Abstract
The aim of the present study was to characterize the phenomenon of resistance in putative preneoplastic hepatocyte nodules. These hyperplastic nodules are generated during the development of liver cancer in response to chemical carcinogens, and comprise a population of cells from which hepatocellular carcinoma can develop. As hepatocyte nodules grow in an environment that is otherwise toxic they possess a resistant phenotype. To understand this resistance phenomenon at the biochemical level, several phase I and II drug-metabolizing enzymes in the nodules were examined. Initial experiments were carried out in rats with nodules produced by initiation with diethylnitrosamine, followed by selection with 2-acetylaminofluorene and carbon tetrachloride. These nodules showed a large decrease in phase I enzymes and enzymic activities, such as the cytochromes P-450, cytochrome b[5], total microsomal haem, aminopyrine N-demethylase and ethoxyresorufin 0-deethylase, but glutathione and the phase II enzymes, namely, glutathione S-transferase, UDP-glucuronyl transferase, DT-diaphorase and gamma-glutamyltransferase were significantly increased. The pattern of changes of these drug-metabolizing enzymes of the nodules was similar when the nodules were produced by different initiation-promotion treatments, including diethylnitrosamine plus a choline/methionine-deficient diet, 2-acetamidofluorene plus phenobarbi-tone, or diethylnitrosamine plus orotic acid. In addition, the resistance phenotype was maintained when these nodules were transferred into the spleen of a rat not exposed to chemical carcinogens, and allowed to grow for several months, thus indicating that the newly acquired biochemical pattern in the nodules had become constitutive. Unlike the hepatic nodules generated by previous initiation-pro-motion treatments, nodules generated by the hypolipidemic agent, ciprofibrate, exhibited only a decrease in phase I components of the drug-metabolizing enzymes, with no increase in the phase II components. Similarly, hyperplastic nodules in liver mouse showed a decrease in phase I components, but no increase in phase II components. In addition to cytochrome P-450 and cytochrome b5, the total haem and two other haem containing proteins, namely, catalase and tryptophan 2,3-dioxygenase were also decreased in the nodules. A deficiency in hepatic iron, and a decrease in the activity of delta-ALA-synthetase, the first rate limiting enzyme in haem synthesis, were also apparent. Characterization of the phase II components revealed the presence of a new glutathione-S-transferase polypeptide, which has been shown to be identical to a placental form of the transferase. This polypeptide, although present to a minimal extent, or absent, in normal rat liver, is present in normal male mouse liver. Administration of lead nitrate to rats induces a biochemical pattern in the liver similar to that seen in the hepatocyte nodules, including a decrease of phase I components and an increase in phase II components of the drug-metabolizing enzymes, and the induction of the novel glutathione S-transferase. Further studies with lead nitrate may yield new insights into the mechanisms of production of the biochemical changes induced in the nodules, as this agent generates the same changes within 30 hours. Furthermore, the lead nitrate-induced changes in phase I and phase II enzymes are reversible, while the changes seen in the hyperplastic nodules are not. Thus this study has characterized one pattern of biochemical changes exhibited by the resistant phenotype of hyperplastic hepatic nodules, and a model system has been developed which induces the same changes, more rapidly and in a reversible fashion. One of the important questions yet to be answered however is the biological significance of the resistant phenotype in cancer development. Is the acquisition of resistance only important in expanding the initiated cell population to generate nodules or does it also have a more direct role in the progression of nodules to cancer? This is highly relevant to the clarification of the carcinogenic process in the liver and perhaps in other organs as well.