Modulation by indomethacin or prostaglandin E2 of the incidence of diethylnitrosamine-induced gamma-glutamyltranspeptidase-positive foci in rat liver

We investigated the effect of a pretreatment with indomethacin (IMC, ip 3.6 mg/kg body weight (bw)) or dimethylprostaglandin E2 (PGE2, ip 10 micrograms/kg bw) on the incidence and development of gamma-glutamyltranspeptidase (GGT)-positive foci of altered hepatocytes, scored 8 or 14 weeks after ip injection of diethylnitrosamine (DENA, 50 mg/kg bw) to rats submitted to two-thirds hepatectomy (PH) or sham operation (Sh). IMC reduced by about 4 times the incidence of DENA-induced GGT-positive foci per cm3 of liver tissue in sham-operated as well as in two-thirds hepatectomized rats, compared to the respective unpretreated controls. In contrast, PGE2 pretreatment increased the incidence of DENA-induced foci in both groups, this effect, in terms of absolute numbers of foci, being additive to that of PH alone. IMC pretreatment resulted in foci with lower average size in the Sh but not in the PH animals, whereas with PGE2 pretreatment the mean volume of the foci was increased in the two groups of rats. At the dose used, IMC did not modify the proliferative response of hepatocytes to PH, and PGE2 did not stimulate proliferation in the sham-operated animals. Altogether, these results indicate that: 1, the incidence of DENA-induced foci can be negatively modulated by interfering with the prostaglandins pathway through a mechanism that does not involve an action either on proliferative activity or on any other process that would be specific to the post-hepatectomy regenerative state; 2, positive modulation of the incidence of DENA-induced foci does not necessarily require stimulation of proliferation.     

Modulation by Indomethacin or Prostaglandin E2 of the Incidence of Diethylnitrosamine-induced γ-Glutamyltranspeptidase-positive Foci in Rat Liver

We investigated the effect of a pretreatment with indomethacin (IMC, ip 3.6 mg/kg body weight (bw)) or dimethylprostaglandin E₂ (PGE₂, ip 10 μg/kg bw) on the incidence and development of γ-glutamyl-transpeptidase (GGT)-positive foci of altered hepatocytes, scored 8 or 14 weeks after ip injection of diethylnitrosamine (DENA, 50 mg/kg bw) to rats submitted to two-thirds hepatectomy (PH) or sham operation (Sh). IMC reduced by about 4 times the incidence of DENA-induced GGT-positive foci per cm³ of liver tissue in sham-operated as well as in two-thirds hepatectomized rats, compared to the respective unpretreated controls. In contrast, PGE₂ pretreatment increased the incidence of DENA-induced foci in both groups, this effect, in terms of absolute numbers of foci, being additive to that of PH alone. IMC pretreatment resulted in foci with lower average size in the Sh but not in the PH animals, whereas with PGE₂ pretreatment the mean volume of the foci was increased in the two groups of rats. At the dose used, IMC did not modify the proliferative response of hepatocytes to PH, and PGE₂ did not stimulate proliferation in the sham-operated animals. Altogether, these results indicate that: 1, the incidence of DENA-induced foci can be negatively modulated by interfering with the prostaglandins pathway through a mechanism that does not involve an action either on proliferative activity or on any other process that would be specific to the post-hepatectomy regenerative state; 2, positive modulation of the incidence of DENA-induced foci does not necessarily require stimulation of proliferation.     

Induction of gamma-glutamyltransferase-positive and nonspecific esterase-positive foci in rat liver by partial hepatectomy following single injection of N-nitrosodiethylamine

The effect of partial hepatectomy (PH) on alteration of liver foci induced by N-nitrosodiethylamine (DENA) was studied in inbred F344 male rats. As early as 2 weeks after PH was performed 6 hours after an injection of 100 mg DENA/kg, gamma-glutamyltransferase-positive hepatocellular foci were induced, whereas DENA alone induced no foci until 12 weeks after PH. The focus counts of the group with PH performed 6 hours after an injection of 100 mg DENA/kg were consistently greater than those of a group with PH performed at 24 hours following DENA injection. At 3 and 6 weeks after PH was done at 12 weeks following treatment with 100 or 200 mg DENA/kg, the focus count was significantly increased compared with that in nonhepatectomized groups. The results indicate that increased liver cell proliferation resulting from PH enhances the conversion of persisting DNA damage to a permanent alteration in DNA. The effect at 12 weeks after exposure supports the concept that DNA damage in hepatocytes is highly persistent.     

Promotion by polychlorinated biphenyls of enzyme-altered foci in rat liver

Aroclor 1254 is a complex mixture of polychlorinated biphenyls (PCB) that upon prolonged administration has been reported to produce hepatic tumors in mice and rats. The ability of Aroclor 1254 to promote enzyme-altered foci was determined in an initiation/promotion bioassay in rat liver. Initiation was accomplished in rats that received a 2/3 partial hepatectomy followed in 24 h by diethylnitrosamine (DENA). Aroclor 1254 was administered to each rat 7, 28 and 49 days after the DENA and some of the rats were killed 21 days after each dose of Aroclor. The liver of rats that received Aroclor 1254 on either day 7 or on day 7 and 28 contained an increased incidence of gamma-glutamyltranspeptidase (GGTase)-positive foci compared to partial hepatectomized and DENA treated rats given tricaprylin (the solvent for Aroclor 1254). Therefore, Aroclor 1254 was demonstrated to enhance the appearance of enzyme-altered foci after only a single oral dose.     

Protooncogene methylation and expression in regenerating liver and preneoplastic liver nodules induced in the rat by diethylnitrosamine: effect of variations of S-adenosylmethionine:S-adenosylhomocysteine ratio

S-adenosylmethionine:S-adenosylhomocysteine (SAM/SAH) ratio, 5-methylcytosine (5mC) DNA content, and methylation and expression of c-myc, c-Ha-ras and c-Ki-ras have been studied in liver nodules, induced by diethylnitrosamine according to the 'resistant hepatocyte' model, and in regenerating liver (RL) between 0.5 and 72 h after partial hepatectomy (PH). Nodules, 11, 13 and 21 weeks after initiation, grew actively, showed a low tendency to remodel (persistent nodules), and did not exhibit carcinomatous changes. They underwent extensive remodeling after a 1-week SAM treatment (64 mumol/kg/day), and decreased in size and number after a 3-11-week treatment. A low SAM/SAH ratio was coupled, in nodules, with a high labeling index (LI), 2-fold fall in 5mC DNA content, increase in c-myc, c-Ha-ras and c-Ki-ras expression and hypomethylation of CCGG sequences in the DNA hybridizing with the three protooncogenes. In RL a low SAM/SAH ratio, overall DNA hypomethylation and enhanced c-myc expression were first observed 0.5 h after PH, reached a peak at 5 h and progressively returned to pre-PH levels later on. Maximum expression of c-Ha-ras and c-Ki-ras occurred 24-30 h after PH, roughly coincident with the LI peak. However, no great modifications of the methylation pattern of protooncogene CCGG sequence occurred at any time after PH, indicating the presence of hypomethylated genes and/or DNA sequences different from those investigated in this paper. SAM injection to nodule-bearing rats, for 1-11 weeks before killing, and to hepatectomized rats, 2 days before PH and then up to killing, largely prevented decrease in the SAM/SAH ratio and overall DNA methylation and inhibited LI and protooncogene expression. In nodules these effects were proportional to the treatment length and coupled with methylation of CpG residues in the CCGG sequence of the three protooncogenes studied. SAM treatment left the methylation pattern of these genes unchanged in RL. Kinetics of increase in protooncogene expression suggest a role in the regulation of cell cycle in RL. However, decrease in the SAM/SAH ratio, protooncogene hypomethylation and enhanced expression are apparently stable in nodules 11-21 weeks after initiation and could be implicated in continuous nodule growth and progression. Control of DNA methylation and gene expression by exogenous SAM could be a mechanism of the SAM anti-progression effect.     

Effects of strain,sex, route of administration and partial hepatectomy on the induction by chemical carcinogens of gamma-glutamyltranspeptidase foci in rat liver

The incidence of gamma-glutamyltranspeptidase (GGT)-positive foci induced by 0.3 mmol/kg diethylnitrosamine (DENA) followed by promotion with 500 ppm sodium phenobarbital in drinking water and was the same in Fischer 344, Sprague-Dawley and Wistar-Lewis rats. There was no difference in the level of GGT-foci initiated by DENA, 7,12-dimethylbenz[a]anthracene (DMBA), or 1,2-dimethylhydrazine (DMH) followed by promotion with phenobarbital with respect to sex or route of administration including gavage and intraperitoneal injection. Maximal stimulation by partial hepatectomy of DENA initiation of GGT-foci occurred when the DENA was administered 18 h after the operation. Our results indicate that the optimal protocol for the rat liver foci assay consists of using partial hepatectomized rats of 1 of the 3 strains and of either sex. The test substance should be administered by either gavage or intraperitoneal injection so that maximal DNA binding coincides with the maximal rate of DNA replication resulting from partial hepatectomy.     

Increased yield in GST-P-positive liver pre-neoplastic foci induced by dena or enu in rats pre-treated with estradiol or tamoxifen

We investigated the mechanisms by which partial hepatec-tomy (PH) increases the ability of chemical hepatocarcinogens to induce pre-neoplastic liver foci. Comparison of the effects of pre-treatment with PH, estradiol (E2) or tamoxifen (TAM) on the yield in glutathione-S-transferase(GST-P)-positive pre-neoplastic foci in rat liver induced by subsequent treatment with ethylnitrosourea (ENU) or diethylnitrosamine (DENA) showed that pre-treatment with E2 increased the yield in foci induced by subsequent treatment with ENU or DENA, as compared with that in animals not pre-treated, the increase being of similar magnitude with either carcinogen. Compared with that of PH, the effect of the hormone was much more pronounced than would be expected from the relative mito-genic effect of the hormonal and surgical pre-treatments if the mitotic rate were the cause. On the other hand, the average volume of pre-neoplastic liver lesions in rats treated with ENU or DENA was 2.5 to 5.0 times higher than in rats not pre-treated whenever PH was included in the pre-treatment, whereas it was not affected by any other pre-treatment.     

Induction of replicative competence ("priming") in normal liver

We have used a system of nutritional manipulation to investigate whether hepatocytes of the normal liver can be primed for replication in vivo. In this system, rats that are denied protein for 3 days undergo a burst of hepatic DNA synthesis and mitosis when they are refed amino acids, while normally fed or starved rats do not respond. To determine if hepatocytes of protein deprived (PD) rats have been "primed" for replication, we examined changes in protooncogene expression in livers of PD rats to see if they would mimic the pattern of gene expression that is induced early after partial hepatectomy. c-jun, c-myc, and p53 mRNAs were elevated in livers of PD rats, while c-fos and c-ras genes were not expressed. The administration of amino acids to PD rats stimulated hepatic DNA synthesis in a shorter period than is required after partial hepatectomy and induced p53 and c-ras expression. In culture, hepatocytes from PD rats had higher levels of c-myc mRNA, underwent morphological changes more rapidly, and reached maximum rates of DNA synthesis earlier than normal hepatocytes. In both normal and primed hepatocyte cultures, transforming growth factor alpha stimulated DNA synthesis more effectively than epidermal growth factor. We conclude that hepatocytes pass through a priming stage before they proliferate and that replicative competence without DNA synthesis can be induced in hepatocytes in the normal liver.     

Effect of orotic acid on in vivo DNA synthesis in hepatocytes of normal rat liver and in hepatic foci/nodules

One of the proposed mechanisms by which orotic acid (OA) promotesliver carcinogenesis is by differentially mito-inhibiting thenormal hepatocytes while permitting the initiated ones to respondto growth stimuli to form foci/nodules. In an attempt to examinethis hypothesis, the present study was designed to determine(i) whether OA inhibits DNA synthesis in normal hepatocytesin vivo, and (ii) whether hepatocytes from hepatic foci/nodulesare relatively resistant to the mito-inhibitory effects of OA.The results of this study indicate that OA given i.p. as a tabletof 300 mg at the time of partial hepatectomy (PH) almost completelyinhibited liver DNA synthesis. Three days later—a timeperiod by which the implanted tablet disappeared—the hepatocytesresumed DNA synthesis. Exposure to OA results in an accumulationof uridine nucleotides and a decrease in adenosine nucleotides.Creation of such an imbalance in nucleotide pools appears tobe important for OA to inhibit DNA synthesis. Adenine (a tabletof 300 mg), an agent that inhibits the metabolism of OA to uridinenucleotides, counteracted the mito-inhibitory effects of OA.To determine whether the hepatocytes in foci/nodules are resistantto the mito-inhibitory effects of OA, rats were initiated withdiethylnitrosamine (DENA; 150 mg/kg) and promoted by the resistant-hepatocytemodel. Fourteen weeks after the administration of DENA, therats were subjected to PH in the presence or absence of OA (300mg tablet). The results indicated that, in contrast to hepatocytesin normal or surrounding non-nodular liver, a subpopulationof hepatocyte foci/nodules appear to be relatively resistantto the mito-inhibitory effects of OA. These findings supportthe hypothesis that differential mito-inhibition is a possiblecomponent in the promoting effect of OA. However, whether thisis the mechanism by which OA promotes liver carcinogenesis needsto be further investigated.     

Development of resistance during the early stages of experimental liver carcinogenesis.

The present study was designed to determine whether the resistant phenotype is acquired at the initiated cell stage itself or requires further exposure to a promoting regimen to express resistance. Male Fischer 344 rats were initiated with diethylnitrosamine (DENA) (200 mg/kg i.p.) and were subjected to either no further treatment or to the resistant hepatocyte (RH) model of liver tumor promotion. Six weeks later, the resistance of the focal lesions generated in these two groups to the mitoinhibitory effects of 2-acetylaminofluorene (2-AAF) was determined by subjecting the rats to two-thirds partial hepatectomy (PH) in the presence of a mitoinhibitory dose of 2-AAF (5 mg/kg i.p.) given at the time of PH. Labeling index was determined by administering multiple injections of [(3)H]thymidine. All rats were killed 48 h post-PH. While only a small percentage (23%) of the glutathione S-transferase-positive foci generated by DENA in the absence of an exogenous liver tumor promoting regimen were resistant to the mitoinhibitory effects of 2-AAF, a majority (85%) of the foci became resistant to 2-AAF following exposure to the RH model of liver tumor promotion. Further, initiated rats exposed to either 2-AAF or to CCl(4) alone, the two components of the RH model, resulted in 71% of the foci being resistant to the mitoinhibitory effects of 2-AAF. Similar patterns of results were obtained when the resistance of the foci to the mitoinhibitory effects of orotic acid, a liver tumor promoter and an inhibitor of DNA synthesis in normal hepatocytes, was monitored. These results suggest that the majority of initiated hepatocytes are not of resistant phenotype, however, they have acquired a unique ability to express resistance upon exposure to certain agents such as 2-AAF and CCl(4) or to a promoting regimen such as the RH model of liver tumor promotion.     

Expression of the c-myc, c-fos and c-rasHa protooncogenes during sex-differentiated rat liver carcinogenesis in the resistant hepatocyte model

The expression of the protooncogenes c-myc, c-fos and c-rasHa has been studied in rats treated according to the resistant hepatocyte model. Protooncogene expression was studied in male and female rat liver during the selection phase, when the outgrowth of putative preneoplastic foci/nodules is markedly faster in males, and compared with the expression in advanced nodules and hepatocellular carcinomas in males. During the first 16 h after partial hepatectomy the expression of c-fos and c-myc showed transient, 2- to 3-fold, increases in both sexes, both in initiated and in 'control' animals, receiving the selection/promotion regiment but no diethylnitrosamine, with a maximum at 0.5 and 2-4 h respectively. c-rasHa exhibited a moderate increase (1.5-fold) at 16-24 h in all groups. A second increase in c-myc expression (2-fold) started 24 h after partial hepatectomy and lasted over the entire selection period in initiated males, while it was unchanged in females and uninitiated males. The c-fos expression also showed a short-lived increase 24 h post partial hepatectomy in initiated males. The expression of c-myc and c-fos was increased 2- to 4-fold in both preneoplastic nodules and hepatocellular carcinomas, whereas c-rasHa expression was unchanged. In conclusion, sex differences were observed in the expression of c-myc and c-fos during the early outgrowth of preneoplastic lesions, possibly reflecting a connection between the expression of these genes and the sex differentiated response to promotion in the resistant hepatocyte model. Furthermore, an overexpression also in later stages of liver carcinogenesis might indicate that expression of the protooncogenes in question is related to the entire process of multistep carcinogenesis in this model.     

Sex-differentiated deoxycholic acid promotion of rat liver carcinogenesis is under pituitary control

The influence of continuous growth hormone (GH) infusion and of implantation of ectopic pituitary grafts (PG) to male rats on the sex differences (male greater than female) in efficiency of promotion with dietary deoxycholic acid (DCA; 0.5% w/w) was studied in the livers of diethylnitrosamine (DEN)-initiated Wistar rats. For comparison, liver regeneration after partial hepatectomy (PH) was examined in differently treated animals. The endpoints examined included the number and size of enzyme-altered foci, hepatic c-myc expression and liver weight gain. The area per focus was 2- to 3-fold larger in initiated, DCA-treated males than in the corresponding PG-bearing males, GH-treated males and in females. The expression of the c-myc gene was increased approximately 2-fold in initiated and promoted males, while the increase in females was very small. In both groups of hormone-treated males the expression was at the same level as in females, significantly lower than in the corresponding DEN/DCA-treated males. Liver weight gain in response to PH in initiated as well as uninitiated rats of both sexes was significantly stimulated by DCA. No sex differences or effects of PG on regeneration could be discerned. In conclusion, a sex difference, regulated by a pituitary influence, in focal growth and in c-myc expression was observed during DCA promotion of DEN-initiated rats. This might indicate a correlation between the GH-regulated, proliferation-associated c-myc gene and focal growth during sex differentiated promotion in rat liver. Furthermore, the lack of sex differences in liver weight gain in response to PH during DCA treatment suggests that selective mitoinhibition is not involved, and might in this model indicate hormone-dependent selective stimulation of focal growth as a mechanism for tumor promotion.     

Immunocytochemical detection of the onco-developmental protein oncomodulin in pre-neoplastic and neoplastic hepatocellular lesions during hepatocarcinogenesis in rats

Oncomodulin is a calcium-binding protein, detectable in extra-embryonic human and rat placental cells and in a wide variety of tumors, but not in any normal embryonic or adult rodent or human tissues. It is also absent from proliferatively active fetal or regenerating adult rat liver. The presence of this oncodevelopmental marker was investigated in pre-neoplastic and neoplastic liver lesions during hepatocarcinogenesis induced in rats by DENA treatment, using an antibody raised against purified oncomodulin. Positive immunostaining was observed in foci of altered hepatocytes, in neoplastic nodules and in HC, but not in the histologically normal surrounding liver parenchyma. The proportion of oncomodulin-positive foci gradually rose from 20-25% at 2-3 months after DENA treatment, to about 88% at 6 months and later. The proportion of positive neoplastic nodules increased from 50% at 5 months to about 73% (range 36-100) at 9 months and later; 88% of the HC found 10 to 20 months after DENA treatment were also positive. That early neoplastic nodules are oncomodulin-positive in a proportion (50%) similar to that of foci after the same duration of treatment is consistent with a lineage relationship between them but makes it unlikely that oncomodulin expression conditions the focus-nodule transition. The role, if any, of oncomodulin in malignant progression remains to be elucidated. It seems out of the question that it is a simple correlate of proliferative activity.     

Influences of diethylnitrosamine on longevity of surrounding hepatocytes and progression of transplanted persistent nodules during phenobarbital promotion of hepatocarcinogenesis

The possibility that phenobarbital (PB) selectively promotes liver nodule development by decreasing survival of surrounding hepatocytes previously exposed to diethylnitrosamine (DENA) was evaluated. Livers of F-344 rats were labelled with [3H-methyl]-thymidine (3H-TdR) during developmental or regenerative growth. Neonatal rats given 3H-TdR between days 3 and 12 were subjected at 12 weeks of age to partial hepatectomy (PH) followed 24 hr later by DENA (10 mg/kg) or saline. Subsequent administration of PB (0.1% in drinking water) for 28 weeks reduced total liver label to 46 +/- 10% (saline group) or 40 +/- 4% (DENA group). Adult male rats initiated with DENA (200 mg/kg) and later labelled with 3H-TdR after PH also lost total liver label during 28 weeks' promotion with PB (0.05% in water) at rates similar to those exhibited by noninitiated rats given PB, and by DENA-treated or control rats not given PB. Large persistent (12 weeks) liver nodules generated by DENA in the Solt-Farber model were transplanted as small fragments into the spleens of syngeneic rats previously given 0, 100 or 200 mg/kg of DENA. Subsequent exposure to PB (0.05% in drinking water for 40 weeks) or Aroclor 1254 (6 X 300 mg/kg per month) promoted nodule and cancer development only in livers of DENA-initiated recipients. Surviving transplanted nodules remained as small microscopic clusters even after 40 weeks of promotion. However, PB increased transplant survival (50% vs. 21% in controls) whereas Aroclor reduced it to 8%. These findings indicate that promotion of liver nodules by PB occurs without enhanced mortality of surrounding hepatocytes previously damaged by DENA. They further suggest that promoters such as PB and PCBs do not directly influence the progression of established persistent nodules.     

Dose-dependent effects of 2-acetylaminofluorene on hepatic foci development and cell proliferation in rats

Dose-dependent development of pre-neoplastic liver cell foci induced by 2-acetylaminofluorene (2-AAF) was investigated in relation to cell-proliferative activity. Male F344 rats were initially given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) and starting 2 weeks later received diets containing 2-AAF at dose levels of 150, 100, 60, 45, 35 or 30 p.p.m., 500 p.p.m. phenobarbital (PB) or basal diet as a control for 6 weeks. Two-thirds partial hepatectomy (PH) was performed at week 3. The rats were sequentially killed from weeks 0 to 16 and liver sections were analysed by double staining for both BrdU incorporation and glutathione S-transferase placental form (GST-P) expression. 2-AAF increased numbers and areas of GST-P positive (GST-P+) foci in a dose-dependent manner, especially after PH. Proliferation of hepatocytes, as indicated by BrdU labelling indices (LI), was higher in GST-P+ foci than in surrounding hepatocytes in all 2-AAF-treated groups, even after cessation of carcinogen administration. Proliferative response of hepatocytes to PH was delayed in rats treated with the highest dose of 2-AAF in both foci and in surrounding areas possibly due to the 2-AAF toxicity. In the PB treated group, the results were similar to those for the lower dose 2-AAF-treated groups. It is concluded that the development of GST-P+ foci and cell proliferation in GST-P+ foci are directly related to 2-AAF treatment in a dose-dependent manner and the present assay system is reliable for detection of carcinogenicity of chemicals even at low doses.     

Effect of cell proliferation on initiation of aflatoxin B1-induced enzyme altered hepatic foci in rats and hamsters

Rat is susceptible whereas hamster is resistant to aflatoxinB₁ (AFB₁) hepatocarcinogenesis. Effect of cell proliferationon AFB₁ -induced glutathione S-transferase placental form (GST-P)positive foci has been examined in these two species after asingle i.p. dose of AFB₁ and phenobarbital (PB) as a promoterin a 3 week period. Bromodeoxyuridine incorporation as a measureof cell proliferation and GST-P hepatic foci were analyzed byimmunohistochemical methods. Hepatic cell proliferation wasmaximum at 24 h after either partial hepatectomy (PH) or CCl₄(4 mmol/ kg) pretreatment of rats whereas cell proliferationwas maximum at 48 h after PH or CCl₄ (1 mmol/kg) treatment ofhamsters. Enhanced number of GST-P positive hepatic minifoci(two to nine cells) and foci (>100 µ) and focal areawere observed in rats with either AFB₁ (0.5 mg/kg) given 24h after PH or AFB₁ (0.5 or 2.5 mg/kg) given 48 h after CCl₄dosing. In hamsters, 1 or 2 mg AFB₁ treatment produced onlyGST-P positive single hepatocytes without presence of any minifociwhereas 3 or 6 mg AFB1 produced minifoci consisting only ofdoublets. Pretreatment with CCl₄ 48 or 72 h before 1 mg AFB₁dose level increased GST-P positive single cells and minifociseveral fold. PH 24 or 48 h before 1 or 2 mg AFB₁ dose levelincreased minifoci. However, increase in minifoci was higherin PH hamsters at 48 h compared with those at 24 h. These resultsindicate that even though maximum initiation occurs in bothspeices when AFB₁ is administered at the peak of DNA synthesis,rats are more responsive than hamsters to cellular proliferationin the initiation phase of AFB₁ induced hepatocarcinogenesis.     

Lack of hepatogenotoxicity of oral contraceptive steroids

The objective of the present study was to determine whethermestranol could initiate hepatocartinogenesis and whether variousgonadal steroids have detectable hepatogenotoxic potential.To test for initiation potential, female, Sprague-Dawley ratswere partially hepatectomized and intubated with mestranol (100or 500 mg/kg) 24 h later. Twenty-four hours after treatmentall rats were transferred to diet containing 0.05% phenobarbitalto promote hepatocytes initiated by mestranol treatment. Fourmonths later an analysis of putative preneoplastic gamma glutamyltranspeptidase positive foci indicated that mestranol did notcause a significant increase in the number of such foci. Hepatogenotoxicitywas assessed in two ways, first using alkaline elution to detectliver DNA damage after in vivo treatment with mestranol, andsecond by detection of DNA repair synthesis hi primary culturesof hepatocytes treated with various oral contraceptive steroids.The results of both studies were negative. In conclusion, theoral contraceptive steroids do not exhibit strong initiatingor genotoxic potential.     

Immunohistochemical detection of c-Ha-ras oncogene p21 product in pre-neoplastic and neoplastic lesions during hepatocarcinogenesis in rats

An immunohistochemical study of c-Ha-ras expression was performed on preneoplastic and neoplastic stages of diethylnitrosamine (DENA)-induced hepatocarcinogenesis in rats, using an antibody raised against a peptide sequence of the Ha-ras p21 product. Moderate to high immunostaining intensity was observed in the following hepatocytic lesions: (1) hepatocellular carcinomas (14/14) and associated neoplastic nodules (8/8) and foci of phenotypic alterations (35/40) (after 13-20 months of treatment); (2) neoplastic nodules (6/6) and associated foci (42/50) (after 5-9 months); (3) foci (10/10) (after 2 months); and (4) small, slowly growing foci (26/40) found 9 months after treatment with DENA without prior partial hepatectomy, resulting in low number of nodules and no tumor even after 15 months. No c-Ha-ras p21 was detected immunohistochemically in normal nor in regenerating rat liver. Our results indicate that increased Ha-ras expression is an early and stable event in liver lesions associated with hepatocarcinogenesis. They also imply that increased Ha-ras expression is insufficient (if at all implicated) for inducing fully malignant hepatocyte transformation. It might be indicative of cell populations at an increased transformation risk.     

In vitro and in vivo response of hepatocytes from hepatic nodules to the mitoinhibitory effects of phenobarbital

One of the proposed mechanisms by which phenobarbital (PB) promoteshepatocarcinogenesis in the rat is by differential mitoinhibition.However, our earlier studies indicated that PB inhibited DNAsynthesis in vitro in hepatocytes isolated from both surroundingnon-nodular liver and hepatic nodules promoted by orotic acid(OA). Since nodules generated by one promoter need not necessarilybe resistant to another promoter, the present study was undertakento determine whether foci/nodules promoted by PB itself areresistant to the mitoinhibitory effects of PB. Accordingly,rats were initiated with diethylnitrosamine (DENA, 200 mg/kgi.p) and promoted with PB (0.07% of PB as its sodium salt) intheir drinking water for 16 or 33 weeks. In vitro studies indicatedthat PB (3–5 mM) inhibited DNA synthesis induced by epidermalgrowth factor (EGF) in hepatocytes from surrounding non-nodularliver as well as from nodules promoted by PB for 33 weeks. Inanother experiment, initiated rats exposed to PB for 33 weekswere subjected to either two-thirds partial hepatectomy (PH)or sham hepatectomy. Hepatocytes were labelled with tritiatedthymidine in vivo for 48 h. Autoradiographic analysis indicatedthat in the presence of PB, the hepatocytes from both foci/nodulesand the surrounding non-nodular liver responded to PH to thesame extent. In addition, they both responded to PH less efficientlyas compared to the corresponding controls. Further, initiatedrats exposed to PB for 16 weeks when subjected to PH and killed4 weeks thereafter, the percentage area occupied by