Growth kinetics of enzyme-altered liver foci in rats treated with phenobarbital or alpha-hexachlorocyclohexane.

A quantitative method based upon a stochastic model for the appearance of initiated cells and their clonal growth was used to estimate cell birth and death rates in enzyme-altered liver foci (EAF). gamma-Glutamyltranspeptidase (gamma-GT)-positive foci were initiated in livers of female SPF Wistar rats by a single application of N-nitrosomorpholine. Serial terminations during and after stop of promoter treatment with either phenobarbital (PB) or alpha-hexachlorocyclohexane (alpha-HCH) provided information on the growth and regression of the EAF. Simultaneous labeling index (LI) measurements were obtained via single injections with [3H]thymidine. No significant increases of the LI were observed with PB or alpha-HCH treatment. Since both agents are strong liver promoters we conclude that the growth of gamma-GT-positive foci is mainly due to a decrease in the rate of apoptosis. Indeed, our analysis supports this conclusion but determines that the abrogation of homeostatic control during promoter treatment is subtle. The ratio of cell death and cell birth rate is found to be decreased only slightly during promoter treatment and slightly increased after stop of promotion. For the mathematical analysis, two distinct focal growth scenarios were employed: (i) volume growth, i.e., all cells within individual foci cycle actively with the same rate, and (ii) surface growth where only cells on the surface of foci cycle actively while interior cells are resting. The model based upon scenario (ii) provides a better fit to the data and is more consistent with the experimental observations indicating heterogeneity of cell division rates within individual foci.

Peroxisomal enzyme induction uncoupled from enhanced DNA synthesis in putative preneoplastic liver foci of rats treated with a single dose of the peroxisome proliferator nafenopin.

Putative preneoplastic foci of spontaneous origin could be detected in the livers of 2 year old, untreated male Wistar rats. The unaltered and preneoplastic hepatocytes showed an identical expression of the peroxisomal marker enzyme acyl-CoA oxidase, as determined by immunohistochemical staining. A single dose of the peroxisome proliferator (PP) nafenopin (NAF) induced the enzyme predominantly in hepatocytes around the central venules and cell replication mainly in the periportal areas. However, upon one NAF application almost all of the preneoplastic foci showed a considerably weaker immunoreaction for peroxisomal acyl-CoA oxidase than the surrounding tissue. Concomitantly NAF elevated replicative DNA synthesis index in foci up to approximately 40%, while replication of hepatocytes in the unaltered portion of the livers increased only slightly to moderately. In conclusion, NAF-induced peroxisomal acyl-CoA oxidase and replicative DNA synthesis seem not to be necessarily coupled within the same liver cell. Furthermore, preneoplastic foci responded rather to the cell replicative than to the peroxisomal effects of NAF, suggesting that the PP-induced growth stimulus is of particular significance for the carcinogenic action of this class of compounds.

DNA synthesis, apoptosis, and phenotypic expression as determinants of growth of altered foci in rat liver during phenobarbital promotion.

Carcinogenesis was initiated in female rat liver by a single dose of N-nitrosomorpholine; subsequently phenobarbital (PB) was administered via the diet at a daily dose of 50 mg/kg body weight for up to 49 weeks. Subgroups of rats were left untreated after 10 or 28 weeks on PB. PB produced the following changes: (a) accelerated appearance of neoplastic nodules and hepatocellular carcinoma (from 28 weeks onwards); (b) phenotypic changes in altered foci such as a shift from clear to eosinophilic appearance, enhanced expression of gamma-glutamyltranspeptidase and other markers, and more distinct borders from surrounding liver; (c) an increase in foci number; and (d) accelerated foci enlargement. The increase in foci number was found to be due to increased phenotypic expression of foci. DNA synthesis was measured by [3H]thymidine labeling at multiple time points. The rate of DNA synthesis was always approximately 10-fold higher in foci than in surrounding liver tissue. Despite this, after N-nitrosomorpholine alone foci grew little before 18-24 weeks. Continuous treatment with PB did not produce a persistent further increase of DNA synthesis in foci, although it accelerated foci growth. Furthermore, at early stages small and larger foci showed similar DNA synthesis activity. These findings indicate that the rate of cell replication as measured by DNA synthesis is not the only determinant of the growth rate of foci. Further studies showed that foci with indistinct borders (reflecting weak expression of the altered phenotype) grew much less than foci with distinct borders; this was at least in part due to an increased rate of cell death by apoptosis found in foci with indistinct borders. In conclusion, besides cell replication, apoptosis and the extent of phenotypic expression (remodeling) determine the growth rate of foci. Foci with weak phenotypic expression predominated after N-nitrosomorpholine alone; in these, a high incidence of apoptosis counterbalanced cell replication. In contrast, during PB treatment foci with strong phenotypic expression predominated; in these, apoptotic activity was lower and the high replicative activity could manifest itself. Finally, all effects of PB on foci were largely although not completely reversible upon cessation of treatment; as a result phenotypic expression declined, and "remodeling" foci with high apoptotic activity predominated again.

Effects of hypolipidemic drugs nafenopin and clofibrate on phenotypic expression and cell death (apoptosis) in altered foci of rat liver.

Phenotypically altered liver foci were produced in female Wistar rats by a single dose of N-nitrosomorpholine followed by promotion with phenobarbital (PB) for 20 or 28 weeks. Then treatment was changed to either hexachlorocyclohexane (HCH), or cyproterone acetate (CPA), or nafenopin (Naf) or clofibrate (Clof), two hypolipidemic drugs. Foci were identified by a positive reaction for gamma-glutamyl-transpeptidase (GGT) and other cytological markers. HCH and CPA could substitute for PB as foci promoters; in contrast, Naf and Clof decreased expression of GGT in foci resulting in a decline of number and area of detectable foci, effects particularly pronounced with Naf. Immunohistochemical investigations of serial sections revealed that Naf also reduced expression of the altered phenotype when cytochrome P450-PB and pyruvate kinase (type L) were used as foci markers, but not when glutathione-S-transferase B (GST-B) was used. Thus, the number of foci with enhanced GST-B did not decline significantly after the change from PB to Naf treatment. Furthermore, the reduction of GGT and the decrease of foci number during Naf treatment were not associated with increased evidence of cell death by apoptosis in foci, in contrast to the situation after PB withdrawal. These findings strongly suggest that the disappearance of GGT-positive foci after Naf is due to a phenotypic change resulting in a suppression of GGT expression rather than to physical elimination of foci.

Effects of non-genotoxic hepatocarcinogens phenobarbital and nafenopin on phenotype and growth of different populations of altered foci in rat liver.

Non-genotoxic hepatocarcinogens share the ability to induce liver growth in rodents. Phenobarbital (PB), as one prototype compound, promotes the development of liver tumors; altered cell foci of the clear-eosinophilic phenotype, also identified by gamma-glutamyltransferase expression, appear to be precursor lesions. These foci seem to over-respond to the growth-inducing effect of PB. In contrast, the question as to whether peroxisome inducers are also tumor promoters is still unsettled. We will present evidence which strongly suggests that the peroxisome inducer, nafenopin (Naf), promotes tumor development in rat livers by stimulating selective growth of a hitherto undescribed subtype of altered foci. This subtype is characterized by weak diffuse cytoplasmic basophilia of its hepatocytes. Initiation in rats by aflatoxin B1 followed by promotion with Naf produced numerous adenomas and carcinomas; their morphology resembled that of the weakly basophilic foci. Both clear-eosinophilic and weakly basophilic foci appear "spontaneously" in the liver of aging rats. Promotion of such lesions by PB-type compounds or peroxisome inducers may explain cancer formation by these non-genotoxic agents.

Facilitated expression of adaptive responses to phenobarbital in putative pre-stages of liver cancer.

Female rats received N-nitrosomorpholine to produce altered cell foci (potential cancer pre-stages) in the liver, followed by phenobarbital (PB) for 2 weeks. As indicators of adaptive responses we measured DNA synthesis cumulatively by infusion of [3H]thymidine for the entire period of PB treatment, and cytochrome P450-PB by immunocytochemistry on histological liver sections. Altered cell foci were identified by expression of gamma-glutamyltransferase (gamma-GT), and/or altered morphology. The following results were obtained. In normal parts of the liver PB induced DNA replication only in association with expression of P450-PB; both responses were restricted to the pericentral area of the liver lobule. In foci, PB treatment led to enhanced DNA synthesis. Furthermore, PB caused a 3-fold increase in the number of foci expressing cytochrome P450-PB, gamma-GT or both. Cumulative determination of DNA synthesis showed that this increase did not result from selective proliferation of single initiated cells. It is concluded that in foci also PB can induce expression of the adaptive increases in cytochrome P450-PB and DNA synthesis. Foci show the responses to PB more readily than normal hepatocytes, and irrespective of their position within the liver lobule. These findings suggest that expression of adaptive responses to inducing tumor promoters is facilitated in altered foci.

Pyruvate kinase isoenzymes in altered foci and carcinoma of rat liver.

Pyruvate kinase (PK) isoenzymes, rate limiting for the last steps of glycolysis, were studied in normal rat liver, putative preneoplastic foci, neoplastic nodules and hepatocellular carcinoma. These lesions were produced by an initiation-promotion protocol: treatment with a single dose of N-nitrosomorpholine (NNM) was followed by feeding diets containing phenobarbital (PB) or alpha-hexachlorocyclohexane (alpha-HCH), or basal diet. PK was demonstrated (i) by immunocytochemistry on histological sections with antibodies specifically directed against the L and M2 isoenzymes, (ii) by electrophoretic separation of isoenzymes in homogenates from liver and larger tumors, and (iii) by electrophoretic separation of isoenzymes in parenchymal and stromal cells isolated from liver and tumors. Immunocytochemistry showed decreases of L-PK (L-PK-) in hepatocytes of most of the foci, nodules and carcinomas. Most L-PK- foci showed increases in gamma-glutamyltransferase (gamma-GT) and epoxide hydrolase (EH). PB or alpha-HCH treatment further decreased expression of L-PK in foci, but not in normal liver. Cells and foci with enhanced L-PK (L-PK+) were also found after carcinogen treatment. These did not show increases of gamma-GT or EH or any distinct morphological alterations with the exception of some which were basophilic ('tigroid') in H and E stained sections. No L-PK+ tumors were found. We could not demonstrate the M2-type PK in parenchymal cells of liver or any of the lesions described above. This isoenzyme was restricted to stromal cells in normal rat liver and in all stages of carcinogenesis as shown by immunohistology and by electrophoresis of preparations from isolated cell populations. However, stromal cells from hepatocellular carcinomas exhibited a 3-fold increase of M2-PK compared with stromal cells from normal liver. These results do not support an isoenzyme shift from L to M2-PK in the course of malignant transformation of hepatocytes as suggested previously.

Controlled death (apoptosis) of normal and putative preneoplastic cells in rat liver following withdrawal of tumor promoters.

Numerous drugs, hormones and environmental pollutants induce liver growth by hypertrophy and/or hyperplasia, and promote preferential growth of putative preneoplastic foci in the liver. In the present study the regression of hyperplasia after cessation of inducer/promoter treatment was studied in normal liver and in liver foci. High doses of cyproterone acetate (CPA), a synthetic sex steroid, were administered to rats and produced a doubling of liver size; after cessation of treatment liver size declined, and 27% of the total liver DNA disappeared within 6 days. In histological sections from the involuting liver no necroses, but numerous apoptotic bodies (ABs) were found; retreatment with CPA interrupted the formation of ABs. These findings suggest that elimination of excess liver DNA after cessation of CPA treatment is due to controlled cell death by apoptosis. In a further series of experiments putative preneoplastic foci were produced by a single dose of N-nitrosomorpholine and subsequently stimulated to grow by 10 or 28 weeks of phenobarbital (PB) treatment. After withdrawal of PB numerous ABs were present in normal liver and in the foci; in both, retreatment with PB decreased the appearance of ABs. It appears that inhibition of cell death by PB may contribute to tumour promotion. Under all conditions tested more ABs were found in the foci than in non-focal parts of the liver, suggesting an enhanced cell turnover in foci. The apparent sensitivity of foci to mechanisms controlling cell death might eventually provide a means for elimination of preneoplastic lesions.

Immunocytochemical demonstration of a phenobarbital-inducible cytochrome P450 in putative preneoplastic foci of rat liver.

Putative preneoplastic foci of rat liver, so far believed to be deficient in monooxygenases, are shown to contain a cytochrome P450 isoenzyme inducible by phenobarbital. The isoenzyme is also present and appears catalytically active in liver tumors obtained after promotion with phenobarbital and alpha-hexachlorocyclohexane.

Glutathione S-transferases in chemically induced tumors.

Rats were treated with N-nitrosomorpholin and subsequently with phenobarbital. After several weeks of phenobarbital treatment, foci of altered cells and tumors developed in the livers of the animals. In liver tumors, the activity of glutathione S-transferase was higher than in the surrounding tissue and remained elevated even after the withdrawal of the promoter. Like the glutathione S-transferase from normal liver, tumor glutathione S-transferase activity is composed of several, predominantly basic, isoenzymes with an apparent molecular weight of approximately 45,000.

Aberrant expression of adaptation to phenobarbital may cause selective growth of foci of altered cells in rat liver.

According to the multistage concept of carcinogenesis, promoters induce selective or preferential multiplication of initiated cells, thereby accelerating one of the rate-limiting steps in cancer formation. The driving force for this selective growth of initiated cells is not known. In normal liver, various liver tumour promoters induce expression of adaptive programmes. One such programme, induced by phenobarbital, consists of organ growth and an increase in the activity of various enzymes involved in the metabolism of lipophilic substrates. In foci of altered rat liver cells (the putative progeny of initiated cells), expression of the programme is still possible and is coupled to certain regulatory defects (excessive, unbalanced proliferation; independence from position-specific restrictions within the hepatic acinus; autonomous expression of some adaptive changes). Promoters seem to switch on this aberrant expression of the programme of adaptation, thereby leading to preferential growth of foci.

The role of growth of normal and preneoplastic cell populations for tumor promotion in rat liver.

A number of different compounds, including phenobarbital, hypolipidemic drugs such as clofibrate and nafenopin, the sex steroids progesterone, cyproterone acetate, estradiol and mestranol, chlorinated hydrocarbons such as DDT, hexachlorocyclohexane, and TCDD and the antioxidant butylhydroxytoluene, appears to promote the development of liver tumors from previously induced initiated cells. The mechanisms of tumor promotion by several representative prototypes of these compounds were studied in rat liver in vivo. All liver tumor promoters mentioned above stimulate growth of normal liver. The growth response is due to cellular hypertrophy and/or increased rate of DNA (and cell) replication and/or decreased rate of cell death. Hepatocytes in foci or islands of altered cells (putatively preneoplastic) show higher rates of replication than normal liver cells; various different liver tumor promoters cause a further increase of proliferation of focal cells. The increased proliferative activity is found in different island phenotypes and thus seems to be a useful marker of the putative preneoplastic state. The focal cells respond to several factors limiting proliferation in normal liver, suggesting that they are not autonomous with respect to growth control. Early preneoplastic foci grow slowly without promotion, despite the relatively high rates of cell replication. Thus their cells seem to have a much shorter life-time than normal hepatocytes or to undergo reversion to the normal phenotype. Promoters seem to accelerate island enlargement by increasing cell replication and delaying cell death or remodeling. Thus, tumor promoters enhance the manifestation of the proliferation advantage of the putative initiated cell population. In addition, promoters cause increases in the number of detectable islands. This can partially be explained by enlargement of existing islands, but phenotypic changes that would enhance the probability of detection of remodelling islands and growth of dormant initiated cells, probably contribute to the apparent increase of island number. Putative preneoplastic foci of unknown origin are frequent in the liver of aged Wistar rats. They are morphologically and functionally very similar to those induced by carcinogens and are responsive to the mitogenic effect of tumor promoters. Promotion of these "spontaneous" foci may explain tumor appearance after long-term application of promoters.The findings may provide a basis for improved identification of initiated hepatocytes (and of initiating hepatocarcinogens) and for detection of tumor promoters. All suspected liver tumor promoters tested so far induced enhanced preneoplastic cell proliferation after single doses. The long-term carcinogenicity bioassay as currently performed does not discriminate between initiating and promoting properties of a test compound if the animals used develop spontaneous preneoplastic lesions in the organ affected.

Promotion of spontaneous preneoplastic cells in rat liver as a possible explanation of tumor production by nonmutagenic compounds.

Foci of putative preneoplastic cells were detected in the livers of untreated aged Wistar rats of both sexes. The foci exhibited markers similar to those of their counterparts in carcinogen-treated rats such as increased cytoplasmic basophilia, clearness of cytoplasm, or expression of gamma-glutamyltransferase. Rates of DNA synthesis in foci were higher than in normal liver and were further increased by single doses of liver mitogens assumed to promote liver tumor development (phenobarbital, alpha-hexachlorocyclohexane, cyproterone acetate, nafenopin). Thus, cells in the spontaneous foci appear to possess a defect in growth control, rendering them more susceptible to endogenous and exogenous growth stimuli. This defect has been found previously in carcinogen-induced foci and may be used as a marker for putative preneoplastic cells. The spontaneous foci are present at low incidence in 8-month-old rats; at 2 years, all of 50 rats studied possessed foci. These observations suggest that nongenotoxic compounds can produce liver tumors if they promote tumor development from preneoplastic foci. Therefore, long-term bioassay for carcinogenicity will not discriminate between initiating and promoting compounds if preneoplastic lesions develop in control animals.

Response of liver foci in rats to hepatic tumor promoters.

Putative preneoplastic foci (islands) of altered hepatocytes were induced by single doses of initiating hepatocarcinogens and identified by various histochemical and morphological markers. DNA synthesis, measured by 3H-thymidine and autoradiography, as well as mitotic activity were found to be higher in the foci than in normal hepatocytes. Without promotion this proliferative advantage is partially counterbalanced by reversion of altered cells to the normal phenotype and/or increased incidence of cell death by apoptosis, so that the islands show little growth. Prolonged administration of promoters such as phenobarbital, hexachlorocyclohexane etc. resulted in the appearance of more and larger foci, which exhibited steady growth. Island enlargement was due to an upsurge of cell proliferation (in the initial stage only) and to interference of the promoter with phenotypic reversion and/or apoptosis of island cells. Island multiplication during promotion was essentially due to induction of the histochemical marker (γ-GT) in "latent" islands. Promoter withdrawal led to rapid reductions in size and number of foci suggesting that the effect of promoters on the phenotype of island cells is reversible under the conditions employed. Finally we offer the hypothesis that non-mutagenic compounds may produce liver tumors in experimental animals by promotion of "spontaneous" preneoplastic lesions.

Proliferative liver lesions and sex steroids in rats.

In standard two year tumorigenicity studies some gestagens, alone or in combination with an estrogen have been shown to produce proliferative liver lesions (PLL) in rats. Estrogens in general have not produced PLLs; for ethinyl estradiol the situation is equivocal. The steroids which increase the incidence of PLLs have not been shown to have the characteristics usually associated with classical hepatocarcinogens such as diethylnitrosamine (e.g. negative mutagenicity and cytoxicity, failure to bind covalently to DNA). Therefore, another mechanism must be assumed. A possible explanation is given by the ability demonstrated for a few steroids (mestranol alone or in combination with norethindrone, ethinylestradiol) to promote preneoplastic islands induced by diethylnitrosamine, resulting in an increase in tumor incidence over rats given only the carcinogen. Spontaneous preneoplastic islands in rats occur in various incidence in different strains and we have recently shown that their reaction to steroids-enhanced proliferation-is the same as the effect on preneoplastic islands produced by classical carcinogens. Sex steroids also enhance the growth or normal hepatocytes. It, therefore, seems reasonable to assume that sex steroids produce PLLs by acting on spontaneous preneoplastic lesions and not by the mechanisms usually associated with classical carcinogens. Extrapolating the incidence of PLLs in rats to a risk in humans may be highly misleading because of different pharmacokinetic patterns. Even in rats treated orally for two years with sex steroids dosed as multiples of the human contraceptive dose, resorption and biovailability may vary by a factor of 10 or more. It is, therefore, inappropriate to state that one sex steroid is more active in the production of PLLs than another, unless the data from pharmacokinetic studies are taken into account. Available evidence shows that sex steroids act as tumor promotors and enhance the growth of normal hepatocytes. It is extremely doubtful whether a sex steroid producing PLLs in the rat after two years of oral treatment can be classified as an initiating tumorigen for the rat. Substantial risk for humans cannot be inferred, because of the high incidence of spontaneous preneoplastic lesions in rats, and because of the very high sensitivity of the rat liver to a variety of chemically unrelated compounds including sex steroids.

Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with the tumor promoters phenobarbital, hexachlorocyclohexane, steroid compounds, and nafenopin.

Putative preneoplastic islands were induced in rat liver by diethylnitrosamine or nitrosomorpholine administered either as single high doses or continuously for 40 days at low dose levels. Following recovery periods of 3 weeks to 11 months, islands were identified by means of a positive gamma-glutamyl transferase reaction and/or altered morphology. DNA synthesis, by means of [3H]thymidine autoradiography, as well as mitotic activity were determined. Under all conditions studied, proliferation rates of island cells were significantly higher than those of normal unaltered hepatocytes. Single doses of liver mitogens known or assumed to promote liver tumor development (phenobarbital, alpha-hexachlorocyclohexane, cyproterone acetate, nafenopin, and pregnenolone-16 alpha-carbonitrile) were administered. Twenty-four to 30 hr later, this treatment produced even higher proliferative activities in island cells and increased the DNA synthesis index up to 50%, while proliferation in normal liver cells increased slightly to moderately. Thus, cells of putative preneoplastic islands appear to possess an inherent defect of growth control rendering them more susceptible to endogenous and exogenous growth stimuli. These findings partially explain why the mitogens mentioned induce rapid enlargement of preneoplastic foci and may provide a clue for further studies on the mechanism of tumor promotion in the liver. In addition, the results may form the basis for a short-term test to detect promoting activity of chemical compounds.