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Copenhagen (Cop) rats are completely resistant to the chemical induction of mammary adenocarcinomas, but their susceptibility to hepatocarcinogenesis is virtually unknown. Rat liver is a well-characterized and easily manipulated tissue in which to study carcinogenesis. Therefore, if Cop rats are resistant to hepatocarcinogenesis, studies into resistance mechanisms may be feasible. Male Cop and F344 rats, 7-8 weeks old, were initiated using either N-nitrosodiethylamine (DEN) (200 mg/kg, i.p.) or a two-thirds partial hepatectomy (PH) followed by N-methyl-N-nitrosourea (MNU) (60 mg/kg, i.p.). The rats were then promoted using a modified resistant hepatocyte (RH) protocol (a combination of four doses of 2-acetylaminofluorene (2-AAF) and a single dose of CCl4 that provides a selective mitotic stimulus for initiated cells). Six weeks after initiation the rats were killed and liver sections were stained for glutathione S-transferase 7-7 (GST 7-7), a marker for putative preneoplastic hepatocytes. Cop rats were found to be highly resistant, having a approximately 9- and approximately 27-fold smaller percentage of liver area occupied by GST 7-7-positive foci than susceptible F344 rats following initiation by DEN and MNU respectively. Furthermore, gross liver nodules did not form in any of the Cop rats, whereas all F344 rat livers contained nodules. Hepatic necrosis caused by DEN during initiation, and CCl4 during promotion is necessary to stimulate compensatory hepatocyte division. We demonstrated that these agents do indeed increase serum transaminase levels and produce histologic evidence of necrosis in Cop rats. In order for liver foci to grow rapidly in the RH protocol, the surrounding normal hepatocytes must be mito-inhibited by 2-AAF. We found that the degree of mito-inhibition of normal hepatocytes by 2-AAF is the same in Cop and F344 rats. These results show that the Cop rat is highly resistant to the chemical induction of putative preneoplastic liver foci and nodules. [ABSTRACT FROM PUBLISHER]

Lasiocarpine (LC), a pyrrolizidine alkaloid, is able to induce a series of chronic and progressive lesions in rat liver, including a long-lasting block in the cell cycle, the appearance of enlarged hepatocytes (megalocytosis), fibrosis, cirrhosis and malignant neoplasma. In this study the effect of transplantation of normal hepatocytes on the development of LC-induced chronic lesions in rat liver was examined. Two-month-old male Fischer 344 rats were given a single dose of LC (80 mumol/kg i.p.). Four weeks later all animals were subjected to 2/3 partial hepatectomy (PH). In addition, at the time of PH one group of rats were transplanted with normal hepatocytes isolated from a syngeneic donor (10(6) cells/rats via the portal vein), while the other group received only the culture medium. All rats were killed 14 weeks after the operation. Grossly, the liver of rats exposed to LC followed by PH with no transplantation of normal hepatocytes was small in size (% liver wt/body wt 1.66 +/- 0.08) and exhibited a few whitish nodules. Histologically, approximately 88% of the liver section was occupied by enlarged hepatocytes and hepatocyte nodules composed of smaller hepatocytes developed in every animal in this group. In addition, extensive bile ductular proliferation was present. However, the liver of rats that were similarly treated but received normal hepatocytes were significantly larger in size (% liver wt/body wt 2.16 +/- 0.07) and were almost completely free of megalocytosis, bile ductular proliferation and hepatocyte nodules. These findings indicate that transplantation of normal hepatocytes is able to modulate the development of chronic liver lesions induced by LC and may be relevant to the pathogenesis of progressive liver diseases such as neoplasia and cirrhosis.

Feeding excess orotic acid (OA) in the diet promotes the carcinogenic process in different organs including the liver. A number of metabolic and genetic disorders are associated with increased synthesis of endogenous OA and some of these disorders appear to pose an increased risk of liver cancer development. This study therefore examines whether excess OA of endogenous origin also exerts a promoting effect on hepatocarcinogenesis in the mouse and the rat. Increased endogenous synthesis of OA was achieved by (i) feeding a diet deficient in arginine (AD) and (ii) feeding excess dietary carbamylaspartate (CA), a precursor for the synthesis of OA. A single dose of diethylnitrosamine (DENA) was given i.p. to male Fischer 344 rats (200 mg/kg) or to male DBA/2 mice (90 mg/kg). One week later they were placed on either AD diet or the same diet supplemented with 1.35% arginine (AS) for a total of 4 weeks. Two-thirds partial hepatectomy (PH) was performed at the end of the second week. All animals were then transferred to a control semisynthetic basal diet for a total of 20 weeks before they were killed. The results indicated that AD diet increased the incidence of hepatic nodules in both rats (percentage area occupied by nodules was 4.7 +/- 0.4 in the AD group compared to a control value of 0.7 +/- 0.5) and mice (4/10 mice had nodules > 5 mm diameter in the AD group while none in the AS group had such large nodules). In another experiment male Fischer 344 rats similarly initiated with DENA were exposed to either basal diet or basal diet containing 2% CA for 4 weeks coupled with PH performed at the end of the second week. This regimen was followed by 20 weeks of feeding basal diet to both groups. Rats given CA developed larger hepatic foci and nodules (0.84 +/- 0.56 mm3) compared to the control group, which was fed basal diet throughout the experiment (0.07 +/- 0.03 mm3). Further, both AD diet and dietary CA, like dietary OA, induced an increase in hepatic uridine nucleotides. Taken together, these results suggest that increased levels of endogenously synthesized OA, like exogenously supplied excess OA, can induce an imbalance in hepatic nucleotide pools and can exert a promoting effect on hepatocarcinogenesis.

One of the proposed mechanisms by which phenobarbital (PB) promotes hepatocarcinogenesis in the rat is by differential mitoinhibition. However, our earlier studies indicated that PB inhibited DNA synthesis in vitro in hepatocytes isolated from both surrounding non-nodular liver and hepatic nodules promoted by orotic acid (OA). Since nodules generated by one promoter need not necessarily be resistant to another promoter, the present study was undertaken to determine whether foci/nodules promoted by PB itself are resistant to the mitoinhibitory effects of PB. Accordingly, rats were initiated with diethylnitrosamine (DENA, 200 mg/kg i.p) and promoted with PB (0.07% of PB as its sodium salt) in their drinking water for 16 or 33 weeks. In vitro studies indicated that PB (3-5 mM) inhibited DNA synthesis induced by epidermal growth factor (EGF) in hepatocytes from surrounding non-nodular liver as well as from nodules promoted by PB for 33 weeks. In another experiment, initiated rats exposed to PB for 33 weeks were subjected to either two-thirds partial hepatectomy (PH) or sham hepatectomy. Hepatocytes were labelled with tritiated thymidine in vivo for 48 h. Autoradiographic analysis indicated that in the presence of PB, the hepatocytes from both foci/nodules and the surrounding non-nodular liver responded to PH to the same extent. In addition, they both responded to PH less efficiently as compared to the corresponding controls. Further, initiated rats exposed to PB for 16 weeks when subjected to PH and killed 4 weeks thereafter, the percentage area occupied by gamma-glutamyltranspeptidase-positive foci/nodules in the PB group increased, but to the same extent as in initiated control rats not exposed to PB. The above results raised an interesting possibility that the lack of resistance of the PB-promoted nodules to the mitoinhibitory effects of PB may be because the PB-promoted nodule does not express a resistant phenotype. To examine this aspect, the response of hepatocytes from 33 week PB-promoted nodules to the mitoinhibitory effects of OA was examined. The results indicated that OA (60-120 microM) inhibited EGF-induced DNA synthesis in hepatocytes isolated from both nodules as well as from surrounding non-nodular liver. These results suggest that PB is a mitoinhibitor but may not provide a strong differential growth advantage to foci/nodules in response to a proliferative stimulus. Further, the nodules promoted by PB do not appear to express the resistant phenotype, defined as being resistant to the mitoinhibitory effects of OA and PB.

It has been hypothesized that orotic acid (OA) promotes rat liver carcinogenesis by a differential mitoinhibitory mode. Consistent with this hypothesis, hepatic nodules are relatively resistant to OA-induced mitoinhibition. OA-induced mitoinhibition is dependent on the metabolism of OA to uridine nucleotides. The present studies investigate the uptake and metabolic pathway of OA, both in vivo and in vitro, as a possible basis for the resistance of hepatic nodules to OA-induced mitoinhibition. Rats bearing hepatic nodules exposed to 1% dietary OA exhibited increased levels of uridine nucleotides in the surrounding non-nodular liver (from 0.44 to 0.70 mg/g liver) but not in the hepatic nodules. Further, following administration of [3H]OA i.p., nodules have significantly lower levels of acid-soluble radioactivity compared to the non-nodular surrounding tissue. Furthermore, most of the acid-soluble radioactivity was present as uridine nucleotides, suggesting that the OA taken up was converted to uridine nucleotides. Similarly, hepatocytes from nodules in primary culture incubated with radiolabeled OA, have significantly lower levels (46-60%) of acid-soluble radioactivity. These results suggest that the decreased uptake of OA by hepatic nodules may be a factor contributing to the observed resistance of hepatic nodules to the mitoinhibitory effects of OA.

Previous work from this laboratory has revealed that a minimum of 10-20 weeks of continuous exposure to 1% dietary orotic acid (OA) is necessary for this regimen to exert a significant promoting effect on the carcinogenic process in rat liver. The present study investigates the effect of partial hepatectomy (PH), given during a short-term exposure (4 weeks) to OA, on the development of hepatocyte nodules (HN) and hepatocellular carcinoma (HCC) initiated by diethylnitrosamine (DEN). Male Fischer 344 rats (130-150 g) were given a single dose of DEN (200 mg/kg body wt i.p.). Starting a week later they were fed either a semisynthetic basal diet (BD) or the same diet containing 1% OA for 2 weeks; two-thirds PH was then performed followed by another 2 weeks of BD or OA diet respectively. At the end of this treatment some animals from both groups were killed while the rest were continued on BD and killed at 20 or 56 weeks thereafter. The results showed no difference between the two groups in the incidence of gamma-glutamyltransferase-positive foci when rats were killed at 2 weeks after PH. However, 4 week exposure to OA coupled with PH significantly enhanced the incidence of HN and HCC when this protocol was followed by 20 or 56 weeks of BD feeding respectively, leading to 63% incidence of HCC in the OA-fed group, while no HCC was observed in control animals. It is concluded that a type of stable or permanent change(s) ('imprinting' or 'memory effect') is induced in the initiated rat liver by this treatment, which imposes a promoting environment in the liver even after withdrawal of the promoter.

The present study was designed to determine the long-term effects of orotic acid (OA), a multi-organ tumor promoter, in rats not exposed to any carcinogen. Male Fischer 344 rats (130-150 g) were divided into two groups and given either a semisynthetic basal diet (BD) or the same diet containing 1% OA. Animals from both groups were killed after 1 or 2 years of treatment. Foci of placental glutathione-S-transferase (GST 7-7) positive hepatocytes were observed in the livers of both BD and OA fed rats killed after 1 year. However, they were more in number in animals receiving OA (156 +/- 21 versus 51 +/- 11/cm3). After 2 years, hepatic nodules were seen in almost all the animals given OA and in approximately 30% of the rats given BD. The nodules were of two main types: (i) a reddish-brown type, present in 85% of rats exposed to OA and in 27% of rats given BD, and (ii) a greyish-white type, found in 50% of animals fed OA and in none of the animals fed BD. These two types of lesions were also histologically different. Reddish-brown nodules were composed of slightly enlarged hepatocytes resembling normal surrounding tissue, while greyish-white nodules were similar in structure and are indistinguishable from hepatic nodules induced by genotoxic chemical carcinogens. The results are interpreted to suggest that the foci/nodules seen in OA-fed rats are due to a promoting effect of OA on spontaneously arising and/or diet-induced altered cells.

Our earlier studies indicated that orotic acid, a precursor for pyrimidine nucleotide biosynthesis, exerts a promoting effect on rat hepatocarcinogenesis. The present study was designed to determine the optimum conditions of exposure to orotic acid required for promotion of hepatocarcinogenesis in the initiated rats. The first series of experiments was designed to determine the optimum dose of orotic acid needed to exert its liver tumor promoting effect. Accordingly male Fischer rats were given diethylnitrosamine (200 mg/kg, i.p.) or 0.9% NaCl. One week later carcinogen-injected rats were divided into six groups and fed either basal diet or the same diet containing 0.1, 0.5, 1, 2 or 4% orotic acid. Rats given 0.9% NaCl were fed 4% orotic acid. Two-thirds partial hepatectomy was performed on all animals 10 weeks after starting on their respective diets, and all groups were killed 3 weeks thereafter. Analysis of gamma-glutamyltransferase-positive foci and nodules revealed that 0.5-1% orotic acid in the diet is sufficient to exert a significant promoting effect on the selective growth of initiated hepatocytes, while higher concentrations of orotic acid were only marginally more effective. No gamma-glutamyltransferase-positive foci were observed in animals given 4% orotic acid diet following saline injection. Using 1% orotic acid as the promoting regimen, in the next series, the minimum exposure time required for dietary orotic acid to promote liver carcinogenesis was determined. Male Fischer 344 rats were given i.p. either 1,2-dimethylhydrazine dihydrochloride (100 mg/kg) or 0.9% NaCl 18 h after 2/3 partial hepatectomy. After 1 week of recovery one group of rats was continued on a semisynthetic basal diet, while others were transferred to the same basal diet containing 1% orotic acid. Rats that were on the 1% orotic acid diet were progressively transferred to the basal diet after 5, 10, 20, 29 and 40 weeks of exposure. All rats were sacrificed 54 weeks after the beginning of the experiment. The results indicate that 100% of the initiated rats developed hepatic nodules whether or not they were exposed to an orotic acid-containing diet. However, the incidence of hepatocellular carcinoma was greatly increased in animals exposed to the orotic acid diet, with 42% incidence in initiated rats given orotic acid diet for 10 weeks and up to 75% in those exposed to this diet for 40 weeks. Further, promotion by orotic acid exhibited a high metastatic potential with 33-60% metastasis to the lungs.(ABSTRACT TRUNCATED AT 400 WORDS)

The present study was designed to determine whether a delay in the start of the promoting regimen after the administration of a carcinogen would influence the promoting efficacy of orotic acid on the development of hepatocellular carcinoma in rats. Male Fischer 344 rats weighing 130-150 g were injected with a single dose of diethylnitrosamine (200 mg/kg body wt i.p.) then divided into 3 groups: groups 1 and 2 were given semi-synthetic basal diet or the same diet containing 1% orotic acid (OA) respectively starting 1 week after the carcinogen; group 3 received the OA diet starting 5 weeks after the administration of diethylnitrosamine. Animals from these 3 groups were sacrificed after 25, 32, 42 and 60 weeks of being fed their respective diets. The results indicated that delaying the start of the OA diet after the carcinogen resulted in about a 50% decrease in the incidence of hepatic nodules and/or hepatocellular carcinomas at various time points during the experiment. This decrease in promoting efficacy of OA was not apparently explainable by lack of metabolic effects of OA, at least in terms of induction of nucleotide pool imbalance, a condition that appears to be important for OA to exert its tumor promoting effects.

One of the proposed mechanisms by which orotic acid (OA) promotes liver carcinogenesis is by differentially mito-inhibiting the normal hepatocytes while permitting the initiated ones to respond to growth stimuli to form foci/nodules. In an attempt to examine this hypothesis, the present study was designed to determine (i) whether OA inhibits DNA synthesis in normal hepatocytes in vivo, and (ii) whether hepatocytes from hepatic foci/nodules are relatively resistant to the mito-inhibitory effects of OA. The results of this study indicate that OA given i.p. as a tablet of 300 mg at the time of partial hepatectomy (PH) almost completely inhibited liver DNA synthesis. Three days later--a time period by which the implanted tablet disappeared--the hepatocytes resumed DNA synthesis. Exposure to OA results in an accumulation of uridine nucleotides and a decrease in adenosine nucleotides. Creation of such an imbalance in nucleotide pools appears to be important for OA to inhibit DNA synthesis. Adenine (a tablet of 300 mg), an agent that inhibits the metabolism of OA to uridine nucleotides, counteracted the mito-inhibitory effects of OA. To determine whether the hepatocytes in foci/nodules are resistant to the mito-inhibitory effects of OA, rats were initiated with diethylnitrosamine (DENA; 150 mg/kg) and promoted by the resistant-hepatocyte model. Fourteen weeks after the administration of DENA, the rats were subjected to PH in the presence of absence of OA (300 mg tablet). The results indicated that, in contrast to hepatocytes in normal or surrounding non-nodular liver, a subpopulation of hepatocyte foci/nodules appear to be relatively resistant to the mito-inhibitory effects of OA. These findings support the hypothesis that differential mito-inhibition is a possible component in the promoting effect of OA. However, whether this is the mechanism by which OA promotes liver carcinogenesis needs to be further investigated.

One of the many hypotheses put forward to explain the mechanism by which phenobarbital (PB) promotes hepatocarcinogenesis is by differential mitoinhibition of surrounding hepatocytes while allowing the initiated hepatocytes to respond to growth stimuli and form foci and nodules. Given the similarity in structures between PB and orotic acid (OA), another rat liver tumor promoter, the present investigation was designed to determine (i) whether PB, like OA, exerts its mitoinhibitory effect at a site beyond the growth factor receptor and receptor mediated early events; and (ii) whether PB exerts a differential mitoinhibitory effect by selectively inhibiting the non-initiated hepatocytes but not the initiated hepatocytes in vitro. Our studies demonstrate that, like OA, PB also inhibits DNA synthesis in hepatocytes from normal rat liver in a dose dependent manner with 80-90% at a dose of 6 mM. One target site may lie beyond the growth factor receptor mediated early events because PB inhibited DNA synthesis in hepatocytes primed with the growth factor 24 h earlier. Interestingly, PB inhibited DNA synthesis not only in hepatocytes from non-nodular surrounding liver but also in hepatocytes from persistent hepatic nodules initiated with 1,2-dimethylhydrazine and promoted with OA. Therefore, our results suggest that although PB is a mitoinhibitor of DNA synthesis in hepatocytes, it does not appear to create as strong a differential mitoinhibition between non-nodular surrounding and initiated hepatocytes as is evident in the resistant hepatocyte and OA models. These results raise the question whether differential mitoinhibition is the major contributing factor in the PB mediated rat liver tumor promotion.

D-Galactosamine is a known hepatotoxin which induces liver cell necrosis via depletion of UTP and other uridine nucleotides. Our previous work indicated that nodular hepatocytes have higher levels of total uridine nucleotides compared to normal liver, and in the present study we investigate the effect of galactosamine treatment on hepatocyte nodules and surrounding liver. Hepatic nodules were generated in male Wistar rats according to the Solt and Farber protocol. Six months after initiation animals received a single injection of D-galactosamine (500 mg/kg i.p.) and were then killed 1, 2, 4 or 7 days later. Histological analysis of liver revealed the presence of extensive liver cell necrosis in normal tissue 1 and 2 days after galactosamine treatment. However, very little or no necrosis was detectable inside hepatic nodules at any time point, indicating that these focal areas are resistant to the cytotoxic effect of galactosamine. This type of resistance could be the expression of a new component in the resistant phenotype of hepatic nodules.

Our earlier studies had demonstrated that inhibition of DNA methylation following carcinogen treatment potentiated initiation of the carcinogenic process in the rat liver system. The hepatic nodules developed by initiation-promotion protocols showed a characteristic hypomethylation in the cell-cycle-related genes c-fos, c-myc and c-Ha-ras. In the present study we have found that the gene for beta-hydroxy-beta-methyl glutaryl coenzyme A reductase, a major rate-limiting enzyme in the biogenesis of mevalonate, is also hypomethylated at both CCGG and GCGC sites and expressed in hepatic nodules. This gene, however, did not exhibit hypomethylation in CCGG sequences in non-nodular surrounding liver, livers from rats subjected to two-thirds partial hepatectomy, or exposed to initiator alone (1,2-dimethylhydrazine given 18 h after partial hepatectomy) or to diets containing 1% orotic acid alone (promoting regimen). The activity of the enzyme and mevalonate formation are positively correlated with DNA synthesis and cell proliferation--two key components of the carcinogenic process. Taken together, the results suggest that hypomethylation of specific genes occurs in the carcinogenic process and this altered pattern of DNA methylation may play a role in the growth of the nodules.

Experiments were designed to determine the efficacy of different types of liver cell proliferative stimuli given during exposure to several liver tumor-promoting regimens, on the formation of foci of enzyme-altered hepatocytes. Male Wistar rats were initiated with diethylnitrosamine (150 mg/kg body wt). After a 2 week recovery period animals were subjected to promoting regimens, the resistant hepatocyte model, the phenobarbital model and the orotic acid model. While the rats were on these regimens they were given liver cell proliferative stimulus, either a compensatory type (two-thirds partial hepatectomy or a necrogenic dose of carbon tetrachloride) or a direct hyperplastic stimulus such as that induced by the primary mitogen, lead nitrate. Initiated cells so promoted by these regimens were monitored as foci of enzyme-altered hepatocytes positive for gamma-glutamyltransferase and placental glutathione S-transferase or deficient for adenosine triphosphatase. While carbon tetrachloride and partial hepatectomy-induced compensatory regeneration stimulated the promoting ability of the regimens used, direct hyperplasia could not stimulate the formation of foci and/or nodules from initiated hepatocytes. Evaluation of thymidine incorporation indicated that there was no significant difference in the extent of DNA synthesis in both the proliferative stimuli irrespective of the promoting procedure used.

Hepatocellular carcinomas from rats of different strains, subjected to a variety of carcinogenic treatment regimens in different laboratories (initiation by diethylnitrosamine or dimethylhydrazine, promotion by phenobarbital, 2-acetylaminofluorene, nafenopin, orotic acid or deoxycholic acid, growth stimulation by partial hepatectomy or necrogenic CCl4 treatment), were all found to be predominantly diploid by flow cytometric analysis, in contrast to normal liver tissue in which polyploid nuclei were predominant. A switch from polyploidization to diploid growth would thus seem to be a common property of malignant liver tumours. Benign neoplastic liver nodules were likewise predominantly diploid, with the exception of nodules induced by long-term deoxycholic acid treatment in Fischer rats. In addition to containing a majority of polyploid cells, the latter nodules failed to progress to the carcinoma stage.

Orotic acid (OA), a promoter of liver carcinogenesis, inhibited proliferation of primary hepatocytes in culture as monitored by labelling index, mitotic index and total DNA content. The mitoinhibitory effect of OA was seen even in the presence of a strong mitogen such as epidermal growth factor (EGF). The growth inhibitory effect of OA was not due to cell killing. Upon exposure to OA the hepatocytes exhibited an increase in the ratio of uridine nucleotides to adenosine nucleotides, and as this ratio increased the response of hepatocytes to proliferate in the presence or absence of EGF decreased. Washing the hepatocytes free of added OA resulted in a gradual decrease in the ratio of uridine nucleotides to adenosine nucleotides, paralleled by an increase in hepatocytic proliferation. Adenine, an agent that inhibits the metabolism of OA to uridine nucleotides, not only inhibited the increase in the ratio of uridine nucleotides to adenosine nucleotides but also counteracted the OA-induced mitoinhibitory effect. These results, together with our earlier observations, suggest that an imbalance in nucleotide pools composed of an increase in uridine nucleotides and a decrease in adenosine nucleotides appears to be important for OA-induced mitoinhibition.

Experiments were designed to investigate the expression of three cell-cycle-dependent proto-oncogenes in response to two different types of proliferative stimuli: compensatory cell proliferation after partial hepatectomy (PH) or CCl4 and liver hyperplasia induced by the mitogens ethylene dibromide (EDB) and cyproterone acetate (CPA). Steady-state levels of messenger RNAs for c-fos and c-myc were found to be elevated after PH or CCl4 with a maximum increase between 0.5 and 2 h for c-fos and at 2-3 h for c-myc and a rapid decline after 3 h. However, when liver cell proliferation was induced by mitogens, no increase in the expression of c-fos mRNA was observed with both EDB or CPA during the first 24 h. In addition, elevated expression of c-myc was found only in liver hyperplasia induced by EDB, but not with CPA. While the expression of c-myc mRNA and c-fos mRNA was different in the two types of proliferative stimuli, that of c-Ha-ras and c-Ki-ras was similar in all the experimental groups. Cell proliferation monitored by means of incorporation of labelled thymidine into DNA or mitotic index at 24 h following PH, EDB and CPA occurred at a similar extent in all the experimental groups. Our data indicate that the transient and sequential expression of cell-cycle-related genes may vary in response to proliferative stimuli of different nature and suggest that increased expression of cell-cycle-related genes may not be a necessary prerequisite for the entry of the cells into the cell cycle.

Our earlier studies have revealed that direct hyperplasia induced by liver mitogens such as lead nitrate, ethylene dibromide, nafenopin and cyproterone acetate, unlike compensatory cell proliferation induced by partial hepatectomy and CCl4, does not support the formation of enzyme-altered islands induced by chemical carcinogens in the liver. In the previous studies carcinogens were given at the peak of DNA synthesis induced by the liver mitogens. If the mitogens have altered the sensitive phase of the hepatocyte to the carcinogenic attack, administering the carcinogen at one time point following the mitogenic stimulus might have missed the sensitive phase. In order to overcome this possibility in the present study male Wistar rats weighing 200-250 g were given N-methyl-N-nitrosourea (MNU; 60 mg/kg, i.p.) at three points representing G1, S and G2/M phases of the cell cycle following different types of liver cell proliferative stimuli. In another experiment MNU (60 mg/kg, i.p.) and diethylnitrosamine (15 mg/kg, i.p.) were given prior to the administration of proliferative stimuli. The initiated hepatocytes were also assayed following promotion by two different promoting regimens, namely phenobarbital and the resistant-hepatocyte model. Further, the initiated hepatocytes were monitored not only by using the appearance of islands of enzyme-altered hepatocytes but also using the incidence of hepatocellular carcinoma. The results of this study clearly revealed that irrespective of the protocol used, only the compensatory liver cell proliferation but not the mitogen-induced direct hyperplasia supported the formation and the growth of enzyme-altered islands in the liver induced by chemical carcinogens.

Feeding male Fischer F-344 rats for 5 weeks a diet containing 1% orotic acid, a precursor for pyrimidine nucleotide biosynthesis, resulted in an increased incidence of gamma-glutamyltransferase (EC 2.3.2.2) positive foci induced by chemical carcinogens including 1,2-dimethylhydrazine, diethylnitrosamine, benzo[a]pyrene, and aflatoxin B1. This unique effect of orotic acid can be accentuated by supplying a liver cell proliferative stimulus. The enzyme altered hepatocytes have a higher labelling index (4.4%) compared with that of the hepatocytes in the surrounding liver (0.26%). The effect of orotic acid on the increased incidence of foci cannot be attributed to either the induction of liver cell proliferation or the imposition of a preferential inhibitory effect on the proliferation of normal hepatocytes while permitting the carcinogen-modified hepatocytes to respond to an endogenous or exogenous liver cell proliferative stimulus and grow to form foci. Orotic acid also did not behave like some of the promoters of liver carcinogenesis such as phenobarbital and polychlorinated biphenyls in that it did not induce either the phase I or phase II components of hepatic drug metabolizing enzyme systems. Some of the possible mechanisms by which orotic acid enhances the incidence of gamma-glutamyltransferase positive foci by carcinogens are discussed.

Orotic acid, a precursor of pyrimidine nucleotide biosynthesis and a promoter for liver carcinogenesis, when fed at 1% level in a diet for 5 weeks resulted in liver DNA damage. The damage can be monitored as alkali-labile lesions using alkaline sucrose gradients as well as alkaline elution technique. Furthermore, the induced DNA damage persists for up to three weeks after withdrawal of the orotic acid diet. The fact that several skin-tumour promoters also induce DNA damage raises the question whether DNA damage is a component in tumour promotion.

To test the validity of the hypothesis that hypomethylation of DNA plays an important role in the initiation of carcinogenic process, 5-azacytidine (5-AzC) (10 mg/kg), an inhibitor of DNA methylation, was given to rats during the phase of repair synthesis induced by the three carcinogens, benzo[a]-pyrene (200 mg/kg), N-methyl-N-nitrosourea (60 mg/kg) and 1,2-dimethylhydrazine (1,2-DMH) (100 mg/kg). The initiated hepatocytes in the liver were assayed as the gamma-glutamyltransferase (gamma-GT) positive foci formed following a 2-week selection regimen consisting of dietary 0.02% 2-acetylaminofluorene coupled with a necrogenic dose of CCl4. The results obtained indicate that with all three carcinogens, administration of 5-AzC during repair synthesis increased the incidence of initiated hepatocytes, for example 10-20 foci/cm2 in 5-AzC and carcinogen-treated rats compared with 3-5 foci/cm2 in rats treated with carcinogen only. Administration of [3H]-5-azadeoxycytidine during the repair synthesis induced by 1,2-DMH further showed that 0.019 mol % of cytosine residues in DNA were substituted by the analogue, indicating that incorporation of 5-AzC occurs during repair synthesis. In the absence of the carcinogen, 5-AzC given after a two thirds partial hepatectomy, when its incorporation should be maximum, failed to induce any gamma-GT positive foci. The results suggest that hypomethylation of DNA per se may not be sufficient for initiation. Perhaps two events might be necessary for initiation, the first caused by the carcinogen and a second involving hypomethylation of DNA.

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

Orotic acid has been shown to promote carcinogenesis in the liver and the intestine of the rat. In an attempt to determine whether orotic acid promotes liver carcinogenesis by creating differential mitoinhibition, experiments were designed to study the effect of orotic acid on the labeling index of isolated hepatocytes in response to epidermal growth factor. The results indicated that orotic acid added in vitro inhibited epidermal-growth-factor-induced labeling index of isolated hepatocytes. In addition, isolated hepatocytes from rats exposed to orotic acid under promoting conditions also exhibited a decreased response to epidermal growth factor. These data suggest that orotic acid may exert its promoting effect by differentially inhibiting the response of normal hepatocytes to one or more endogenous growth stimuli while permitting the initiated hepatocytes to respond to such stimuli and grow to form hepatic nodules.

Hepatocyte nodules in the rat exhibit a unique biochemical pattern which is characterized by a decrease in Phase I and an increase in Phase II components of the drug-metabolizing system. The present study was designed to determine whether this biochemical pattern is unique for rat hepatocyte nodules or is a property of the liver cell, but expressed only when the liver cell is perturbed. The results obtained indicate that lead nitrate (5 or 10 mumol/100 g body wt), an inducer of liver cell proliferation, caused a decrease in Phase I components such as microsomal cytochromes P-450 and in the activity of aminopyrine N-demethylase, while it caused an increase in Phase II components such as glutathione, and in the activities of glutathione-S-transferase and DT-diaphorase in rat liver. Of particular interest was the finding in liver cytosol of lead-treated rats of an increased content of a polypeptide which cross-reacts with the anti-rat placental form of glutathione-S-transferase. Recently, it has been shown that rat hepatocyte nodules exhibited an increased content of the placental form of glutathione-S-transferase. Thus, the results suggest that some chemicals, such as lead nitrate, can induce in rat liver a biochemical pattern similar in certain respects to that exhibited by hepatic nodules. These chemicals may be used as model compounds to understand the molecular mechanism(s) underlying the induction of new and unique biochemical machinery seen in hepatic nodules.