Keiichirou Morimura

Role of CYP2E1 in Diethylnitrosamine-Induced Hepatocarcinogenesis In vivo

CYP2E1 metabolizes many low-molecular weight toxins and carcinogens. Some in vitro experiments suggest that CYP2E1 may be involved in the metabolic activation of diethylnitrosamine. However, there has been no direct evidence demonstrating a role for CYP2E1 in diethylnitrosamine-mediated carcinogenesis in vivo. To clarify this, we carried out a diethylnitrosamine-induced hepatocarcinogenesis experiment using Cyp2e1-null mice. Male 14-day-old wild-type and Cyp2e1-null mice were treated with diethylnitrosamine (10 mg/kg of body weight) and killed at weeks 24 and 36 after diethylnitrosamine treatment for investigation of tumors and at 6, 24, and 48 h for examination of apoptosis and gene expression. Liver weights of Cyp2e1-null mice were significantly different at weeks 24 and 36 compared with wild-type mice (P < 0.01). Liver tumor incidences of Cyp2e1-null mice were significantly decreased at weeks 24 and 36 compared with wild-type mice (P < 0.01). Cyp2e1-null mice showed significant decrease in the multiplicities of hepatocellular adenoma at weeks 24 and 36 (P < 0.05 and P < 0.01, respectively), and of hepatocellular carcinoma at week 36 (P < 0.01) compared with wild-type mice. Apoptotic index and caspase-3 and/or Bax mRNA expression of Cyp2e1-null mice were significantly different at 6, 24, and 48 h after diethylnitrosamine treatment compared with wild-type mice (P < 0.05). We conclude that Cyp2e1-null mice show lower tumor incidence and multiplicity compared with wild-type mice in diethylnitrosamine-induced hepatocarcinogenesis. It is suggested that CYP2E1 completely participates in diethylnitrosamine-induced hepatocarcinogenesis, and high frequency of tumors in wild-type mice could be associated with the increased apoptosis.

Hepatocyte nuclear factor 4α in the intestinal epithelial cells protects against inflammatory bowel disease

Background: Hepatocyte nuclear factor 4&agr; (HNF4&agr;; NR2A1) is an orphan member of the nuclear receptor superfamily expressed in liver and intestine. While HNF4&agr; expression is critical for liver function, its role in the gut and in the pathogenesis of inflammatory bowel disease (IBD) is unknown. Methods: Human intestinal biopsies from control and IBD patients were examined for expression of mRNAs encoding HNF4&agr; and other nuclear receptors. An intestine‐specific HNF4&agr; null mouse line (Hnf4&agr;&Dgr;IEpC) was generated using an Hnf4&agr;‐floxed allele and villin‐Cre transgene. These mice and their control floxed counterparts (Hnf4&agr;F/F), were subjected to a dextran sulfate sodium (DSS)‐induced IBD colitis protocol and their clinical symptoms and gene expression patterns determined. Results: In human intestinal biopsies, HNF4&agr; was significantly decreased in intestinal tissues from Crohns disease and ulcerative colitis patients. HNF4&agr; expression was also suppressed in the intestine of DSS‐treated mice. In Hnf4&agr;&Dgr;IEpC mice, disruption of HNF4&agr; expression was observed in the epithelial cells throughout the intestine. In the DSS‐induced colitis model Hnf4&agr;&Dgr;IEpC mice showed markedly more severe changes in clinical symptoms and pathologies associated with IBD including loss of body weight, colon length, and histological morphology as compared with Hnf4&agr;F/F mice. Furthermore, the Hnf4&agr;&Dgr;IEpC mice demonstrate a significant alteration of mucin‐associated genes and increased intestinal permeability, which may play an important role in the increased susceptibility to acute colitis following an inflammatory insult. Conclusions: While HNF4&agr; does not have a major role in normal function of the intestine, it protects the gut against DSS‐induced colitis.

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.

Role of CYP2E1 in Thioacetamide-induced Mouse Hepatotoxicity

Previous experiments showed that treatment of mice and rats with thioacetamide (TAA) induced liver cell damage, fibrosis and/or cirrhosis, associated with increased oxidative stress and activation of hepatic stellate cells. Some experiments suggest that CYP2E1 may be involved in the metabolic activation of TAA. However, there is no direct evidence on the role of CYP2E1 in TAA-mediated hepatotoxicity. To clarify this, TAA-induced hepatotoxicity was investigated using Cyp2e1-null mice. Male wild-type and Cyp2e1-null mice were treated with TAA (200 mg/kg of body weight, single, i.p.) at 6 weeks of age, and hepatotoxicity examined 24 and 48 h after TAA treatment. Relative liver weights of Cyp2e1-null mice were significantly different at 24 h compared to wild-type mice (p<0.01). Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in Cyp2e1-null mice were significantly different at both time points compared to wild-type mice (p<0.01). Histopathological examination showed Cyp2e1-null mice represented no hepatototoxic lesions, in clear contrast to severe centriobular necrosis, inflammation and hemorrhage at both time points in wild-type mice. Marked lipid peroxidation was also only limited to wild-type mice (p<0.01). Similarly, TNF-alpha, IL-6 and glutathione peroxidase mRNA expression in Cyp2e1-null mice did not significantly differ from the control levels, contrasting with the marked alteration in wild-type mice (p<0.01). Western blot analysis further revealed no increase in iNOS expression in Cyp2e1-null mice. These results reveal that CYP2E1 mediates TAA-induced hepatotoxicity in wild-type mice as a result of increased oxidative stress.

Lack of a Dose-response Relationship for Carcinogenicity in the Rat Liver with Low Doses of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline or N-Nitrosodiethylamine

For a long period, it has been generally considered that carcinogens, particularly genotoxic ones, have no threshold in exerting their potential for cancer induction. However, the non‐threshold theory can be challenged with regard to assessment of cancer risk to humans. Here we show that a food‐derived, genotoxic hepatocarcinogen, 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline, forms DNA adducts at low doses, but does not induce glutathione S‐transferase placental form (GST‐P)‐positive foci (considered to be preneoplastic lesions) or 8‐hydroxy‐2′‐deoxyguanosine in rat liver. Moreover a N‐nitroso compound, N‐nitrosodiethylamine, at low doses was also found not to induce GST‐P‐positive foci in rat liver. These results imply that there is a no‐observed effect level for hepatocarcinogenesis by these genotoxic carcinogens.

Promotion of Skin Carcinogenesis by Dimethylarsinic Acid in Keratin (K6)/ODC Transgenic Mice

Dimethylarsinic acid (DMA) is a major metabolite of inorganic arsenicals in mammals, and arsenic exposure is associated with tumor development in a wide variety of human tissues, particularly the skin. Transgenic mice with ornithine decarboxylase (ODC) targeted to hair follicle keratinocytes are much more sensitive than littermate controls to carcinogens. In this study we investigated the promoting effect of DMA on skin carcinogenesis in such K6/ODC transgenic mice. The back skin of female C57BL/6J K6/ODC transgenic mice, 10 to 14 weeks old, was initiated with topical application of 7,12‐dimethylbenz[α]anthracene (DMBA) at a dose of 50 μg or acetone alone on day 1 of the experiment, followed by treatment with 3.6 mg of DMA, 5 μg of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or neutral vehicle (control) twice a week for 18 weeks. Mice were killed 1 week after the end of the treatment. Induction of skin tumors was significantly accelerated in the DMA‐treated group, as well as in the TPA‐treated group, indicating that DMA has a promoting effect on skin tumorigenesis in K6/ODC transgenic mice.

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.

Elevated oxidative stress and DNA damage and repair levels in urinary bladder carcinomas associated with schistosomiasis

To cast light on mechanisms underlying development of urothelial carcinomas (UCs) of the urinary bladder associated with Schistosomiasis, we immunohistochemically analyzed the relationship between oxidative stress markers, DNA single strand breaks (ssDNA) which could also measure the levels of base damage and apoptosis in DNA, and expression of DNA repair genes with levels of nitric oxide synthases in bladder carcinomas of Egyptian patients with or without Schistosoma hematobium infection. Marked elevation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) levels was found in squamous cell carcinomas and UCs associated with Schistosomiasis when compared with non‐Schistosomal carcinomas. This was accompanied by strong over expression of the DNA‐repair genes, 8‐oxoguanine‐DNA‐glycosylase and apurinic/apyrimidinic endonuclease, as well as increased formation levels of ssDNA. Expression levels of inducible nitric oxide synthase (iNOS) which is known to be indirectly related to oxidative stress was higher in Schistosomal than in the non‐Schistosomal carcinomas. However, expression of endothelial nitric oxide synthase was slightly stronger in non‐Schistosomal than in the Schistosomal carcinomas. In conclusion, these findings suggest a strong correlation between Schistosoma haematobium infection and increased levels of oxidative stress accompanied by a continuous DNA damage and repair in UCs, all directly correlating with elevated iNOS.

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 initiation activity in rat liver of low doses of 2-amino-3,8- dimethylimidazo(4,5-f )quinoxaline

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.