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We investigated the effects of bovine LF (bLF) on different phases of NNK-induced lung tumorigenesis in A/J mice. Mice were orally administered 0.02, 0.2 and 2% bLF during the initiation phase, and 2% bLF during the whole tumorigenesis phase or post-initiation phase. Administered bLF during the post-initiation phase showed significant reduction of macroscopical lung nodules, and immunohistochemically decreased expression levels of cell proliferation marker and increased expression levels of apoptosis marker in lung proliferative lesions. bLF might inhibit NNK-induced mouse lung tumorigenesis, only when given limited to the post-initiation phase, through modification of cell proliferation and/or apoptosis.

We have reported that pretreatment by stomach tube with 8-methoxypsoralen (methoxsalen; 8-MOP), a potent human CYP2A6 inhibitor, strongly suppresses lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female A/J mice (Cancer Res. 2003). Here, we examined inhibitory effects with administration in the diet. When the mice were 7 weeks of age, they received dietary supplementation with 8-MOP at concentrations of 1, 10 or 100 ppm for 3 days prior to a single dose of NNK (2mg/0.1 ml saline/mouse, i.p.) or an equal volume of saline (vehicle control). The experiment was terminated 16 weeks after the first 8-MOP treatment and lung proliferative lesions were analyzed. The incidences and multiplicities in the 8-MOP 100 ppm-treated group were significantly reduced as compared with values for the NNK alone group (P<0.001). Multiplicities of NNK-induced lung proliferative lesions were also reduced in a dose dependent manner (Spearman rank correlation coefficient; rho=-0.806, correction P<0.0001). Mouse CYP2A4 and CYP2A5 differ from each other only 11 amino acids, and are closely related to the human CYP2A6. One hour after the last of three daily doses of 8-MOP (0.5, 5 or 50mg/kg body weight in 0.2 ml corn oil, given by stomach tube) or an equal volume of corn oil (vehicle control), given to the mice at 7 weeks of age, isolation of lung and liver RNAs demonstrated no effects on CYP2A4 and CYP2A5 mRNA levels with 8-MOP. In conclusion, the results of this study showed that clear dose response inhibitory effects of 8-MOP on NNK-induced lung tumorigenesis in female A/J mice fed diets containing 8-MOP, due to inhibition of enzyme activity of CYP2A4 and CYP2A5, rather than their gene expression.

Arctiin, a plant lignan that can be extracted from the Arctium lappa (burdock) seeds, is a possible environmental endocrine disruptor compounds and have been shown to influence sex hormone metabolism as well as protein synthesis, steroid biosynthesis. Modifying effects of arctiin on prostate carcinogenesis in probasin/SV 40 T antigen (Tag) transgenic (TG) rats were examined. A total of 64 male TG rats, 6 weeks old, were randomly divided to three experimental groups (soybean free Oriental MF diet with 0.1, 0.02, or 0.004% arctiin) and a control group (soybean free Oriental MF diet). Animals were killed at the end of week 18. Histopathological evaluation of prostate revealed that all the rats in any group developed adenocarcinoma in dorsolateral lobe of prostate, except two rats in 0.1% arctiin treated and one rat in 0.002% arctiin treated groups without prostate adenocarcinoma development. However, there were no definite treatment-related changes with statistical significance in all parameters for prostate carcinomas measured in this experiment. These results indicated that arctiin might not exert significant modifying effect on prostate carcinogenesis in SV 40 Tag TG rats at least under the present experiment.

Induction of liver lesions in male F344 rats by the genotoxic and carcinogenic N-nitrosodimethylamine (NDMA) was studied at a wide range of dose levels, i.e. from 0.001 to 10 ppm, in drinking water for 16 weeks. Dose related and statistically significant increase of glutathione S-transferase placental form-positive foci, endpoint markers for hepatocarcinogenesis in rats, at 1 and 10 ppm dose groups was obtained, but no increment in foci could be detected with the lower doses (0.001, 0.01, and 0.1 ppm). This finding of a no-observed effect level supports our hypothesis that a threshold, at least in practical terms, exists in carcinogenesis proposed on the basis of extensive wide range dose-dependence studies of other genotoxic carcinogens.

The modifying effects of late neonatal administration of p-nonylphenol (NP), a suspected xenoestrogen, on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced prostatic carcinogenesis were investigated in male F344 rats. Three-week-old rats received 25, 250 or 2000 ppm of NP in the diet for 3 weeks prior to DMAB treatment and were sacrificed at 67 weeks of age for histopathological assessment of lesions and Ki-67 immunohistochemical analysis of cell cycle kinetics. Dietary administration of NP during the sexually immature period had no effects on maturation of male sex organs. Incidence, multiplicity and areas of neoplastic lesions in the prostate and seminal vesicles, and Ki-67 labeling indices in normal-looking epithelium were not significantly different among the experimental groups. These results indicate that late neonatal treatment with NP has no modulating effects on DMAB-induced rat prostatic carcinogenesis.

It has been generally accepted that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged for cancer risk assessment in humans. Here we examined low dose carcinogenicity of a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), using an in vivo medium-term bioassay to detect initiating activity for rat hepatocarcinogenesis. With MeIQx initiation at various doses followed by administration of phenobarbital, a well known hepatopromoter, no induction of glutathione S-transferase placental form-positive foci, assessed as preneoplastic lesions, was noted at doses of 0.001-1 ppm. The results imply a no-observed effect level for hepatocarcinogenicity with this genotoxic agent.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in cooked meat and fish, is carcinogenic to the mammary glands of rats. Mutations in the H-ras gene were here examined in PhIP-induced mammary tumors of female F344 rats by the polymerase chain reaction followed by single strand conformation polymorphism analysis (PCR-SSCP) and restriction enzyme length polymorphism analysis (RFLP). Mutations in codon 12 of the H-ras gene were detected in four out of 13 tumors by PCR-SSCP. Three of them were GGA to GAA, and one was GGA to GTA. However, by RFLP analysis, four additional mutations in codon 13 were also detected in the same samples. Two had a GGC to CGC mutation, and the other shifts were GGC to GAC and GGC to GTC. Therefore, overall eight out of 13 cases had H-ras gene mutations. These results indicate that changes in H-ras function may play important roles in PhIP-induced mammary carcinogenesis.

We have previously shown that chronic administration of a pharmacological dose of testosterone propionate (TP) after treatment with the carcinogen, 3,2'-dimethyl-4-aminobiphenyl (DMAB), results in development of invasive and metastatic adenocarcinomas arising from the dorso-lateral and anterior prostate, as well as the seminal vesicles. Co-administration of ethinyl estradiol (EE) with TP increased the yield of carcinomas in the lateral and anterior lobes. In the present experiment, male F344 rats were treated with DMAB for 20 weeks and then co-administered a pharmacological dose of TP together with various doses of EE for 40 weeks. Without hormone(s) administration, carcinomas were confined to the ventral prostate and all were of intra-acinar type. TP administration suppressed development of the ventral prostate carcinomas but caused invasive carcinomas of the lateral and anterior lobes and of seminal vesicles and intra-acinar carcinomas in the dorsal prostate. The appearance of carcinomas in the lateral and anterior prostate was increased by co-administration of EE in a dose-related fashion but carcinomas of the seminal vesicles were inversely reduced. The suppressive influence of TP on ventral carcinoma development was overcome by only the highest dose of EE. It is concluded that estrogen can modify the enhancing effects of TP on induction of rat prostate and seminal vesicle carcinomas in a dose-related fashion with lobe specificity.

Group-A xeroderma pigmentosum (XPA) gene-deficient mice are defective in nucleotide-excision repair and highly susceptible to ultraviolet-B-, and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA)-induced skin carcinogenesis. In this study, changes of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP)-DNA adduct formations in the liver, colon and lung, as assessed by the 32P-postlabeling method and immunohistochemical analysis, and carcinogenic and/or toxic susceptibility of both sexes of XPA-deficient mice (XPA-/-) to PhIP, which is a carcinogenic heterocyclic amine, was examined. Levels of PhIP-DNA adduct formations in the liver, colon and lung, were almost twice as high in XPA-/- as in wild type mice (XPA+/+) mice, 7 days after a single i.g. administration of PhIP, and their delay in recovery was observed in XPA-/- mice. For the long-term experiment, XPA-/- and XPA+/+ type mice were treated with 80 ppm PhIP in the diet for the first 4 weeks followed by 40 ppm after a 2-week recovery period (long-term experiment I), or 40 ppm PhIP throughout the experiment (long-term experiment II). Severe toxicity, as evidenced by body weight retardation and poor survival, was observed in the PhIP treated XPA-/- mice of both sexes, but not in the XPA+/+. At week 40 the experiments were terminated and histopathological examinations were performed after complete autopsy. Only lymphomas/leukemias were observed as neoplastic lesions, but no significant differences were observed between the groups. As non-neoplastic lesions, degenerating changes, for example in the pancreatic acinar cells, were observed with XPA-/- mice tending to be more sensitive than XPA+/+ mice. The present study demonstrated that PhIP-DNA adduct formations in the liver, colon and lung of XPA-/- mice were demonstrated and their recovery rate was more delayed than XPA+/+ mice, and furthermore, more severe toxicity to PhIP in XPA-deficient mice was observed, but they were not susceptible to PhIP carcinogenicity under the conditions of the experiment.

We previously showed that 5% sodium L-ascorbate (Na-AsA) in the diet promotes rat urinary bladder carcinogenesis, whereas 5% ascorbic acid (AsA) and 1% sodium chloride (NaCl) in the diet do not. In order to cast light on basic properties of these compounds regarding the rat urinary bladder, we examined membrane potential levels in the bladder epithelium of F344 rats treated with 5% Na-AsA, 5% AsA or 1% NaCl in the diet for 2, 4 or 8 weeks. Microelectrode measurement showed Na-AsA to induce hyperpolarization of the membrane potential, which was, however, lacking with AsA and NaCl from week 2 until the end of experiment. Thus a good correlation between the effects on membrane potential and tumor promoting activity could be established.

Using the Solt and Farber model (Nature, 263 (1976) 701), dose-response relationships between initiating agents and the induction of hyperplastic nodules in rat liver were investigated. Male Fischer 344 rats were initiated by a single application of 1 of 5 carcinogens at 3 doses: 200.0, 50.0 and 12.5 mg/kg of diethylnitrosamine (DEN); 1.00, 0.25 and 0.062 mg/kg of aflatoxin B1 (Af-B1); 60.0, 15.0 and 3.75 mg/kg of N-2-fluorenylacetamide (2-FAA); 600.0, 150.0 and 37.5 mg/kg of 3'-methyl-4-dimethylaminobenzene (3'-me-DAB); 40.0, 10.0 and 2.5 mg/kg of dimethylnitrosamine (DMN) and their vehicles. Two weeks after initiation, animals were placed on a 0.02% 2-FAA diet for 2 weeks. Partial hepatectomy was performed at the end of the third week of the experiment. All rats were killed 4 weeks after the initiation, and the hyperplastic nodules of the liver were counted and their areas measured. Dose responses were observed in both numbers and areas of hyperplastic nodules per unit area of sections with all of the 5 carcinogens examined.

The effect of ligation of the left ureter on the development of lesions in the urinary bladder were investigated in male F344 rats treated with 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) for 2 weeks followed by 0.002% N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) for 10 or 22 weeks. The lesions of the urinary bladder found in weeks 12 and 24 in rats treated with BBN and EHBN were preneoplastic papillary or nodular hyperplasia (PN hyperplasia), papilloma and cancer. The incidences and numbers of these lesions per 10 cm of basement membrane of urinary bladder were increased significantly by ureteric ligation. Treatment with BBN or EHBN only induced no remarkable change and thus the doses used were subcarcinogenic in the conditions used. The results show that unilateral ureteric ligation enhanced two-stage bladder carcinogenesis.

The initiating and promoting activities of several mycotoxins were investigated in F344 male rats. N-2-Fluorenylacetamide (2-FAA) was used as an initiator or a promoter and citrinin, ochratoxin A, sterigmatocystin, (+)rugulosin or patulin was administered at a fixed dose for the same period in the initiation stage or the promotion stage. Partial hepatectomy and administration of carbon tetrachloride (CCl4) were examined to increase the induction of hyperplastic nodules. Ochratoxin A, sterigmatocystin and (+)rugulosin administered in either the initiating or the promoting stage significantly increased the induction of hyperplastic nodules. Citrinin and patulin administered in the initiating stage significantly increased the induction of hyperplastic nodules, but were not effective when administered during the promoting stage. The theoretical classification of the mycotoxins examined is discussed.

Effectiveness of two different chemically induced stimuli for hepatocellular proliferation was compared with regard to that of commonly used partial hepatectomy (PH), for the purpose of developing short-term protocol for the assay of promoting agents of hepatocarcinogenesis. Enhancing effect of D-galactosamine (DGA) and carbon tetrachloride (CCl4) given during the promotion procedure by 2-acetylaminofluorene (2-AAF) was compared along with PH in rats initiated by diethylnitrosamine (DEN), using preneoplastic glutathione S-transferase positive (GST-P+) hepatocyte foci as an end-point marker lesion. The number of GST-P+ foci per cm2 was largest in the group given CCl4 followed by DGA, no treatment (2-AAF alone) and PH. In contrast, the area (mm2) per cm2 and mean diameter of the focus were largest in the PH group then DGA followed by CCl4 and no treatment. The results indicate that the number of GST-P+ foci were not clearly affected by 3 different treatments whereas area and size of foci which represented the result of promoting effect were clearly influenced by those treatments, indicating they caused differential proliferation of initiated cells. In this respect, even though PH is the most potent procedure, DGA is also efficient and preferred to CCl4 for the non-surgical enhancing method.

L-Ascorbic acid, citric acid or their sodium salts (at levels equivalent to 5% sodium L-ascorbate) were fed in the diet simultaneously with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) or N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) (0.025% BBN or 0.021% EHBN) in the drinking water to male F344 rats for 20 weeks to determine whether urinary pH changes affect the carcinogenicity of BBN or EHBN. In the urine, pH was decreased in rats fed the acidic chemicals and increased in rats fed their corresponding sodium salts. Histopathologically, the incidences and numbers of preneoplastic and neoplastic lesions in groups treated with each test chemical were not different from those in control groups except for sodium citrate-treated groups in which induction of carcinomas was higher, resulting from increased intake of either carcinogen and also from increased urinary excretion of main carcinogenic metabolites. These results show that the test chemicals do not affect the carcinogenicity of BBN or EHBN on the rat urinary bladder when simultaneously administered despite significant differences in urinary pH.