Albrecht Buchmann

Heterogeneity of enzyme-altered foci in rat liver

Enzyme-altered foci (EAF) in liver are assumed to be precursor lesions for tumors in this organ. Results obtained with selected hepatocarcinogens which produce lesions of differing phenotype and growth behavior indicate that not the total number of enzyme-altered cells but rather the proliferation of individual cell clones is of major importance for additional changes leading to malignancy. Analyses including multiple marker enzymes demonstrate a relationship between foci phenotype and proliferation. The inducibility of certain downregulated enzymes in EAF indirectly suggests disturbances in the expression of regulatory genes such as proto-oncogenes. Data on Ha-ras and c-myc proto-oncogene expression in EAF are presented.

Expression and inducibility of drug-metabolizing enzymes in preneoplastic and neoplastic lesions of rat liver during nitrosamine-induced hepatocarcinogenesis

The expression, inducibility, and regulation of four different cytochrome (cyt.) P-450 isoenzymes (PB1, PB2, MC1, and MC2) NADPH-cytochrome P-450 reductase, the glutathione transferases (GSTs) B and C and microsomal epoxide hydrolase (mEHb) have been studied during nitrosamine-induced hepatocarcinogenesis using immunohistochemical techniques. The investigations revealed basic differences in the expression of the individual drug metabolizing enzymes in the course of neoplastic development. While the two GSTs and mEHb were increased in all preneoplastic and benign neoplastic lesions, the levels of the distinct cyt. P-450 isoenzymes were characteristically different from each other. Following initial changes in the expression of these enzymes in early preneoplastic lesions (i. e., increase of cyt. P-450 PB1 versus slight decrease of the other cyt. P-450 isoenzymes), a continuous reduction of all cyt. P-450 isoenzymes was observed during the further course of hepatocarcinogenesis. In progressed neoplastic nodules, all cyt. P-450-isoenzymes and NADPH cyt. P-450 reductase were decreased to varying extents.Treatment of animals with inducers of the monooxygenase system, such as phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls, led to a rather heterogenous pattern of enzyme alterations in preneoplastic and neoplastic lesions. Following administration of phenobarbital, some islets responded to the same degree as the surrounding tissue, others were less or not at all inducible and a few of the lesions showed a prominent increase in cyt. P-450 PB2 and NADPH-cyt. P-450 reductase levels. The interesting finding that these two enzymes always showed concurrent changes may be indicative of a common regulation. Similar to phenobarbital, an induction of cyt. P-450 isoenzymes within carcinogen-induced lesions was also observed following administration of 3-methyl-cholanthrene or polychlorinated biphenyls.The results demonstrate that drug-metabolizing enzymes are abnormally regulated in carcinogen-induced lesions. The multiplicity of enzyme deviations within individual lesions and especially the enzyme inducibility strongly suggest that the focal enzyme alterations result from genotoxic effects of the carcinogen on regulatory systems of a higher order rather than from mutational events in individual structural genes.

The Use of Enzyme-Altered Foci for Risk Assessment of Hepatocarcinogens

Chemically-induced hepatocarcinogenesis is characterized by the sequential appearance of phenotypically altered cell populations which can be identified by changes in the expression of a variety of markers such as canalicular adenosine triphosphatase (ATPase), γ-glutamyl transpeptidase, glucose-6-phosphatase and others (for review see Peraino et al., 1983). There is increasing evidence to suggest that at least some of these early enzyme-altered foci are precursor lesions which are causally related to the malignant transformation. This is substantiated by the sequential appearance of enzyme-altered foci and liver tumors and by the observation that neoplastic nodules and hepatocellular carcinoma show enzyme-patterns similar to those seen in preneoplastic foci (Fried- rich-Freksa et al., 1969; Goldfarb and Pugh, 1981; Bannasch et al., 1986). Moreover, strong quantitative relationships between the total volume of enzyme-altered tissue in liver and the subsequent development of liver tumors have been established (Emmelot and Scherer, 1980; Kunz et al., 1983, 1985). Enzyme-altered foci are monoclonal in origin (Rabes et al., 1982; Williams et al., 1983) and show a growth advantage over the surrounding normal hepatocytes (Rabes et al., 1979). The analysis of multiple marker enzymes within individual foci points towards a marked heterogeneity of phenotypes which is also reflected by differences in the proliferation rates of the foci (Buchmann et al., 1987; Peraino et al., 1984). The analysis of number and size of enzyme altered foci in liver can yield quantitative data on the effects of hepatocarcinogens at low, relevant dose levels. Moreover, these data inherit information on mechanistic aspects of carcinogenesis which may be of importance for the improvement of risk assessment of carcinogens and tumor promoting agents in liver.

Cell Proliferation and Hepatocarcinogenesis

Chemically-induced hepatocarcinogenesis in rodents is a very useful tool to study qualitatively and quantitatively critical changes occurring during the carcinogenic process. There is strong evidence to suggest that enzyme-altered foci in liver are precursor lesions causally related to malignant transformation. In experiments with continuous exposure of rats to different doses of hepatocarcinogens the existence of quantitative relationships between the development of enzyme-altered foci and subsequent tumor manifestation in liver was demonstrated. The analysis of multiple enzyme markers indicated a marked heterogeneity of phenotypes between individual foci. Such diversity was also observed with respect to the growth properties of individual lesions and relationships between foci phenotype and proliferation rate were demonstrated. Our results suggest that not only the total number of enzyme-altered cells in liver but also the proliferation rate of individual cell clones is of major relevance for the transition(s) leading to malignant cell populations. In initiation-promotion experiments quantitative relationships between promoting activity of various xenobiotics and their potency to induce adaptive liver growth were established. Induction of cell proliferation in normal liver and in preneoplastic liver cells is therefore assumed to play an important role during the carcinogenic process.