Enhancement by ethyl alcohol of experimental hepatocarcinogenesis induced byN-nitrosomorpholine

Abstract

The effects of ethyl alcohol (EtOH) during or after treatment with N-nitrosomorpholine (NNM) on hepatocarcinogen-esis, ornithine decarboxylase (ODC) activity and the labeling index of the liver were investigated in male Sprague-Dawley rats. Rats were given drinking water containing NNM for 8 weeks and received i.p. injections of 1 g EtOH/kg body weight every other day during or after treatment with NNM. Pre-neoplastic and neoplastic lesions staining positively for gluta-thione-S-transferase, placental type (GST-P), were examined immunohistochemically. At the end of experiment at week 16, administration of EtOH after NNM treatment had no significant effect on the number and size of GST-P-positive hepatic lesions, whereas administration of EtOH during NNM treatment significantly increased the number and percentage area but not the mean area of GST-P-positive hepatic lesions. EtOH caused significant increases in the ODC activity of the liver and in the labeling indices of enzyme-altered lesions and the adjacent hepatocytes after the cessation of EtOH administration but not during EtOH treatment. Our findings indicate that EtOH enhances hepatocarcinogenesis and suggest that this effect may be closely related to the increases in ODC activity and cell proliferation in enzyme-altered lesions and the adjacent liver after EtOH treatment. Int. Consumption of alcoholic beverages has been linked to an increased risk of hepatocellular carcinoma (HCC) in humans. Case-control studies (Oshima et al., 1984; Tsukuma et al., 1990) of HCC and alcohol consumption have shown a significantly increased risk of HCC in heavy drinkers. Ethanol (EtOH) also has been found to enhance hepatic tumorigenesis (Yamagiwa et al., 1994). However, the mechanism by which alcohol induces cancers in humans is not clear. Epidemiologically, Tsukuma et al. (1993) have reported that the risk of HCC is significantly increased in former heavy drinkers but is small and insignificant in current heavy drinkers. These findings suggest that alcohol at an initiation phase may have an important role in hepatocarcinogenesis. Therefore, to investigate this possibility , in the present work, we examined the effect of EtOH given during or after administration of the carcinogen N-nitrosomorpho-line (NNM) on hepatocarcinogenesis in rats. MATERIAL AND METHODS Animals Ninety inbred Sprague-Dawley rats, initially weighing 150 g, were purchased from Japan SLC (Shizuoka, Japan). Rats were housed in suspended cages with wire mesh bottoms in a room with controlled temperature (21 6 1°C) and humidity (40 6 10%) and a 12:12 light:darkness cycle and given free access to regular chow pellets (Nihon-Nosan, Yokohama, Japan). Experimental design Animals were randomly divided into 3 groups of 30 rats each and from the beginning of the experiment were given drinking water containing 175 mg/l of NNM (Sigma, St. Louis, MO) for 8 weeks. NNM was dissolved in distilled water at a concentration of 50 g/l and stored in a cool place. The stock solution was diluted to 175 mg/l with tap water just...

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“…GST-P is a marker of preneoplastic and neoplastic cells (36,37), not only at the protein level but also at the mRNA level (38). GST-P is a marker of preneoplastic and neoplastic cells (36,37), not only at the protein level but also at the mRNA level (38).…”

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“…A low dose intervention increased the number of small MeIQx-induced GST-P+ foci by enhancing proliferating cell nuclear antigen (PCNA) immunohistochemical staining and the levels of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage [ 188 ]. Similar effects on the enhancement of GST-P+ foci emergence were also observed when ethanol was co-administered with MeIQx or N-nitrosomorpholine [ 189 , 190 ].…”