Anna Kinoshita

Promoting effects of monomethylarsonic acid, dimethylarsinic acid and trimethylarsine oxide on induction of rat liver preneoplastic glutathione S-transferase placental form positive foci: A possible reactive oxygen species mechanism

Dimethylarsinic acid (DMA) is a major metabolite of inorganic arsenicals, which are epidemiologically significant chemicals in relation to liver cancer in mammals. The present study was conducted to determine the promoting effects of organic arsenicals related to DMA [monomethylarsonic acid (MMA) and trimethylarsine oxide (TMAO)] on rat liver carcinogenesis using a liver medium‐term bioassay (the Ito test). Male, 10‐week‐old, F344 rats were given a single i.p. injection of diethylnitrosamine at a dose of 200 mg/kg b.w. as an initiator. Starting 2 weeks thereafter they received 100 ppm of MMA, DMA or TMAO in their drinking water, or no supplement as a control, for 6 weeks. All animals underwent 2/3 partial hepatectomy in week 3 after initiation. Quantification of glutathione S‐transferase placental form (GST‐P)‐positive foci as preneoplastic lesions in liver sections revealed significantly increased numbers and areas in all 3 treated groups compared with controls. Hepatic microsome cytochrome P‐450 content was markedly increased with all 3 arsenic treatments. Markedly elevated CYP 2B1 protein levels and CYP 2B1/2 mRNA levels were thus observed in all cases. The potency of promotion was similar for MMA, DMA and TMAO. Since hydroxyradicals were found to be generated in the relatively early phase while methylated arsenicals were metabolized in liver, the resultant oxidative stress might have promoted lesion development.

Detailed low-dose study of 1,1-B is(p-chlorophenyl)-2,2,2-trichloroethane carcinogenesis suggests the possibility of a hormetic effect

To obtain information on the effects of nongenotoxic carcinogens at low doses for human cancer risk assessment, the carcinogenic potential of the organochlorine insecticide, 1,1‐bis(p‐chlorophenyl)‐2,2,2‐trichloroethane (DDT), in the liver was assessed in F344 rats. In experiment 1, 240 male animals, 21 days old, were administered 0, 0.5, 1.0, 2.0, 5.0, 20, 100 and 500 ppm DDT in the diet for 16 weeks. Experiment 2 was conducted to elucidate the carcinogenic potential of DDT at lower levels using 180 rats given doses of 0, 0.005, 0.01, 0.1, 0.2 and 0.5 ppm. The livers of all animals were immunohistochemically examined for expression of glutathione S‐transferase placental form (GST‐P), putative preneoplastic lesions. Quantitative values for GST‐P‐positive foci in the liver were increased dose‐dependently in rats given 20 ppm DDT and above with statistical significance as compared with the concurrent control value. In contrast, doses of 0.005 and 0.01 ppm were associated with a tendency for decrease below the control value, although not significantly. Western blotting analysis show that cytochrome P‐450 3A2 (CYP3A2) protein expression tended to decrease at 0.005 and 0.01 ppm, a good correlation being observed with the change in the number of GST‐P‐positive foci. These findings suggest that a DDT hepatocarcinogenicity may show nonlinear response, that is, hormetic response at low doses. Furthermore, since CYP3A2 protein expression appears to be important for the effects of phenobarbital and the α‐isomer of benzene hexachloride, mRNAs for IL‐1 receptor type 1 (IL‐1R1) and TNF‐α receptor type 1 (TNFR1) whose ligands have roles not only in downregulating CYP3A2 expression but also in inducing antiproliferative effect or apoptosis in hepatocyte were examined. Increase was observed at low doses of DDT. Oxidative stress in liver DNA, assessed in terms of 8‐hydroxydeoxyguanosine as a marker, was also decreased. These findings suggest that the possible hormetic effect that was observed in our detailed low‐dose study of DDT carcinogenesis, although not statistically significant, may be linked to levels of oxidative stress and proinflammatory cytokines.

Increased oxidative stress with gene alteration in urinary bladder urothelium after the Chernobyl accident.

We have previously shown that bladder urothelium of people living in the cesium‐137 (137Cs)–contaminated areas of Ukraine demonstrates accumulation of stable p53 and p53 mutational inactivation, preferentially through G:C to A:T transition mutations at CpG dinucleotides, with a codon 245 hot spot. In the present study, we analyzed immuno‐histochemically the relationship between oxidative stress markers and over‐expression of p53 and H‐ras in urinary bladder urothelium from 42 men with benign prostatic hyperplasia. Bladder mapping biopsies were obtained from 15 patients from a highly radiocontaminated area (group I), 14 patients from the less contaminated city of Kiev (group II) and 13 patients as a control group from “clean” (without radiocontamination) areas of Ukraine (group III). Irradiation cystitis with multiple foci of severe dysplasia and carcinoma in situ were observed in 15 of 15 (100%, group I) and 9 of 14 (64%, group II) cases, with 4 small transitional‐cell carcinomas incidentally detected in groups I and II. Markedly elevated levels of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX‐2) and 8‐hydroxy‐2`‐deoxyguanosine (8‐OHdG) were noted in these bladder urothelial lesions from groups I and II, accompanied by strong over‐expression of p53 and less H‐ras expression. These findings support the hypothesis that iNOS, COX‐2 and 8‐OHdG in bladder urothelium are induced by long‐term exposure to low‐dose radiation with a close relationship to p53 over‐expression that could predispose to bladder carcinogenesis. Int. J. Cancer 86:790–798, 2000.

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 (DMAV), 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 DMAV in their drinking water at a dose of 200 p.p.m. for up to 72 weeks. All DMAV‐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 DMAV treatment. Marked induction of Pola1, Cyp7b1, Ndfua3, Mmp13 and other genes specific to cell proliferation, cell signaling and xenobiotic metabolism in the lungs of DMAV‐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 DMAV‐exposed Ogg1−/– mice. Therefore, we strongly suggest that DMAV exerts carcinogenicity in the lungs of Ogg1−/– mutant mice, with a possible role for persistent accumulation of DNA oxidative adducts. (Cancer Sci 2007; 98: 803–814)

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/cm2 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.

High susceptibility of p53 knockout mice to esophageal and urinary bladder carcinogenesis induced by N, N-dibutylnitrosamine

In human cancer, alterations in the p53 tumor suppressor gene are the most common genetic alterations. The aim of the present study was to detect sensitivity of the p53 (+/-) mice and their littermates p53 (+/+) mice to N, N-dibutylnitrosamine (DBN) carcinogenicity. In experiment 1, 6-7-week-old p53 (+/-) and p53 (+/+) mice were treated with 0, 0.025 and 0.05% DBN, respectively, in drinking water for 20 weeks. Esophageal squamous cell and urinary bladder transitional cell carcinomas (TCCs) and fibrosarcomas were found to be significantly increased in p53 (+/-) mice treated with doses of DBN compared to p53 (+/+) mice administered similar doses. In experiment 2, 6-7-week-old p53 (+/-) and p53 (+/+) mice were administered 0 or 0.05 % DBN in drinking water for 8 weeks. Immunohistochemical staining revealed a significant increase in numbers of p53 and bromodeoxyuridine (BrdU) positive cells in the esophageal and urinary bladder epithelia of DBN-treated p53 (+/-) mice compared to p53 (+/+) mice administered DBN. Molecular analysis revealed point mutations in the residual p53 allele in four of eight (50%) esophageal mucosa of DBN-treated p53 (+/-) mice, and in three of eight (38%) of treated p53 (+/+) mice. The results show that p53 (+/-) mice were sensitive to DBN treatment with respect to esophageal and bladder tumor development, with a mechanism that could be confined to early mutations of the residual p53 allele and increased cellular proliferation in the target organs.

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. (Cancer Sci 2005; 96: 747–757)

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.

Aberrant expression of E-cadherin and β-catenin in association with transforming growth factor-β1 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/β‐catenin complex, and transforming growth factor‐β1 (TGF‐β1) 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‐β1 in the urothelium and lamina propria, iNOS in the urothelium and both β‐catenin and E‐cadherin in the cytoplasm were observed between groups 1 and 2 with marked expression in group 1. Furthermore, TGF‐β1 overexpression and alteration in E‐cadherin/β‐catenin complexes in bladder urothelium might play a crucial role in urinary bladder carcinogenesis in humans exposed to long‐term low‐dose ionizing radiation. (Cancer Sci 2005)