S-adenosylmethionine:S-adenosylhomocysteine (SAM/SAH) ratio, 5-methylcytosine (5mC) DNA content, and methylation and expression of c-myc, c-Ha-ras and c-Ki-ras have been studied in liver nodules, induced by diethylnitrosamine according to the 'resistant hepatocyte' model, and in regenerating liver (RL) between 0.5 and 72 h after partial hepatectomy (PH). Nodules, 11, 13 and 21 weeks after initiation, grew actively, showed a low tendency to remodel (persistent nodules), and did not exhibit carcinomatous changes. They underwent extensive remodeling after a 1-week SAM treatment (64 mumol/kg/day), and decreased in size and number after a 3-11-week treatment. A low SAM/SAH ratio was coupled, in nodules, with a high labeling index (LI), 2-fold fall in 5mC DNA content, increase in c-myc, c-Ha-ras and c-Ki-ras expression and hypomethylation of CCGG sequences in the DNA hybridizing with the three protooncogenes. In RL a low SAM/SAH ratio, overall DNA hypomethylation and enhanced c-myc expression were first observed 0.5 h after PH, reached a peak at 5 h and progressively returned to pre-PH levels later on. Maximum expression of c-Ha-ras and c-Ki-ras occurred 24-30 h after PH, roughly coincident with the LI peak. However, no great modifications of the methylation pattern of protooncogene CCGG sequence occurred at any time after PH, indicating the presence of hypomethylated genes and/or DNA sequences different from those investigated in this paper. SAM injection to nodule-bearing rats, for 1-11 weeks before killing, and to hepatectomized rats, 2 days before PH and then up to killing, largely prevented decrease in the SAM/SAH ratio and overall DNA methylation and inhibited LI and protooncogene expression. In nodules these effects were proportional to the treatment length and coupled with methylation of CpG residues in the CCGG sequence of the three protooncogenes studied. SAM treatment left the methylation pattern of these genes unchanged in RL. Kinetics of increase in protooncogene expression suggest a role in the regulation of cell cycle in RL. However, decrease in the SAM/SAH ratio, protooncogene hypomethylation and enhanced expression are apparently stable in nodules 11-21 weeks after initiation and could be implicated in continuous nodule growth and progression. Control of DNA methylation and gene expression by exogenous SAM could be a mechanism of the SAM anti-progression effect.

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Liver ornithine decarboxylase (ODC) activity and content of S-adenosyl-L-methionine (SAM) and its catabolite 5'-methylthioadenosine (5'-MTA) were determined in the late stages of hepatocarcinogenesis. Wistar rats received one diethylnitrosamine dose, followed by a partial hepatectomy at the midpoint of a 15-day treatment with 2-acetylaminofluorene (2-AAF), and then by an 18-week phenobarbital (PB) treatment. Thirty-eight per cent of liver was gamma-glutamyltranspeptidase (GGT)-positive and no visible nodules and hepatocellular carcinomas developed 16 weeks after starting 2-AAF feeding. Hyperplastic nodules and hepatocellular carcinomas were found on weeks 24 and 56 respectively. On weeks 24 and 56 only approximately 10% of liver was occupied by GGT-positive foci. At all times studied the foci exhibited a low labeling index (LI), and liver ODC activity was near control values. By contrast, a high ODC activity and LI and a low SAM and 5'-MTA levels were found in hyperplastic nodules and neoplasia. These tissues exhibited a high 5'-MTA phosphorylase activity. SAM administration during PB treatment, caused a 25-36% fall of GGT-positive liver and prevented the development of hyperplastic nodules and hepatocellular carcinomas. This was coupled to a sharp increase of SAM and 5'-MTA liver contents. SAM and 5'-MTA inhibited hepatocyte DNA synthesis in vitro. The addition of 5'-MTA to the reaction mixture for the ODC assay strongly inhibited ODC activity. However, the preincubation of SAM with liver homogenates or hepatocytes, used to prepare crude ODC, was necessary to inhibit ODC activity by SAM. Adenine, an inhibitor of 5'-MTA-phosphorylase, enhanced inhibition of DNA synthesis and ODC activity by SAM and 5'-MTA. Thus, during a prolonged promoting treatment a selected population of GGT-positive foci appears to acquire a stable phenotype characterized by a high DNA and polyamine synthesis. The development of nodules and carcinomas is associated with low SAM and 5'-MTA contents and high ODC activity and LI. 5'-MTA accumulation, during SAM administration, is probably responsible for the inhibition of promotion by SAM.

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The effect of dehydroepiandrosterone (DHEA) on the activity of NADPH-producing enzymes and the development of enzyme-altered foci has been investigated in the liver of female Wistar rats subjected to an initiating treatment (a necrogenic dose of diethylnitrosamine) followed, 15 days later, by a selection treatment [a 15-day feeding of a diet containing 0.03% 2-acetylaminofluorene (2-AAF), with a partial hepatectomy at the midpoint of this feeding]. At the end of the selection treatment all rat groups received, for 15 days, a basal diet containing, when indicated, 0.05% phenobarbital (PB) and/or 0.6% DHEA. The effect of DHEA on the activity of NADPH-producing enzymes was also studied in normal rats fed, for 15 days, a diet containing 0.6% DHEA and in their pair-fed controls. DHEA caused a 43-58% inhibition of glucose-6-phosphate dehydrogenase (G6PD) and, respectively, 338-420% and 21-24% increases in malic enzyme (ME) and isocitric dehydrogenase activities in all rat groups. This was coupled with a great fall in the production of ribulose-5-phosphate, while no change in NADP+/NADPH ratio occurred. Hepatocytes, isolated from DHEA-treated rats, exhibited a very low activity of hexose monophosphate shunt (HMS), which was not stimulated by methylene blue, an exogenous oxidizing agent that markedly stimulated HMS activity in control hepatocytes. DHEA caused a great fall in the percentage of liver occupied by gamma-glutamyltranspeptidase (GGT)-positive foci, in the rats subjected to the initiation-selection treatments. PB enhanced the development of these foci, an effect which was completely overcome by DHEA. In addition, focal cells no longer expressed a G6PD activity higher than that of surrounding liver in DHEA-treated rats, but exhibited a high histochemical reaction for ME. DHEA also caused a great fall in labelling index of GGT-positive foci. Starting at the end of 2-AAF feeding, a mixture of ribonucleosides (RNs) of adenine, cytosine, guanine and uracil and of deoxyribonucleosides (DRNs) of adenine, cytosine, guanine and thymine were injected i.p. every 8 h for 12 days to the rats subjected to the initiation-selection treatments plus PB. Rats were killed 3 days after the end of RN and DRN treatments. These treatments completely overcome the DHEA effect on the development of GGT-positive foci and DNA synthesis by the focal cells, without affecting G6PD activity of both whole liver and putative preneoplastic foci. Experiments with labeled nucleosides revealed that RNs and DRNs produced derivatives that were incorporated into liver DNA.(ABSTRACT TRUNCATED AT 400 WORDS)