This paper reviews the evidence for thresholds for DNA-reactive carcinogens and presents dose response data for effects in rat liver of two DNA-reactive hepatocarcinogens, diethylnitrosamine and 2-acetylaminofluorene, which is consistent with thresholds.

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Kojic acid (KA) has been used as a food additive for preventing enzymatic browning of crustaceans and as a cosmetic agent for skin whitening. To date, it has been reported that female B6C3F1 mice receiving 3% KA in the diet were found to develop hepatocellular tumors, but the underlying mechanism of liver tumorigenicity is still not clear. In the present experiments, in order to investigate possible liver initiation activity, partially hepatectomized male F344 rats received a single oral dose of 0, 1000 and 2000 mg/kg body weight of KA followed by dietary administration of 0.015% of N-2-acetylaminofluorene (2-AAF) for 2 weeks and a single 0.8 mL/kg body weight dose of CCl₄. Furthermore, male F344 rats were fed a diet containing 0 or 2% KA for 3, 7 and 28 days, and the 8-oxodeoxyguanosine (8-OxodG) levels in nuclear DNA were measured to examine the formation of oxidative DNA adduct and cell proliferating activities of hepatocytes in the liver. In the liver initiation assay, there were no significant differences in the number or area of glutathione S-transferase placental form (GST-P) positive foci, putative preneoplastic lesions, between the KA-treated and control groups. Cell proliferation of hepatocytes in rats given KA for 3 and 7 days was significantly increased as compared with the relevant control values, but no significant elevation in 8-OxodG levels was apparent. The results of the present study suggest that KA has neither liver initiation activity nor capability of 8-OxodG formation, but some evidences suggestive of liver tumor promoting effects in rats.

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D-psicose, a C-3 epimer of D-fructose, is present in very small quantities in commercial carbohydrate complexes and agricultural products, and is therefore called a rare sugar. The effects of D-psicose on diethylnitrosamine (DEN)-induced hepatocarcinogenesis were examined in male F344 rats using a rat medium-term bioassay based on the two-step model of hepatocarcinogenesis. The modifying potential was determined by comparing the numbers and areas/cm² of induced glutathione S-transferase placental form (GST-P) positive foci in the liver with those of a corresponding group (control) of rats given DEN alone. Increased relative liver weights were found in the 1% D-psicose treatment group as compared with the basal diet group, while no significant change occurred in the 0.1% D-psicose, 0.01% D-psicose, and 1% D-fructose groups. D-psicose did not significantly alter the numbers and area/cm² of GST-P positive liver cell foci observed after DEN initiation. The results thus demonstrate that D-psicose shows neither promoting nor preventive potential for liver carcinogenesis in our medium-term bioassay.

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We previously demonstrated that rat offspring exposed perinatally to methoxychlor (MXC) still exhibited immunotoxic changes in young adulthood at 10 weeks of age. That result led us to further investigate whether the influence of perinatal exposure to MXC on the rat immune system persistently remains in adult life. Sprague-Dawley rat offspring of both sexes from dams receiving MXC at dietary dose levels of 0, 30, 100, 300, and 1000 ppm were used for the present study. The pups exposed to MXC through the placenta, milk, and/or direct intake during the gestation and lactation periods were maintained on a normal diet from weaning up to 52 weeks of age. At the termination, a significant increase in plasma chloride was noted in both sexes at 300 and 1000 ppm MXC exposure. Females at 1000 ppm MXC exposure showed increases in serum IgM and urinary protein and had significantly increased relative weights (ratio to body weight) of the spleen and kidneys. An increased relative kidney weight was also noted in females at 300 ppm MXC exposure. Histopathologically, the incidence and severity of chronic nephropathy tended to be higher in females at 1000 ppm MXC exposure, and their kidneys had enlarged glomeruli with increased IgG and IgM deposits. In the immune system, however, there were neither notable histological changes nor significant alterations in the splenic lymphocyte subsets for any dose group of either sex. These results indicate that the immunotoxic damage caused by pre- and post-natal MXC exposure appears to be repaired during the process of growth, although the effect seems to remain in females even at 52 weeks after birth and consequently may accelerate the progression of chronic nephropathy.

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The modifying potentials of two heterocyclic amines, harman and norharman, as well as sodium nitrite (NaNO₂), on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced rat liver carcinogenesis were investigated using a medium-term liver bioassay system. Harman (500 ppm in diet), norharman (500 ppm in diet) or NaNO₂ (0.1% in drinking water) were given with or without MeIQx (300 ppm in diet) for 6 weeks after initiation with a single dose of dimethylnitrosamine (DEN). The appearance of MeIQx-induced glutathione S-transferase placental form positive foci in the liver was significantly decreased in the harman and norharman cases (p< 0.001), but it was significantly increased by NaNO₂ (p< 0.001). These chemicals, however, did not modify DEN-liver carcinogenesis in the absence of MeIQx. Neither harman nor norharman affected mRNA expression of CYP1A1 and 1A2 in the liver, with or without MeIQx administration, whereas NaNO₂ significantly enhanced CYP1A1 mRNA levels together with MeIQx.

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To assess sensitivity to hepatocarcinogenesis by BBN, formalin-fixed and paraffin-embedded rat liver tissues from two initiation/promotion (I/P) bioassays for urinary bladder carcinogenesis were sectioned, immunohistochemically stained for glutathione S-transferase placental form (GST-P), and quantitatively analyzed. In Experiment 1, male Sprague-Dawley (SD)/cShi strain rats (highly sensitive to urinary bladder carcinogenesis by BBN) and SD/gShi rats (insensitive), were treated with 0.05% BBN in the drinking water for 4 weeks and then housed for up to week 36, with or without exposure to bladder tumor promoters. Slightly higher quantitative values for small GST-P positive (GST-P⁺) foci were found in SD/gShi rats (8.57/cm²) compared to SD/cShi rats (2.48/cm²). In Experiment 2, F344 and Lewis rats, subjected to a similar I/P protocol, were maintained on MF or CA-1 diet for 36 weeks. Higher quantitative values of GST-P ⁺ hepatocytic foci (more than 0.1 mm in diameter) were found in Lewis rats (9.51/cm² for MF diet; 7.95/cm ² for CA-1 diet) compared to F344 rats (2.82/cm² and 1.30/cm², respectively). Slight inhibiting effects of GST-P⁺ foci were apparent in rats receiving uracil, but not sodium L-ascorbate, in the present experiment. Thus, it was confirmed that the bladder carcinogen BBN also targets the liver. The results, from quantitative analysis of small GST-P⁺ foci as end-point marker lesions, indicate considerable strain differences, but that liver tumor modifying potential of test chemicals can be evaluated in I/P protocols for urinary bladder carcinogenesis using any strain of rat.

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In order to examine the modifying effects of co-administration of t-butylhydroquinone (TBHQ) and sodium nitrite (NaNO₂) on forestomach carcinogenesis, a total of 50 male transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice) received a single intraperitoneal injection of 60 mg/kg body weight of N-ethyl-N-nitrosourea (ENU), and starting 1 week later, they were given diet containing 0 (control group) or 0.5% TBHQ (TBHQ alone group), drinking water containing 0.2% NaNO₂ (NaNO₂ alone group) or diet containing 0.5% TBHQ in combination with 0.2% NaNO₂ in drinking water (TBHQ+NaNO₂ group) for 20 weeks. Squamous cell hyperplasias, papillomas and carcinomas were induced in all of the ENU treated groups, and there were no significant differences in the incidence, multiplicity and PCNA labeling index of these forestomach proliferative lesions between the NaNO₂ alone, TBHQ alone, TBHQ+NaNO₂ and control groups. These results suggest that the co-administration of TBHQ and NaNO₂ does not cause any modifying effects on ENU-induced forestomach carcinogenesis in rasH2 mice.

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A female BALB/c:Slc mouse was administered two intravenous injections of doxorubicin (5 mg/kg) at the ages of 22 and 24 weeks. When this mouse was sacrificed at the age of 30 weeks, a tumor was found in the left mandible. The tumor was composed of dental tissues at varying stages of differentiation, ranging from primitive tooth germ-like structures to mature tooth-like tissue. Amyloid deposition was noted in a part of the tumor. Based on these histopathological findings, the tumor was diagnosed as an ameloblastic odontoma with amyloid deposition.

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