Sensitive quantitative assay for point mutations in the rat H-ras gene based on single nucleotide primer extension.

Point mutations in oncogenes and tumor suppressor genes occur at early stages in the carcinogenic process. Point mutations in ras family oncogenes are the most common mutational events in several types of human cancer, and are available as molecular markers for the detection of cancer cells in carcinogenicity bioassay systems as well as in clinical samples. Although several techniques are utilized to detect point mutations in carcinogenicity bioassay systems, the sensitivity is too low to determine a small number of mutations. In order to overcome the disadvantage and to sensitively determine gene mutation rates for in vivo carcinogenicity bioassays of presumptive carcinogens, we established a Thermosequenase Cycle End Labeling (TCEL) method, a sensitive approach based on single nucleotide primer extension. One of the characteristics of the method is a high sensitivity of 1:100,000, ten times the sensitivity of the mutant allele-specific amplification now commonly employed. Using TCEL, we here quantified H-ras mutations in the livers of rats treated with a genotoxic carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. Our findings suggest that this method may be applied for many genetic targets as a component in vivo.

Elevation of 8-hydroxydeoxyguanosine and cell proliferation via generation of oxidative stress by organic arsenicals contributes to their carcinogenicity in the rat liver and bladder.

Monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)) and trimethylarsine oxide (TMAO(V)) are well-documented inorganic arsenic (iAs) methylated metabolites. In our previous studies, DMA(V) and TMAO(V) were shown to exert carcinogenicity in the rat bladder and liver, respectively. Furthermore, MMA(V), DMA(V) and TMAO(V) exhibited promoting activity on rat hepatocarcinogenesis. To clarify mechanisms of arsenical carcinogenicity and compare biological responses in the liver and bladder, male F344 rats were sequentially treated for 5, 10, 15, 20 days with MMA(V), DMA(V) and TMAO(V) in their drinking water at a dose of 0.02%. Significant increase of P450 total content and generation of hydroxyl radicals in the liver were observed from 10 and 15 days of treatment with arsenicals, respectively, with the highest levels induced by TMAO(V). Similarly, elevation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation was found in the DNA with significant increase by TMAO(V) treatment in the liver at days 15 and 20, and DMA(V) in the bladder after 20 days treatment. In addition, cell proliferation and apoptosis indices were significantly increased by TMAO(V) in the liver and by DMA(V) in the bladder of rats. These events were accompanied by differential up-regulation of phase I and II metabolizing enzymes, cyclins D1 and E, PCNA, caspase 3 and FasL. The results indicate that early elevation of 8-OHdG and cell proliferation via generation of oxidative stress by TMAO(V) and DMA(V) contributes to their carcinogenicity in the rat liver and bladder.

Carcinogenicity of dimethylarsinic acid in Ogg1-deficient mice.

Oxidative stress to DNA is recognized as a mechanism underlying carcinogenic effects of some environmental agents. Here, we hypothesized that dimethylarsinic acid (DMA(V)), an organic metabolite of inorganic arsenic in humans, might exert carcinogenic potential in a mouse line carrying a mutant Mmh allele of the Mmh/OGG1 gene encoding the enzyme 8-hydroxyguanine DNA glycosylase 1 (OGG1). Ogg1 mutant and wild type mice were treated with DMA(V) in their drinking water at a dose of 200 p.p.m. for up to 72 weeks. All DMA(V)-treated Ogg1(-/-)animals developed tumors, with a tendency for lower total incidences in the Ogg1(+/+) cases. Lung tumors in particular were induced as compared to the lack in non-carcinogen controls and were significantly more frequent in the homozygotes. At week 4, the levels of DNA 8-OH-dG and cell proliferation were significantly elevated in the lungs of non-treated Ogg1(-/-) as compared to Ogg1(+/+) mice and were strongly enhanced by DMA(V) treatment. Marked induction of Pola1, Cyp7b1, Ndfua3, Mmp13 and other genes specific to cell proliferation, cell signaling and xenobiotic metabolism in the lungs of DMA(V)-treated Ogg1(-/-) mice was found. Electron microscopic examination revealed the growth of microvilli, with increased numbers of mitochondria only in lungs and lung tumors of DMA(V)-exposed Ogg1(-/-) mice. Therefore, we strongly suggest that DMA(V) exerts carcinogenicity in the lungs of Ogg1(-/-) mutant mice, with a possible role for persistent accumulation of DNA oxidative adducts.

Analysis of gene expression in different stages of MeIQx-induced rat hepatocarcinogenesis.

To clarify hepatocarcinogenesis by the heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), we investigated the global expression of genes in rat liver. Rats were continuously fed MeIQx 100 ppm in their diet, and were sacrificed at weeks 4 and 16 for early time points, and week 104 for tumor sampling. Global expression analysis using oligonucleotide microarrays (Affimetrix Gene Chip, Rat Genome 230 2.0 Array) was carried out to detect altered genes in MeIQx-treated liver at 4 and 16 weeks (n=5, each), MeIQx-induced hepatocellular adenomas (HCA; n=3), and hepatocellular carcinomas (HCC; n=3), compared with age-matched normal livers (n=5). To investigate functional networks and gene ontology, two clusters were analyzed by Ingenuity Pathway Analysis. Clustering analysis of global genes demonstrated gene profiles of HCA and HCC to greatly differ from those of age-matched normal liver. However, after treatment with MeIQx for 4 or 16 weeks, no major differences were apparent. Ingenuity Pathway Analysis suggested pathways related to the cell cycle and glutathione metabolism may be involved in MeIQx-induced hepatocarcinogenesis. Real-time PCR analysis confirmed elevation of cyclin B1, cell division cycle 2, glutathione peroxidase 2 and glutathione S-transferase A2 in tumors, but not in early stage livers. In conclusion, molecular signatures of MeIQx-induced tumors clearly vary from that of age-matched normal liver, but no such shift is evident at early stages of hepatocarcinogenesis.

Evaluation of the toxicity of mastic gum with 13 weeks dietary administration to F344 rats.

Dietary toxicity of mastic gum, a natural food additive, was studied in male and female F344 rats fed 0%, 0.22%, 0.67% and 2% levels mixed into powdered basal diet for 13 weeks. No mortality or obvious clinical signs were observed in any of the animals throughout the experimental period. Body weights were significantly reduced in the high dose-treated group from week 2 to the end of the experiment in males, and at weeks 8 and 13 in females. There were increased absolute and relative liver weights in a dose-related manner or limited to the high dose group males or females, along with changes in hematological parameters, including increased WBC and platelet in high dose males. Altered serum biochemistry parameters included increases of total proteins, albumin, and total cholesterol in both sexes, and gamma-GTP in females only. However, macroscopic examination at necropsy revealed no gross lesions, and microscopic examination also revealed no treatment-related findings in any organs examined. As dietary treatment of mastic gum for 13 weeks in the present study caused decreased body weights at the high dose, especially in males, and increased liver weights in a dose-related manner in both genders without any morphological findings, it is concluded that the administration of it has a no observed adverse effect level (NOAEL) of 0.67% in the diet.

Upregulation of fibroblast growth factor receptor 3 and epidermal growth factor receptors, in association with Raf-1, in urothelial dysplasia and carcinoma in situ after the Chernobyl accident.

The present study was carried out in order to examine the molecular status of selected growth factor receptors (GFR) in urinary bladder lesions, recently described by our group as representing 'Chernobyl cystitis'. Fibroblast growth factor receptor 3 (FGFR3), epidermal growth factor receptor 1 (EGFR1), EGFR2neu (a member of the same family), p53 and Raf-1 serine/threonine kinase expression were evaluated immunohistochemically in urinary bladder biopsies from 22 men with benign prostate hyperplasia (group 1). For comparison, 16 men with benign prostate hyperplasia and five women with chronic cystitis living in non-radio-contaminated areas of the country were also investigated as controls (group 2). Additionally, 14 patients with dysplasia, carcinoma in situ (CIS) and primary urothelial carcinoma (UC) operated before the Chernobyl accident as well as 23 patients with UC living in the radio-contaminated areas were included as pre- and post-Chernobyl UC groups 1 and 2, respectively. Chronic proliferative atypical cystitis ('Chernobyl cystitis') was observed in group 1 patients. Foci of dysplasia and CIS were found in 22 (100%) and 19 (86%) of the 22 cases, respectively; moreover, two small UC were also detected. Elevated levels of FGFR3, EGFR2/neu, p53 and to a lesser extent EGFR1 and Raf-1 expression in the urothelial dysplasia and CIS were evident for patients of group 1. Statistically significant differences in immunohistochemical scores for FGFR3, EGFR1, p53 and Raf-1 were observed between groups 1 and 2 and between group 1 and the post-Chernobyl UC group 2, where a change in expression of EGFR2/neu was also noted. A significant decrease in FGFR3 expression in additional pre-Chernobyl UC group 1 with dysplasia, CIS and UC compared with group 1 Chernobyl cystitis cases was detected. Our findings suggest that FGFR and EGFR signaling pathways, associated with p53 and Raf-1 activation, may contribute to multistage urothelial carcinogenesis caused by irradiation, through autocrine or paracrine growth stimulation.

Lack of promoting effects of phenobarbital at low dose on diethylnitrosamine-induced hepatocarcinogenesis in TGF-alpha transgenic mice.

Phenobarbital (PB), a rodent non-genotoxic carcinogen, showed hormesis, biphasic effects on rat liver carcinogenesis. To test the hypothesis that the hormesis earlier observed for PB induced hepatocarcinogenesis might also exist in the TGF-alpha transgenic mice model, one which is highly susceptible to carcinogenesis, the carcinogenic or promotion effects of a wide range of phenobarbital (PB) concentrations were investigated. Two weeks after a single i.p. dose of 5 mg /kg bw of diethylnitrosamine (DEN) to 15 day old mice, animals were treated with diet containing PB at doses of 0, 2, 15 or 500 ppm. The incidence and multiplicity of tumors, including hepatocellular adenomas and carcinomas, were significantly increased by the high dose of PB, but no significant difference among the groups receiving 2 and 15 ppm for liver tumors when compared to DEN alone group. The proliferating cell nuclear antigen indices for liver tumors and surrounding hepatocytes in high dose PB treated mice were significantly increased, but no change was noted at the lower doses. The total cytochrome P450 content in the liver was also elevated by 500 ppm of PB, while hepatic 8-OHdG levels demonstrated no significant change. In conclusion, PB at high dose enhances DEN-induced hepatocarcinogenesis in TGF-alpha transgenic mice, but low doses lack any significant effects. One possible mechanism of phenobarbital carcinogenicity might be influenced by cytochrome P450 system exhibiting a strong promoting activity for liver of mice.

Existence of no hepatocarcinogenic effect levels of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline with or without coadministration with ethanol.

There is increasing evidence of the existence of no effect levels for genotoxic carcinogens. However, only limited information is available regarding dose-response curves for combination effects of multiple carcinogens at low dose. In the present study, 280 male F344 rats were divided into 14 groups to determine the effects of co-administration of various doses of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 10% ethanol on the development of glutathione S-transferase placental form (GST-P)-positive foci in the liver. The results provided concrete evidence for the existence of no effect levels for hepatocarcinogenicity of MeIQx either in presence or absence of ethanol and, therefore, for a practical threshold for this genotoxic carcinogen.

Inhibition of rat urinary bladder carcinogenesis by the antiangiogenic drug TNP-470.

Potential inhibitory effects of the antiangiogenic drug TNP-470 on rat urinary bladder carcinogenesis were investigated in F344 male rats initiated with 0.05% BBN in the drinking water for 8 weeks. Group 1 was then continuously treated with TNP-470 by subcutaneous injection using osmotic minipump until the end of the experiment; group 2 served as the control with only initiation. The incidences and multiplicities of papillomas and carcinomas in the TNP-470-treated group were significantly decreased compared to the control group values along with the tumor vascular density. In conclusion, TNP-470 can inhibit rat urinary bladder carcinogenesis, presumably through its effects on angiogenesis.

Effects of cessation of alcohol exposure on rat hepatocarcinogenesis.

Cessation of long-term alcohol exposure is reported to enhance rat hepatocarcinogenesis. The purpose of the present study was to assess this possibility using glutathione-S transferase placental form (GST-P) positive foci as end point lesions. All rats were treated with a single i.p. injection of diethylnitrosamine (DEN) (200 mg/kg body weight) and then given a MF pellet diet for 2 weeks. Thereafter, the animals were maintained on: alcohol liquid diet in which 36% of total calories were provided by alcohol (5% Al diet) for 6 weeks (group 1); control liquid diet (C diet) for 6 weeks (group 2); 5% Al diet for 6 weeks and subsequently C diet for 4 weeks (group 3); 5% Al diet for 10 weeks (group 4); or C diet for 10 weeks (group 5). All rats were subjected to two thirds partial hepatectomy at 3 weeks after DEN injection. The number and area of GST-P positive foci per cm2 of liver tissue were slightly increased in group 1 compared to the group 2 and significantly elevated in the group 4 compared to group 5. However, numbers in group 3 were significantly lower in group 4 and similar to the group 5 values. PCNA positive cells in the GST-P positive foci in the group 1 and group 4 were significantly increased as compared with respective controls (groups 2 and 5, respectively), while indices in the group 3 were again similar to values for group 5. Cessation of short-term alcohol administration thus had no promoting effects on development of GST-P foci, suggesting that the duration of alcohol treatment may be important. The results also imply the existence of a cumulative exposure time or dose threshold for alcohol if promoting effects of cessation are to be seen on rat hepatocarcinogenesis.

Involvement of ubiquitination and sumoylation in bladder lesions induced by persistent long-term low dose ionizing radiation in humans.

PURPOSE: We determined whether ubiquitination and sumoylation processes are up-regulated in bladder urothelium by chronic, long-term, persistent low doses of ionizing radiation in male patients with benign prostate hyperplasia and females with chronic cystitis living more than 19 years in 137Cs contaminated areas after the Chernobyl accident in Ukraine. MATERIALS AND METHODS: Bladder urothelial biopsies from 45 patients were subjected to histopathological and immunohistochemical study of Ub, SUMO1, SUMO E2 conjugating enzyme Ubc9, and the cell cycle inhibitors p53 and p27(Kip1). RESULTS: Of 25 group 1 patients from radio contaminated areas chronic proliferative atypical cystitis (Chernobyl cystitis), featuring multiple foci of dysplasia, and carcinoma in situ were observed in 23 (92%) and 19 (76%), respectively, in addition to 1 small pTa grade 1 urothelial carcinoma. Chronic cystitis with areas of dysplasia and urothelial hyperplasia were detected in 2 (10%) and 3 (15%), respectively of the 20 patients in control group 2 from clean (without radio contamination) areas of Ukraine. Greatly increased levels of Ub, SUMO1, Ubc9 and p53 as well as decreased levels of p27(Kip1) were evident in patients in group 1 compared to those in group 2 (all p <0.001). CONCLUSIONS: These findings support the hypothesis that up-regulated ubiquitination and sumoylation processes might be an adaptive response to unscheduled proteolysis of aberrant p53 and p27(Kip1) cell cycle regulators occurring with long-term low dose rate ionizing radiation exposure with a possible contribution to urothelial carcinogenesis.

Aberrant expression of E-cadherin and beta-catenin in association with transforming growth factor-beta1 in urinary bladder lesions in humans after the Chernobyl accident.

This study examines the molecular pathways of cell-cell communication in chronic inflammatory processes associated with long-term low-dose urinary bladder exposure to ionizing radiation in people without major disease living more than 19 years in radio-contaminated areas of Ukraine after the Chernobyl accident. Patterns of components of the E-cadherin/beta-catenin complex, and transforming growth factor-beta1 (TGF-beta1) and inducible nitric oxide synthase (iNOS) expression were immunohistochemically evaluated in urinary bladder biopsies from 52 males with benign prostate hyperplasia and 8 females with chronic cystitis (group 1). For comparison, 25 males and 6 females living in non-contaminated areas of Ukraine were also investigated (group 2). Fourteen patients with primary urothelial carcinomas, which were operated on before the Chernobyl accident, were included as a carcinoma group. Chronic proliferative atypical cystitis ('Chernobyl cystitis') was observed in group 1 patients. Foci of dysplasia and carcinoma in situ were found in 51 (85%) and 34 (57%) of the 60 cases, respectively. Chronic cystitis with areas of dysplasia was detected in only 4 (13%) cases of 31 group 2 patients. Statistically significant differences in immunohistochemical scores for TGF-beta1 in the urothelium and lamina propria, iNOS in the urothelium and both beta-catenin and E-cadherin in the cytoplasm were observed between groups 1 and 2 with marked expression in group 1. Furthermore, TGF-beta1 overexpression and alteration in E-cadherin/beta-catenin complexes in bladder urothelium might play a crucial role in urinary bladder carcinogenesis in humans exposed to long-term low-dose ionizing radiation.

Alpha-benzene hexachloride exerts hormesis in preneoplastic lesion formation of rat hepatocarcinogenesis with the possible role for hepatic detoxifying enzymes.

Recently there has been a shift in the prevailing paradigm regarding the dose dependence of carcinogen action with increasing acceptance of hormesis phenomenon, although underlying mechanisms remain to be established. To ascertain whether alpha-benzene hexachloride (alpha-BHC) might act by hormesis, rats were initiated with diethylnitrosamine and then alpha-BHC ranging from 0.01 to 500 ppm was administered in the diet for 10 weeks. The highest concentration of alpha-BHC significantly increased the number and area of glutathione S-transferase placental form (GST-P) positive foci, preneoplastic lesions in the liver, but its low dose, 0.05 ppm, caused significant reduction, showing a J-shape dose-response curve. The proliferating cell nuclear antigen positive index for GST-P positive foci in the low dose-treated group was significantly reduced. The dose response curves of CYP450 content, NADPH-P450 reductase activity and 8-hydroxydeoxyguanosine formation revealed the same pattern as GST-P positive foci data. The response curves of CYP2B1 and 3A2 in their activities, protein and mRNA expression showed a threshold but CYP2C11 activity exhibited an inverted J-shape. These results might suggest the possibility of hormesis of alpha-BHC at early stages of rat hepatocarcinogenesis. The possible mechanism involves induction of detoxifying enzymes at low dose, influencing free radical production and oxidative stress, and consequently pathological change in the liver.

N-acetylcysteine and S-methylcysteine inhibit MeIQx rat hepatocarcinogenesis in the post-initiation stage.

N-acetylcysteine (NAC) and S-methylcysteine (SMC), water soluble organosulfur compounds contained in garlic, were evaluated for chemoprevention of hepatocarcinogenesis after 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) initiation in rats. Intergastric treatment with NAC or SMC five times a week resulted in decreased numbers and areas of preneoplastic, glutathione S-transferase placental form (GST-P) positive foci of the liver in a dose-dependent manner. Moreover, cell proliferation was reduced in GST-P positive foci by NAC and SMC. Insulin-like growth factor I (IGF-I) and inducible nitric oxide synthase (iNOS) mRNA expressions were found downregulated in the liver by NAC. The studies indicate that NAC can serve as a chemopreventive agent for rat hepatocarcinogenesis induced by MeIQx by reducing cell proliferation, which may involve IGF-I and iNOS downregulation.

A comparative study of the sub-chronic toxic effects of three organic arsenical compounds on the urothelium in F344 rats; gender-based differences in response.

Epidemiological studies indicated that human arsenic exposure can induce urinary bladder cancer. Methylation of inorganic arsenic can generate more reactive and toxic organic arsenical species. In this regard, it was recently reported that the methylated arsenical metabolite, dimethylarsinic acid [DMA(V)], induced urinary bladder tumors in rats. However, other methylated metabolites, like monomethylarsonic acid [MMA(V)] and trimethylarsine oxide (TMAO) were not carcinogenic to the urinary bladder. In order to compare the early effects of DMA(V), MMA(V), and TMAO on the urinary bladder transitional cell epithelium at the scanning electron microscope (SEM) level, we investigated the sub-chronic (13 weeks) toxicological effects of MMA(V) (187 ppm), DMA(V) (184 ppm), TMAO (182 ppm) given in the drinking water to male and female F344 rats with a focus on the urinary bladder in this study. Obvious pathological changes, including ropy microridges, pitting, increased separation of epithelial cells, exfoliation, and necrosis, were found in the urinary bladders of both sexes, but particularly in females receiving carcinogenic doses of DMA(V). Urine arsenical metabolic differences were found between males and females, with levels of MMA(III), a potential genotoxic form, higher in females treated with DMA(V) than in males. Thus, this study provides clear evidence that DMA(V) is more toxic to the female urinary bladder, in accord with sensitivity to carcinogenesis. Important gender-related metabolic differences including enhanced presentation of MMA(III) to the urothelial cells might possibly account for heightened sensitivity in females. However, the potential carcinogenic effects of MMA(III) need to be further elucidated.

Dose-dependence of promotion of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline-induced rat hepatocarcinogenesis by ethanol: evidence for a threshold.

Although ethanol is thought to be a tumor-promoter, there are conflicting results concerning its effects on experimental hepatocarcinogenesis. Furthermore, the relationship between the amount of ethanol consumed and tumor promoting effects has hitherto not been investigated in detail. In the present study, 21-day-old F344/DuCrj rats were fed 200 p.p.m. 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in their diet for 8 weeks and thereafter received ethanol at doses of 0, 0.1, 0.3, 1, 3, 10 and 20% in drinking water ad libitum for 16 weeks. The incidences of hepatocellular adenoma and total tumors increased dose-dependently with statistical significance at doses of 10% and 20%, compared to the initiated control value. Similarly, dose dependence was observed for the incidence of hepatocellular carcinoma, which was elevated significantly at the dose of 20%. No alteration in development of preneoplastic glutathione-S-transferase placental form positive foci or tumors was observed with 0.1-1%. Cell proliferation also increased dose-dependently and CYP2E1 protein induction was recognized in centrilobular regions without alteration in mRNA levels, but no effects were evident on formation of 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage marker, or lipid peroxidation in any of the initiated groups. The mRNA expression of cyclin D1 increased dose dependently. The results demonstrated that ethanol dose-dependently promotes hepatocarcinogenesis induced by MeIQx, but with no adverse influence at doses of 1% or less, comparable to sensible drinking levels in humans.

Current and emerging challenges in toxicopathology: carcinogenic threshold of phenobarbital and proof of arsenic carcinogenicity using rat medium-term bioassays for carcinogens.

For the last 25 years, Prof. Nobuyuki Ito and his laboratory have focused on the development of liver medium-term bioassay system for detection of carcinogens in F344 rats utilizing glutathione S-transferase placental form (GST-P)-positive foci as an end point marker. In this presentation, the outline and samples of medium-term bioassay systems were described. Furthermore, our data demonstrated the presence of a threshold for the non-genotoxic carcinogen, phenobarbital (PB), and the lack of linearity in the low-dose area of the dose-response curve, providing evidence for hormesis. In addition, the establishment and applications of multiorgan carcinogenicity bioassay (DMBDD model), used for the examination of the carcinogenicity of genotoxic and non-genotoxic chemicals, are discussed. Dimethylarsinic acid, one of organic arsenics, was found to be carcinogenic in rat bladder using DMBDD model and carcinogenicity test.

Hormesis and dose-response-mediated mechanisms in carcinogenesis: evidence for a threshold in carcinogenicity of non-genotoxic carcinogens.

Recently the idea of hormesis, a biphasic dose-response relationship in which a chemical exerts opposite effects dependent on the dose, has attracted interest in the field of carcinogenesis. With non-genotoxic agents there is considerable experimental evidence in support of hormesis and the present review highlights current knowledge of dose-response effects. In particular, several in vivo studies have provided support for the idea that non-genotoxic carcinogens may inhibit hepatocarcinogenesis at low doses. Here, we survey the examples and discuss possible mechanisms of hormesis using phenobarbital, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), alpha-benzene hexachloride (alpha-BHC) and other non-genotoxins. Furthermore, the effects of low and high doses of non-genotoxic and genotoxic compounds on carcinogenesis are compared, with especial attention to differences in mechanisms of action in animals and possible application of the dose-response concept to cancer risk assessment in humans. Epigenetic processes differentially can be affected by agents that impinge on oxidative stress, DNA repair, cell proliferation, apoptosis, intracellular communication and cell signaling. Non-genotoxic carcinogens may target nuclear receptors, cause aberrant DNA methylation at the genomic level and induce post-translational modifications at the protein level, thereby impacting on the stability or activity of key regulatory proteins, including oncoproteins and tumor suppressor proteins. Genotoxic agents, in contrast, cause genetic change by directly attacking DNA and inducing mutations, in addition to temporarily modulating the gene activity. Carcinogens can elicit a variety of changes via multiple genetic and epigenetic lesions, contributing to cellular carcinogenesis.

Low dose DDT inhibition of hepatocarcinogenesis initiated by diethylnitrosamine in male rats: possible mechanisms.

Previously we reported a tendency for reduction of the development of glutathione-S-transferase placental form (GST-P) positive foci, recognized as preneoplastic changes in rat liver, by a low dose of 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), which belongs to the same group of hepatic cytochrome P-450 inducers as phenobarbital and is itself a non-genotoxic hepatocarcinogen. In order to clarify the biological significance of this phenomenon, we investigated the reproducibility and changes in other parameters using an initiation-promotion model in which male F344 rats were treated with DDT at doses of 0, 0.005, 0.5, 500 ppm in the diet for 11 or 43 weeks after initiation of hepatocarcinogenesis with N-diethylnitrosamine (DEN). When 500 ppm DDT was applied, the formation of GST-P positive foci and tumor were markedly elevated. In contrast, induction of GST-P positive foci and liver tumors tended to be inhibited at a dose of 0.005 ppm, correlating with protein levels of cytochrome P450 2B1 and 3A2 (CYP2B1 and 3A2) and generation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage. mRNA levels for 8-oxoguanine glycosylase 1 (OGG1), an 8-OHdG repair enzyme, connexin 32 (Cx32), a major component of Gap junctions, and hepatic nuclear factor 1alpha (HNF-1alpha), a Cx32 regulator, were inversely correlated with GST-P positive foci and tumor formation. These results indicate that low dose DDT may indeed exhibit inhibitory effects on chemically initiated-rat hepatocarcinogenicity, in contrast to the promotion observed with high doses, and that this is related to changes in metabolizing enzymes, cell communication, and DNA damage and its repair.

Lack of large intestinal carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine at low doses in rats initiated with azoxymethane.

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), an abundant food-derived heterocyclic amine (HCA), has attracted particular attention as a human colon carcinogen. Humans are in fact exposed to continuous low doses of HCAs during lifetime. Therefore, we focused on rat large intestinal carcinogenicity of PhIP at levels that mimic practical human exposure. A total of 192 6-week-old male F344 rats were subcutaneously injected twice with 15 mg/kg body weight azoxymethane (AOM), then continuously fed various doses (0, 0.001, 0.01, 0.1, 1, 10, 50 and 200 ppm) of PhIP in the diet. At week 16, aberrant crypt foci (ACF) were quantitatively analyzed. At week 36, tumor occurrence was pathologically analyzed. Then immunohistochemical examinations were performed. PhIP was found to enhance strongly AOM-initiated rat large intestinal tumorigenesis at high doses (50 and 200 ppm), while lower doses (0.001-10 ppm) had no apparent effects. High doses also caused variation in tumor histologic types and their distribution throughout the large intestinal segments. Frequencies of ACF/cm(2) did not meaningfully vary between the groups. Cellular proliferation activity in normal-appearing colonic mucosa was significantly increased at high doses. These novel findings may provide evidence of a low-dose potential for PhIP, with a no-observed effect level speculated to be 10 ppm in the present initiation-promotion experimental model.