Inhibition of dimethylnitrosamine-induced strand breaks in liver DNA and liver cell necrosis by diethyldithiocarbamate.

Diethyldithiocarbamate (DEDTC) prevented dimethylnitrosamine (DMN)-induced strand breaks in liver DNA and liver cell necrosis in male Wistar rats. In contrast, DEDTC did not inhibit the fragmentation of liver DNA caused by several other chemical carcinogens (N-hydroxy-2-acetylaminofluorene, 3-hydroxyxanthine, aflatoxin B1, N-acetoxy-2-acetylaminofluorene, methyl methanesulfonate, methylnitrosourea, and methylazoxy-methanol acetate), whether or not they required metabolic activation. Aminoacetonitrile exerted an action similar to that of DEDTC. The inhibitory effect was transitory, lasting at least for 4 hours, and protection for longer than 4 hours required multiple administrations of DEDTC. DEDTC also inhibited the serum clearance of DMN, methylation of liver DNA, and oxidative demethylation of DMN in the in vitro hepatic microsomal system prepared from either male Wistar rats or from hamsters. Interference of the metabolism of DMN appeared to be the mechanism by which DEDTC arrested DMN-induced biochemical and biologic effects.

Requirement of cell proliferation for the initiation of liver carcinogenesis as assayed by three different procedures.

Experiments were designed to determine the role of cell proliferation in the initiation of liver carcinogenesis induced by chemicals. To investigate this, two methylating carcinogens, N-methyl-N-nitrosourea and 1,2-dimethylhydrazine, were used as the initiating carcinogens. The initiated hepatocytes were monitored by selectively stimulating them to grow into focal islands of presumptive preneoplastic hepatocytes. The experimental approach in brief consisted of the following. Rats received a nonnecrogenic dose of the carcinogen; at a time period when the carcinogen could no longer be detected in the system, they were subjected to either partial or sham hepatectomy. The initiated cell thus formed were selectively stimulated to grow into foci of preneoplastic hepatocytes using three different selection regimens: (a) feeding a diet containing 0.02% 2-acetylaminofluorene plus one administration of carbon tetrachloride (2 ml/kg body weight) intragastrically; (b) feeding a diet containing 0.05% phenobarbital; and (c) feeding a choline-deficient diet. The foci were quantitated by staining them for the presence of gamma-glutamyltransferase. The results obtained indicate that irrespective of the type of selection procedure used foci of preneoplastic hepatocytes were seen only in rats that received the carcinogen coupled with a cell-proliferative stimulus such as partial hepatectomy. Very few or no foci were seen in rats that received the carcinogen plus sham hepatectomy. These results suggest that cell proliferation plays an important role in the initiation of liver carcinogenesis by chemicals.

Role of acute hepatic necrosis in the induction of early steps in liver carcinogenesis by diethylnitrosamine.

Experiments were performed to assess the role of liver cell necrosis in the induction of early steps in liver carcinogenesis with diethylnitrosamine, as measured by the appearance of foci of resistant hepatocytes that stain for gamma-glutamyl transpeptidase and that are presumptive preneoplastic lesions in the rat. With the use of a necrogenic dose of diethylnitrosamine and an assay for the carcinogen-induced early stages or resistant hepatocytes, the number of enzyme-altered foci was decreased to a major extent (up to 62%) by posttreatment with diethyldithiocarbamate, a derivative of disulfiram. Such posttreatment decreased to a large degree (78%) the cumulative labeling index of hepatocytes following an initial exposure to diethylnitrosamine. The performance of partial hepatectomy up to 68 hr after such posttreatment restored the level of induction of the resistant hepatocytes. Nonnecrogenic doses of diethylnitrosamine or dimethylnitrosamine induced virtually no foci of resistant hepatocytes but did so when coupled with cell proliferation. These results establish clearly an important role for liver cell necrosis in the production of early steps in liver carcinogenesis in one model. The mechanism for this effect appears to be by the induction of compensatory liver cell proliferation.

Sequential alterations in growth control and cell dynamics of rat hepatocytes in early precancerous steps in hepatocarcinogenesis.

This set of experiments is the second of a series designed to explore alterations in cell dynamics and growth control of new populations of hepatocytes that appear to play a role in the carcinogenic process induced in the liver by chemical carcinogens. This is part of an ongoing study of the biochemical and molecular basis for cancer development. A rat model for hepatocarcinogenesis, the resistant hepatocyte model, was chosen with its synchrony of several steps in the process. Carcinogenesis was initiated by the administration of a single necrogenic dose of diethylnitrosamine. Resistant hepatocytes so induced were stimulated to proliferate rapidly to form nodules by a mitogenic stimulus in the presence of a brief exposure to dietary 2-acetylaminofluorene sufficient to inhibit the proliferation of the majority of uninitiated hepatocytes, the nonresistant population. A small subset of these hepatocyte nodules, the persistent nodules, was examined at 2, 4, and 6 mo postinitiation. Duration of phases of the cell cycle, growth fraction, doubling time, cell death, and cell loss and the responses and subsequent recovery after the application of a strong mitogenic stimulus, partial hepatectomy, were measured. The first precancerous hepatocyte nodule, at 2 mo, showed a "normal" duration of phases of the cell cycle. The growth fractions were about 4,4, and 8% at 2, 4, and 6 mo, respectively, as compared to 0.4% in the surrounding hepatocytes. Accompanying the increased growth fractions were considerable levels of cell loss, measuring about 3% at 2 mo and 7% at 6 mo. At 6 mo, the hepatocyte nodule population, unlike the hepatocytes in the surrounding liver, shows a failure to return to its base-line level after stimulation of cell proliferation by partial hepatectomy. The results of this study have identified two new steps in the early precancerous phase of hepatocarcinogenesis relating to alterations in the control of cell proliferation and are consistent with the hypothesis that new and evolving cell populations may play an important role in the step-by-step carcinogenic process. These new populations appear to acquire alterations in growth control in a seriatim fashion, with retention of some "normal" properties.

Persistence of DNA damage during development of liver angiosarcoma in rats fed dimethylnitrosamine.

Chronic feeding of a diet containing dimethylnitrosamine (50 ppm) to rats resulted in liver DNA damage monitored as slow sedimentation of the DNA compared to that of the control in alkaline sucrose gradients. The damage in rat liver DNA could be seen within 2 days after beginning the feeding of diets containing the carcinogen and was progressive with the time of feeding, up to 8 weeks. Extended feeding up to 15 or 31 weeks did not result in a proportionate increase in the damage to the DNA. The DNA damage observed at 8 weeks persisted until 31 weeks, at which time liver angiosarcoma was present. Despite the fact that the DNA damage induced by dimethylnitrosamine appears to involve the bulk of the liver DNA, the tumors developed were entirely from vascular endothelium. The implication of these results in the initiation of carcinogenesis is discussed.

Nonrandom nature of in vivo methylation of dimethylnitrosamine and the subsequent removal of methylated products from rat liver chromatin DNA.

This investigation was designed to study whether methylation of liver chromatin DNA by dimethylnitrosamine (DMN) and the subsequent in vivo removal of DNA-bound methylated products are random. Liver chromatin DNA was fractionated into nuclease-digestible and nondigestible material 4 hr following the administration of [3H]DMN (0.5 mg/250 muCi/100 g body weight). Digestion of such methylated liver chromatin with pancreatic DNase I or micrococcal nuclease and analysis of nuclease-digested acid-soluble products revealed a discrepancy between the radioactivity released (72%) and the nucleotides released (50%) as measured by the absorbance at 260 nm. This discrepancy disappeared, and the rate and extent of release of both the radioactivity and the absorbance at 260 nm were identical when the total purified DNA isolated from methylated chromatin was used as the substrate instead of chromatin DNA in the nuclease reaction. These results, together with the fact that guanine contents of the DNA of the two fractions of the chromatin isolated by nuclease digestion were identical, suggest that methylation of the nuclease-accessible region of hepatic chromatin DNA is relatively greater than that of the inaccessible region. The study of the removal of methylated products in the accessible region of the chromatin DNA further reveals that, of the methylated products present at 4 hr, 62% is lost by 3 days, 87% is lost by 1 week and 94% is lost by 2 weeks. However, loss from the nuclease-inaccessible region of chromatin DNA is only 27% by 3 days, 49% by 1 week, and 86% by 2 weeks, thereby suggesting that the removal of methylated products from this region of chromatin DNA is relatively slower compared with that from the nuclease-accessible region of chromatin-DNA. The results of this study thus indicated (a) an increased methylation and faster rate of removal of DMN-induced methylated products in nuclease-accessible regions of chromatin DNA and (b) decreased methylation and slower rate of removal from the nuclease-inaccessible regions of chromatin DNA. It is concluded that the distribution and removal of DMN-induced methylated products in liver chromatin DNA is nonrandom as measured by this technique.

Promotion by orotic acid of liver carcinogenesis in rats initiated by 1,2-dimethylhydrazine.

Our earlier experiments revealed that orotic acid, a precursor for pyrimidine nucleotides, selectively stimulated the growth of carcinogen-modified liver cells to grow into enzyme-altered hepatocytes (Cancer Lett., 16: 191-196, 1982). The present study was designed to determine whether prolonged feeding of orotic acid will result in hepatocellular carcinoma in initiated rats. Accordingly, groups of rats were given i.p. either 1,2-dimethylhydrazine dihydrochloride (100 mg/kg) or an equivalent volume of 0.9% sodium chloride solution 18 hr after two-thirds partial hepatectomy. After 1 week of recovery, they were continued on either the basal diet or the basal diet containing 1% orotic acid for 10 to 13 months. Some groups of rats, in addition, received a single necrogenic dose of CCl4 8 weeks following exposure to orotic acid diet. The results obtained indicated that 87.5% of initiated rats exposed to orotic acid developed hepatocellular carcinomas in 10 months and 100% in 13 months. Initiated rats exposed to orotic acid diet coupled with a single administration of CCl4 developed 100% hepatocellular carcinoma by 10 months. In contrast, the incidence of hepatocellular carcinoma in initiated rats fed basal diet alone for 13 months was 37.5%, while, in those that received CCl4 in addition, the incidence was 25% in 10 months. Interestingly, a significant number of liver cancers (29 to 36%) in the orotic acid-fed group metastasized to lungs, whereas none of the liver cancers in rats exposed to basal diet metastasized.(ABSTRACT TRUNCATED AT 250 WORDS)

A common biochemical pattern in preneoplastic hepatocyte nodules generated in four different models in the rat.

Hepatocyte nodules, structures consistently seen in every model of liver carcinogenesis well before the first appearance of cancer, were examined with respect to some Phase I and Phase II components considered to be important in the metabolism of carcinogens and other xenobiotics. Phase I components are those related to the metabolism of xenobiotics and include microsomal cytochromes P-450 and mixed-function oxygenase activities. Phase II components are those related to the conjugation and detoxification reactions of xenobiotics and their metabolites and include glutathione S-transferases and glutathione. Nodules were induced by the resistant hepatocyte, choline-deficient, methionine-low diet, phenobarbital and orotic acid models of liver carcinogenesis. Also, nodules generated by the resistant hepatocyte model were examined after transplantation to the spleen of syngeneic animals. The hepatocyte nodules show a common biochemical pattern, consisting of decreased microsomal cytochromes P-450, cytochrome b5, and aminopyrine N-demethylase activity and increased glutathione and gamma-glutamyltransferase in whole homogenates and glutathione S-transferase activity in the cytosol. This similarity, appropriate to a resistance phenotype, adds additional support for the hypothesis that hepatocyte nodules may be a common step in liver carcinogenesis in several different models.

Persistence of resistant putative preneoplastic hepatocytes induced by N-nitrosodiethylamine or N-methyl-N-nitrosourea.

The administration of either N-nitrosodiethylamine (diethyl-nitrosamine) to intact male F-344 rats or N-methyl-N-nitrosourea to similar animals 18 hr after partial hepatectomy results in the induction of altered resistant hepatocytes that persist for up to 36 weeks with no perceptible decrease in their number. The criterion for resistance was the ability to proliferate rapidly and to develop into foci or nodules when exposed to a level of dietary 2-acetylaminofluorene that inhibits the proliferation of the vast majority of hepatocytes when liver cell proliferation is stimulated by surgical or chemical partial hepatectomy. Since this selection procedure, when coupled with a single dose of diethylnitrosamine, is associated with a high incidence of liver cancer as compared to appropriate controls, and since similar foci and nodules were shown previously to be one site of origin for hepatocellular carcinoma in this model, the induction of resistant hepatocytes is interpreted as initiation. Thus, these results suggest that initiation of liver carcinogenesis in this model is irreversible, at least for a period of 36 weeks.

Effects of delays in the cell cycle on the induction of preneoplastic and neoplastic lesions in rat liver by 1,2-dimethylhydrazine.

This study was designed to explore further the relationship between cell proliferation and the induction of early putative preneoplastic lesions by carcinogens. Rats were given a non-necrogenic dose of 1,2-dimethylhydrazine 24 hr before being subjected to partial hepatectomy. Beginning 4 hr later, hydrocortisone was injected 10 times at 4-hr intervals to delay progression through the cell cycle, including inhibition of DNA synthesis by at least 85% for about 40 hr. At the appropriate time thereafter, the putative preneoplastic hepatocytes were selectively stimulated to grow in vivo into gamma-glutamyltransferase-positive focal lesions. Animals given hydrocortisone showed a large decrease (71%) in the number of gamma-glutamyltransferase-positive foci. In contrast, when hydrocortisone was given at 6 days after partial hepatectomy, no inhibition in the induction of hepatic lesions was observed. In the next experiments, rats were treated with 1,2-dimethylhydrazine and were subjected to partial hepatectomy at 12, 24, or 48 hr or 1 week thereafter. A significant number of gamma-glutamyltransferase-positive foci was found when partial hepatectomy was performed at 12 or 24 hr but far fewer were found when the operative partial hepatectomy was delayed to 48 hr or 1 week later. Similarly, in long-term experiments, six of 14 animals developed primary hepatocellular carcinoma 13 months after the time of injection of 1,2-dimethylhydrazine when partial hepatectomy was performed at 12 hr, while none of the animals developed liver cancer when the operation was performed at 48 hr. These results imply that the majority of biochemical lesions induced by 1,2-dimethylhydrazine that are relevant to the induction of liver preneoplasia and neoplasia are short-lived and that their persistence is associated with some cellular activity closely related to the cell cycle.

Inability of mitogen-induced liver hyperplasia to support the induction of enzyme-altered islands induced by liver carcinogens.

Experiments were designed to determine whether liver cell proliferation induced by direct mitogens is as effective as compensatory cell proliferation consequent to previous cell loss, in supporting the growth of enzyme-altered islands in the liver induced by chemical carcinogens. Male Wistar rats were given injections of a single nonnecrogenic dose of N-methyl-N-nitrosourea or benzo(a)pyrene during the S phase following the administration of four different liver mitogens, namely, lead nitrate, ethylene dibromide, nafenopin, and cyproterone acetate, or during compensatory cell proliferation following partial hepatectomy or a necrogenic dose of CCl4. The carcinogen-altered hepatocytes were monitored as gamma-glutamyltransferase- or placental glutathione S-transferase-positive foci using a 2-wk promoting regimen consisting of 0.03% 2-acetylaminofluorene coupled with a necrogenic dose of CCl4. The results indicate that, unlike compensatory cell proliferation induced by partial hepatectomy or CCl4, the mitogen-induced cell proliferation did not result in a significant number of enzyme-altered islands, despite the fact that the extent of cell proliferation at the time of carcinogen administration, as monitored by the examination of labeled cells, is similar with both types of proliferative stimuli.

Resistance to hepatotoxins acquired by hepatocytes during liver regeneration.

The appearance of resistance to a number of hepatotoxins in primary cultures of hepatocytes prepared at various time intervals up to 2 weeks after partial hepatectomy is the major focus in this study. Resistance to the cytocidal effect of aflatoxin B1, 2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, methotrexate, or methyl methanesulfonate shows a progressive increase until 48 hr and then returns to the resting level of susceptibility by 2 weeks. The genesis of mutagens from 2-acetylaminofluorene and aflatoxin B1 by S-9 liver fractions shows a decrease from and return to control values after partial hepatectomy that parallels the resistance. The levels of total cellular cytochromes P-450 also decrease following partial hepatectomy and remain from 28 to 36% less than those of controls for at least 1 week. The glutathione and total soluble sulfhydryl ("glutathione") content increase following partial hepatectomy, and the pattern is consistent with a partial role for glutathione in the resistance phenomenon as it relates to 2-acetylaminofluorene. The possible relationship between resistance to the cytocidal effects in vitro and the resistance to inhibition of cell proliferation in vivo during liver carcinogenesis is discussed.

Physicochemical alterations in the conformation of rat liver chromatin induced by carcinogens in vivo.

Administration of methylating carcinogens such as methyl methanesulfonate (120 mg/kg), dimethylnitrosamine (5 mg/kg), or methylnitrosourea (80 mg/kg) to rats resulted in an increased ellipticity in circular dichroism spectra and in an enhanced ability to bind ethidium bromide in the liver chromatin. Although shearing of the chromatin preparations increased both the ellipticity and number of binding sites for ethidium bromide, the carcinogen-induced effects were noticeable whether or not chromatin was sheared. Although the doses of the 3 carcinogens used in these studies are equivalent in their ability to induce strand breaks in liver DNA at 4 hr, their effects on the induction of conformational changes in liver chromatin are different. For example, methyl methanesulfonate induced the minimum conformational changes in liver chromatin at 4 hr, whereas methylnitrosourea induced the maximum changes at 4 hr. Methyl methanesulfonate and dimethylnitrosamine, on the other hand, induced maximum changes at 3 days. The conformational changes induced by methyl methanesulfonate and methylnitrosourea, and not by dimethylnitrosamine, tend to be repaired by 14 days.

Orotic acid, nucleotide-pool imbalance, and liver-tumor promotion: a possible mechanism for the mitoinhibitory effects of orotic acid in isolated rat hepatocytes.

This study was designed to determine the possible mechanism by which orotic acid exerts its mitoinhibitory effect on rat hepatocytes in primary culture. Orotic acid inhibited, dose-dependently DNA synthesis in hepatocytes induced by epidermal growth factor, transforming growth factor alpha, hepatocyte growth factor, acidic fibroblast growth factor, or plasma from rats exposed to various liver cell-proliferative stimuli, such as two-thirds partial hepatectomy, lead nitrate, cyproterone acetate, ethylene dibromide, or a diet deficient in choline. Further, orotic acid inhibited DNA synthesis even when added 24 h after the hepatocytes were primed with transforming growth factor alpha. Taken together, these results suggested that the target site may not be at the level of the growth-factor receptor and receptor-mediated early events. In a preliminary experiment, orotic acid inhibited the expression of the ribonucleoside diphosphate reductase gene. Exposure to orotic acid results in an imbalance in nucleotide pools characterized by an increase in uridine nucleotides and a decrease in adenosine nucleotides. It is hypothesized that this imbalance in nucleotide pools inhibits the expression of the ribonucleoside diphosphate reductase gene and, therefore, is a likely target for the mitoinhibitory effect of orotic acid.

Induction of hepatic neoplastic lesions in mice with a single dose of hycanthone methanesulfonate after partial hepatectomy.

Experiments were designed to determine whether hycanthone methanesulfonate (1-([2-(diethylamino)ethyl]amino)-4-(hydroxymethyl)thioxanthen-9-one monomethanesulfonate), an antischistosomal drug, and its analog, IA-4-N-oxide (8-chloro-2-[2-(diethylamino)ethyl]-2H-[1]benzothiopyrano[4,3,2-cd]indazole 5-methanol monomethanesulfonate), will induce neoplastic lesions in the livers of mice not infected with Schistosoma mansoni. All the mice received a single i.m. injection of hycanthone methanesulfonate (76 mg/kg), IA-4-N-oxide (80 mg/kg), or an equivalent volume of the solvent, 0.9% NaCl solution, 42 hr after partial hepatectomy. Of the mice receiving hycanthone methanesulfonate and living 200 days or longer, hepatocellular carcinoma was seen in 11.5% and liver sarcoma was seen in 4.2%. This type of malignant neoplasm was not seen in the animals receiving either IA-4-N-oxide or 0.9% NaCl solution. In addition, mice receiving hycanthone methanesulfonate showed a significantly higher incidence of both type 1 (43% compared to 21% in controls) and type 2 (21% compared to 12% in controls) hepatocyte neoplasms. Mice receiving IA-4-N-oxide showed no increased incidence of neoplasms.

Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques.

The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.

Butyrate mediates Caco-2 cell apoptosis via up-regulation of pro-apoptotic BAK and inducing caspase-3 mediated cleavage of poly-(ADP-ribose) polymerase (PARP).

Butyrate exerts potent anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. Using the Caco-2 cell line, a well established model of colon cancer cells, our data show that butyrate induced apoptosis (maximum 79%) is mediated via activation of the caspase-cascade. A key event was the proteolytic activation of caspase-3, triggering degradation of poly-(ADP-ribose) polymerase (PARP). Inactivation of caspase-3 with the tetrapeptide zDEVD-FMK completely inhibited the apoptotic response to butyrate. In parallel, butyrate potently up-regulated the expression of the pro-apoptotic protein bak, without changing Caco-2 cell bcl-2 expression. Butyrate-induced Caco-2 cell apoptosis was completely blocked by the addition of cycloheximide, indicating the necessity of protein synthesis. However, when this inhibitor was added at a time point where bak expression was already enhanced (12 - 16 h after butyrate stimulation), it failed to protect Caco-2 cells against apoptosis. Taken together, these data provide evidence that the molecular events involved in butyrate induced colon cancer cell apoptosis include the caspase-cascade and the mitochondrial bcl-pathway.

Studies on the effect of dietary orotic acid on mouse liver carcinogenesis induced by diethylnitrosamine.

The present study was designed to determine whether orotic acid, a liver tumor promoter in the rat, also promotes liver carcinogenesis in the mouse. Eight-week-old male BALB/c mice were initiated with diethylnitrosamine (90 mg/kg i.p.). One week later they were divided into 2 groups and given either a basal diet or the basal diet containing 1% orotic acid (OA). They were killed at 6 or 10 months after the administration of the carcinogen. At 6 months, no nodular lesions were seen in mice whether or not they were exposed to OA. However by 10 months 100% of mice in both groups developed hepatic nodules. OA neither shortened the latent period for the appearance of the nodular lesions not did it increase the size of the nodules. Although BALB/c mice exhibited an increase in uridine nucleotides and a decrease in adenosine nucleotides in the liver upon exposure to OA, the magnitude of the change was less compared with that seen in the rat liver. The resistance of BALB/c mouse to the tumor-promoting effects of OA may reflect in part the resistance of the mouse to OA-induced nucleotide pool imbalance.

Sequential histopathological analysis of hepatocarcinogenesis in rats during promotion with orotic acid.

In the present study, sequential histopathological changes during hepatocarcinogenesis promoted by orotic acid were examined. Male Fischer 344 rats were given 1,2-dimethylhydrazine.2HCl (100 mg/kg, i.p.) 18 h after 2/3 partial hepatectomy. After 1 week of recovery, they were divided into 2 groups; group 1 was continued on a semisynthetic basal diet while the group 2 received the basal diet containing 1% orotic acid. Rats were sacrificed after 5, 10, 20, 29, 40 and 53 weeks of promotion. Histopathological analysis indicated that emergence of hepatocellular carcinomas was preceded first by foci of morphologically and histochemically altered hepatocytes and then by the appearance of hepatocyte nodules. Clear cell foci, eosinophilic ground glass foci and gamma-glutamyltransferase positive foci were detectable after 5 weeks in initiated rats fed orotic acid. Hepatocyte nodules developed in 56% of the rats after 20 weeks of promotion, while the first hepatocellular carcinoma was observed in one rat sacrificed after 29 weeks of orotic acid promotion. Cancer incidence steadily increased with the duration of the orotic acid treatment and 59% developed hepatocellular carcinomas with 30% metastasis to lungs by 53 weeks of promotion. A relevant feature of this model is that during exposure to orotic acid no liver hyperplasia, nor bile duct or oval cell proliferation were seen and the liver architecture in the tissue surrounding focal lesions was well preserved throughout the sequence.

Induction of hepatic nodules in the rat by aristolochic acid.

Aristolochic acid (AA), used as an anti-inflammatory agent in the past, is known to be mutagenic and carcinogenic to several organs of the rat, including forestomach, renal pelvis and urinary bladder. However, despite the induction of DNA adducts in the liver, no carcinogenic potential of AA has been reported in the latter organ. The present study was based on the rationale that the lack of carcinogenicity of AA to the liver could be because this chemical may not be necrogenic at the doses examined and liver cell proliferation has been established as an essential component for initiation of liver carcinogenesis in the rat. The results indicated that AA is non-necrogenic to the rat liver. However, a single non-necrogenic dose of AA (10 mg/kg b.w., i.p.) given 18 hours after 2/3 partial hepatectomy initiated liver cell carcinogenesis. The initiated cells are promotable with 1% dietary orotic acid, a liver tumor promoter, to form glutathione-S-transferase 7-7 positive hepatic foci and nodules.