- to 10-fold) in the activity of peroxisomal beta-oxidation enzymes. Feeding ciprofibrate at 0.025% concentration for 15 months followed by a 0.0125% for 6 months led to the development of hepatic adenomas in 8/14 (57%) and hepatocellular carcinomas (HCC) in 3/14 (21%) mice. In mice given 0.0125% ciprofibrate for 18 months 5 of 8 (62%) and 3 of 8 (37%) developed adenomas and HCC respectively. Similar to the findings observed in rats, both the adenomas and HCC were negative for gamma-glutamyltranspeptidase. These results in C57BL/6N mice of hepatocarcinogenic effect of ciprofibrate, a non-genotoxic chemical, indicate that peroxisome proliferation can be used as a reliable parameter to evaluate the carcinogenicity of hypolipidaemic compounds.     

Neoplastic and nonneoplastic lesions in aging (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice.

Neoplastic and nonneoplastic lesions in untreated (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice used as controls in carcinogenesis tests were tabulated and evaluated. The most common neoplasms in 2,543 male mice were hepatocellular adenomas and carcinomas. In 2,522 female mice, common tumors were lymphomas, leukemias, pulmonary adenomas and carcinomas, hepatocellular adenomas and carcinomas, and pituitary adenomas. The risk of developing most neoplasms increased with the age of the mouse. Hepatocellular carcinomas metastasized in 12% of the animals with these tumors. Other than lymphomas and leukemias, few other tumors metastasized. Nonneoplastic lesions included cystic hyperplasia of the uterus, nephritis, ovarian and uterine cysts, inflammatory lesions of the lung, mineralization in the brain, and focal hyperplasias in several tissues. The focal hyperplasias in lung and pituitary, adrenal, and thyroid glands were suggestive of the early stages of neoplasia. Comparative aspects of lesions in aging mice and their interpretation in carcinogenesis tests are discussed.     

Induction of uterine hemangioendothelioma and lymphoma in (C57BL/6N x C3H/2N)F1 mice by oral administration of azathioprine

The tumorigenicity of 6-(1-methyl-4-nitro-5-imidazolyl)mercaptopurine (azathioprine), an immunosuppressant, was examined in (C57BL/6N X C3H/2N)F1 mice. Animals were divided into 6 groups with 50 mice in each group, and they were given powdered diet mixed with 0, 5 or 20 ppm azathioprine starting at 6 weeks and ending at 100 weeks of age. Female mice, given 20 ppm azathioprine, developed lymphomas and uterine hemangioendotheliomas at incidences of 12.5 and 14.6%, respectively, which were significantly higher than the incidences in control mice (P less than 0.05). Lymphoma and uterine hemangioendothelioma developed independently in different mice. On the other hand, the male mice given 20 ppm azathioprine had a significantly (P less than 0.05) lower incidence of hepatic tumor (0.5%) compared to the control mice (16%).     

Renal carcinogenic and nephrotoxic effects of the flame retardant tris(2,3-dibromopropyl) phosphate in F344 rats and (C57BL/6N X C3H/HeN)F1 mice.

Tris(2,3-dibromopropyl) phosphate (TBP) was administered in the feed at one of two concentrations to groups of 55 male and 55 female inbred F344 rats and to 50 male and 50 female (C57BL/6N X C3H/HeN)F1 mice. The high and low dietary concentrations of TBP administered orally were 100 and 50 ppm for the rats, respectively, and 1,000 and 500 ppm for the mice, respectively. For each rodent type, 55 animals of each sex were used as contols. In both rodent types, renal epithelial tumors developed at incidences that were significant for male and female rats and mice that received the doses. These tumors included renal tubular cell adenomas and carcinomas that developed from the proximal convoluted tubular epithelium. Among female mice and rats, hyperplasia and/or dysplasia of the proximal convoluted tubular epithelium with or without cystic dilatation of the tubules and increase in the size of cell nuclei were dose dependent and recognized as preneoplastic and/or toxic lesions. The comparative histogenesis of renal tubular neoplasms was discussed.     

Nasal cavity neoplasia in F344/N rats and (C57BL/6 x C3H)F1 mice inhaling propylene oxide for up to two years

Propylene oxide (CAS: 75-56-9) was studied for potential carcinogenicity and chronic toxicity by inhalation in F344/N rats and (C57BL/6 x C3H)F1 mice. Groups of 50 animals of each sex were exposed to 0, 200, or 400 ppm propylene oxide for 6 hours/day, 5 days/week, for up to 103 weeks. Survival decreased in mice exposed to propylene oxide; the decrease was significant (P less than .005) in mice exposed to 400 ppm. Survival of exposed rats was comparable to that of controls. Mean body weight of rats and mice exposed to 400 ppm propylene oxide decreased, when compared to that of controls, during the 2d year of exposure. Exposure to propylene oxide for up to 2 years induced inflammatory and proliferative responses in nasal cavity of both species. There was clear evidence of carcinogenicity in mice exposed to 400 ppm propylene oxide; 10 of 50 males and 5 of 50 females had hemangiomas or hemangiosarcomas of the nasal submucosa. Papillary adenomas involving the nasal respiratory epithelium and underlying submucosal glands were observed in 3 female rats and 2 male rats exposed to 400 ppm propylene oxide.     

N-Sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-2-acetylaminofluorene in the livers of infant male C57BL/6J x C3H/HeJ F1 (B6C3F1) mice

The hepatic DNA of 12-day-old male B6C3F₁ (C57BL/6J x C3H/HeJ)mice given an i.p. dose of 0.06 or 0.11 µmol/g body weightof N-hydroxy-[³H]-2-acetylaminofluorene (N-hydroxy-AAF) containedat 9 h 3 or 6 pmol of N-(deoxyguanosin-8-yl)-2-aminofluoreneadducts per mg. Together the level of the two acetylated adductsN-(deoxyguanosin-8-yl)-2-acetyl-aminofluorene and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorenewas 10% of this amount. The same doses of unlabeled carcinogeninduced by 10 months a 100% incidence of hepatomas with averagesof 10 and 15 hepatomas per mouse, respectively. Injection of0.04 µmol/g body weight of pentachlorophenol (PCP) 45min before the dose of N-hydroxy-AAF decreased the number ofadducts in the DNA by 90% and the average number of hepatomasper liver by 80–90%. As compared to their normal malelittermates, male brachymorphic B6C3F₂ mice, which are deficientin hepatic 3'-phosphoadenosine-5'-phosphosulfate (PAPS), treatedwith N-hydroxy-AAF formed only 25% as many hepatic DNA adductsand developed only 10% as many hepatomas. Hepatic cytosols from12-day-old B6C3F₁ mice contained PAPS-dependent sulfotransferaseactivity for N-hydroxy-2-aminofluorene (N-hydroxy-AF), a previouslyunrecognized activity, as well as sulfotransferase activityfor N-hydroxy-AAF; both activities were inhibited 60% by 1 µMand 80% by 10 µM PCP. Cytosolic acetyl coenzyme A-dependentacetyltransferase activity for N-hydroxy-AF, cytosolic N,O-acyltransferaseactivity for N-hydroxy-AAF, and microsomal deacetylase for N-hydroxy-AAFwere not significantly inhibited by PCP under these conditions.The above data strongly indicate that N-sulfoöxy-2-aminofluoreneis the major ultimate electrophilic and carcinogenic metaboliteof N-hydroxy-AAF in the livers of infant male B6C3F₁ mice.     

Comparative carcinogenicity of N-butyl-N-(3-carboxypropyl)-nitrosamine and N-butyl-N-(4-hydroxybutyl)nitrosamine for the urinary bladder of (C57BL/6 X DBA/2)F1 mice

The carcinogenicity of N-butyl-N-(3-carboxypropyl)-nitrosamine [CAS: 38252-74-3; 4-(N-butyl-N-nitrosamino)butyric acid] in male and female (C57BL/6 X DBA/2)F1 mice was determined. N-Butyl-N-(3-carboxypropyl)nitrosamine given in the drinking water at a concentration of 3 mM (0.056%) for 13 weeks induced only carcinoma of the urinary bladder in both sexes. At 22-28 weeks, the incidences of bladder cancer in the male and female mice were 100 and 88%, respectively. These bladder tumors were classified histologically according to the frequency (%) of tumor type: pure transitional cell carcinoma, 42%; mixed (transitional cell carcinoma with squamous or glandular differentiation, or both), 28%; squamous cell carcinoma, 27%; and carcinoma in situ, 3%. No significant sex differences were observed. In comparative studies, the incidence of bladder cancer was 100% for both sexes after administration of 3 mM (0.052%) N-butyl-N-(4-hydroxybutyl)nitrosamine [CAS: 3817-11-6; 4-(butylnitrosoamino)-1-butanol] in the drinking water. The frequency of pure transitional cell carcinoma was 47%, which was not significantly different from that observed for the carboxypropyl compound. The frequencies of other types of bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine were the following: mixed, 8%; squamous cell carcinoma, 42%; and carcinoma in situ, 3%.     

Structure-activity studies of the hepatocarcinogenicities of alkenylbenzene derivatives related to estragole and safrole on administration to preweanling male C57BL/6J x C3H/HeJ F1 mice

Further information on the structure-activity relationships among the synthetic and naturally occurring alkenylbenzene derivatives was obtained by examining their hepatocarcinogenicities for mice following administration of one or a few doses prior to weaning. Under these conditions preweanling male C3H/HeJ mice were more susceptible than male C57BL/6J mice or females of either strain to liver tumor induction by 1'-hydroxyestragole (1'-hydroxy-1-allyl-4-methoxybenzene) and 1'-hydroxysafrole (1'-hydroxy-1-allyl-3,4-methylenedioxybenzene). Male C57BL/6J X C3H/HeJ F1 mice given a single dose of 1'-hydroxyestragole at 12 days of age developed approximately twice as many hepatomas per liver as did those given the same dose per g of body weight at 1 day of age. The acetylenic compounds 1'-hydroxy-2',3'-dehydroestragole and 1'-hydroxy-2',3'-dehydrosafrole were the most potent derivatives studied; they were 5- and 10-fold more potent (based on the average numbers of hepatomas per liver) than the corresponding allylic benzene derivatives. 1'-Acetoxyestragole and 1'-acetoxysafrole had activities similar to those of their respective 1'-hydroxy derivatives; estragole derivatives were consistently 2- to 3-fold more potent than the related safrole derivatives. 1'-Hydroxyelemicin (1'-hydroxy-1-allyl-3,4,5-trimethoxybenzene), its acetic acid ester 1'-oxoestragole, and 3'-bromo-trans-anethole (3'-bromo-1-trans-propenyl-4-methoxybenzene) each had very weak, but statistically significant, hepatocarcinogenic activity. The propenylic derivatives cis-anethole, trans-isosafrole, 1:1 cis,trans-isosafrole, 3'-hydroxy-trans-anethole, piperine, and trans-cinnamaldehyde showed no hepatocarcinogenic activity at the levels examined. In contrast, the propenylic derivatives cis- and trans-asarone (1-propenyl-2,4,5-trimethoxybenzene) were each active; the hepatocarcinogenicities of the asarones were not inhibited by prior administration of pentachlorophenol, a sulfotransferase inhibitor that abolished the hepatocarcinogenicity of estragole under the same conditions. Furthermore, precocene II (6,7-dimethoxy-2,2-dimethyl-2H-1-benzopyran), a cyclic propenylic plant metabolite and asarone analogue, showed strong hepatocarcinogenic activity similar to that of 1'-hydroxy-2',3'-dehydroestragole and 1'-hydroxy-2',3'-dehydrosafrole; precocene I (the 7-methoxy analogue of precocene II) was less active than precocene II but more active than cis-asarone.