2-( Allylthio ) pyrazine , a Cancer Chemopreventive Agent , Inhibits Liver Fibrosis Induced by Dimethylnitrosamine in Rats : Role of Inhibition of Transforming Growth Factorb Factorb Factorb 1 Expression

Exposure to nitrosamines may be the occupational risk factor for liver cirrhosis. 2-(Allylthio)pyrazine, a chemopreventive agent, inhibits CYP2E1 and induces phase II enzymes. We examined the effects of 2-(allylthio)pyrazine on hepatic fibrosis, a prepathologic state of cirrhosis, and on the expression of transforming growth factor-b1 induced by dimethylnitrosamine. Treatment of rats with dimethylnitrosamine for 4 weeks increased plasma alanine/aspartate aminotransferase and g-glutamyl transpeptidase activities, and bilirubin content, whereas the total plasma protein and albumin levels were decreased. 2-(Allylthio)pyrazine inhibited dimethylnitrosamine-induced increases in the enzyme activities and bilirubin, and restored the plasma protein and albumin contents. Masson’s trichrome staining showed that dimethylnitrosamine induced liver fibrosis, the extent of which was reduced by 2-(allylthio)pyrazine treatments. Reverse transcriptionpolymerase chain reaction analysis revealed that 2-(allylthio)pyrazine inhibited production of transforming growth factorb1 mRNA by dimethylnitrosamine. These results demonstrated that 2-(allylthio)pyrazine might inhibit dimethylnitrosamine-induced liver fibrosis due to suppression of CYP2E1 expression and transforming growth factor-b1 production. 2-(Allylthio)pyrazine has been studied as a cancer chemopreventive agent active against a variety of chemical carcinogens including vinylcarbamate, azoxymethane, benzopyrene and aflatoxin B1 (Kim & Kim 1999). Formation of aflatoxin B1-induced preneoplastic foci is significantly inhibited by 2(allylthio)pyrazine in the rat liver (Ha et al. 1999). 2-(Allylthio)pyrazine also effectively suppresses the liver injury induced by toxicants including acetaminophen, isoniazid and carbon tetrachloride (Kim et al. 1997a). Chemopreventive and hepatoprotective effects of 2-(allylthio)pyrazine may result from the inhibition of cytochrome P450 2E1 (an alcohol-inducible form of P450, CYP2E1) expression and the metabolic activity, and from the induction of phase II detoxifying enzymes such as glutathione S-transferases (Kim & Kim 1999; Kim et al. 1999). Epidemiological studies have shown that rubber workers died excessively from liver cirrhosis, which might be associated with the occupational risk factors such as exposure to N-nitroso compounds (Straif et al. 1999). Case reports and animal data raised the possibility that exposure to nitrosamines may be a risk factor for incidence of liver cirrhosis and the resultant mortality (Chao et al. 1995; Park & Mirer 1996; Straif et al. 1999). Nitrosamine, in particular dimethylnitrosamine, is bioactivated by the catalysis of Author for correspondence: Sang Geon Kim, College of Pharmacy, Seoul National University, Sillim-dong, Kwanak-gu, Seoul 151– 742, Korea (fax π822 872 1795, e-mail sgk/snu.ac.kr). CYP2E1 (Peng & Yang 1982; Koop 1992). Hence, CYP2E1 may be an important target for the prevention of liver cirrhosis inducible by N-nitroso compounds. Cirrhosis plays a role in the carcinogenesis of several types of cancer. In particular, the risk of hepatocellular carcinoma was substantially increased in the patients with liver cirrhosis (Sørensen et al. 1998). Hepatic fibrosis is a prepathologic state of cirrhosis that occurs as a consequence of severe liver damage in diverse chronic liver diseases. The prevention of hepatic fibrosis may serve as an important potential target for chemoprevention (Sakaida et al. 1994). Fibrosis arises from overproduction of extracellular matrix (e.g. type I, III and IV collagens) as a result of activation of hepatic nonparenchymal cells including Kupffer cells and stellate cells (Pinzani et al. 1998). Activation of hepatic stellate cells in the fibrotic liver causes morphological changes in myofibroblast-like cells, which is accompanied by synthesis of large quantities of extracellular matrix (Savolainen et al. 1988; Freidman 1993). Transforming growth factor-b1 (TGF-b1) is involved in the regulation of cell growth and differentiation. TGF-b1 appeared to play a role in fibrogenesis after liver injuries (Cheever et al. 1998; Kimura et al. 1999), although the complex pathogenesis of hepatic fibrosis is poorly understood yet. TGF-b1, as a key fibrogenic mediator, can directly enhance deposition of extracellular matrix and inhibit collagenase activity in the liver (Freidman 1993). Treatment of rats with the adenoviral vector expressing the mutant TGFKEON WOOK KANG ET AL. 24 Fig. 1. Chemical structure of 2-(allylthio)pyrazine. b1 receptor completely inhibited the process of hepatic fibrosis induced by DMN (Qi et al. 1999). Conversely, the transgenic mice overexpressing TGF-b1 developed severe hepatic fibrosis (Clouthier et al. 1997). Hence, inhibition of TGF-b1 production may serve as the potential therapeutic approach against liver fibrogenesis. Considering the chemoprevention and efficient liver protection by 2-(allylthio)pyrazine as an inhibitor of CYP2E1 against chemical carcinogens and toxicants, we determined the antifibrotic effect of 2-(allylthio)pyrazine in rats treated with dimethylnitrosamine activated primarily by CYP2E1. The role of TGF-b1 expression was further investigated as part of mechanistic studies. Materials and methods Materials. 2-(Allylthio)pyrazine was obtained from Yuhan Research Center (Gunpo, Korea). Chemical structure of the agent is shown in fig. 1. The purity of the compound was greater than 99%, as assayed by high-performance liquid chromatography. Reverse transcriptase and Taq. polymerase were obtained from Takara Co. (Tokyo, Japan). Dimethylnitrosamine and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Animals. Animal studies were conducted in accordance with the institutional guidelines for care and use of laboratory animals. Male Sprague-Dawley rats at 6 weeks of age (140–160 g) were supplied from Korea Food and Drug Administration (Seoul, Korea) and maintained under 12 hr light and dark cycles in an air-conditioned room with commercially-available rat chow (Purina, Korea) and water available ad libitum. Dimethylnitrosamine (10 ml/kg) was intraperitoneally injected, as dissolved in sterile saline (10 ml/kg body weight) 3 times a week for 4 weeks, as described previously (Ala-Kokko et al. 1987; Tsukamoto et al. 1990). Data were obtained from two groups of dimethylnitrosamine-treated animals (NΩ16 animals). Control animals received vehicle. 2-(Allylthio)pyrazine was orally administered 24 hr after each treatment with dimethylnitrosamine (3 times per week for 4 weeks). Animals were sacrificed on day 28 under light anaesthesia with diethyl ether. Blood was collected from vena cava. The left lateral lobe was sub-