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     

Influences of different polychlorinated biphenyls on cytocidal, mitoinhibitory, and nodule-selecting activities of N-2-fluorenylacetamide in rat liver

The influences of different polychlorinated biphenyl (PCB) isomers and congeners on distinct hepatotoxic responses to the hepatocarcinogen N-2-fluorenylacetamide [(2-FAA) CAS: 53-96-3] were examined in F344 rats. Cytocidal toxicity of 2-FAA (25-400 microM), determined by lactate dehydrogenase release during 20 hours in primary monolayer cultures of isolated rat hepatocytes, was reduced by in vivo pretreatment with either phenobarbitone [(PB) CAS: 50-06-6] or 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP), a PB-type PCB inducer. However, cytocidal toxicity of 2-FAA was substantially potentiated by either 3-methylcholanthrene [(MCA) CAS: 56-49-5] or 3,3',4,4'-tetrachlorobiphenyl [(TCBP) CAS: 32598-13-3], an MCA-type PCB. In the same cell culture assays, all four pretreatments similarly reduced cytocidal toxicity of N-hydroxy-N-2-fluorenylacetamide (0.32-32 microM; CAS: 53-95-2). By comparison, pretreatments with either the PB-type or MCA-type PCB's (50-200 mumol/kg) diminished mitoinhibitory toxicity of 2-FAA in vivo, as measured by hepatic regenerative growth and hepatocyte labeling indices 7 days after partial hepatectomy (PH) in rats given 3 consecutive daily doses of 2-FAA (20/mg/kg/day) before PH. This regimen of 2-FAA and PH promoted rapid selective growth of gamma-glutamyltranspeptidase-positive (gamma-GT+) nodules at 2 and 4 weeks after PH in rats previously given an initiating hepatocarcinogen, diethylnitrosamine [(DENA) CAS: 55-18-5]. However, various PCB's, including 2,2',4,4',5,5'-HCBP, 3,3',4,4'-TCBP, 2,2',4,4'-TCBP, 2,2',5,5'-TCBP, and the commercial mixture Aroclor 1254, each given as a single dose of 50 mumol/kg by gavage 10 days after DENA and 7 days before 2-FAA, all reduced the size of 2-FAA-selected gamma-GT+ nodules during the 4-week period after PH. These results indicate that, in spite of predictable inducer-specific opposite influences of different types of PCB's on cytocidal toxicity of 2-FAA, all PCB's similarly reduce nodule selection by 2-FAA in initiated livers. Reduced growth of 2-FAA-selected nodules correlated with the consistent ability of all PCB's to enhance regeneration of liver mass after 2-FAA and PH.     

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.     

Initiation and selection of resistant hepatocyte nodules in rats given the pyrrolizidine alkaloids lasiocarpine and senecionine

The biological mechanisms by which pyrrolizidine alkaloids contribute to initiation and nodule selection (promotion) steps in hepatic carcinogenesis were studied in male Fischer 344 rats. Lasiocarpine at single or double dosages (up to 80 mumol/kg) delayed hepatic regeneration for at least 8 weeks after partial hepatectomy (PH). This regimen of lasiocarpine and PH had a strong selective influence on the growth of gamma-glutamyltranspeptidase (gamma-GT)-positive hepatocyte nodules in rats previously initiated with diethylnitrosamine. However, both lasiocarpine (up to 80 mumol/kg) and senecionine (up to 160 mumol/kg) were inactive as initiators of gamma-GT-positive nodules in rats exposed to a similar selection regimen consisting of 2-acetylaminofluorene and PH. When lasiocarpine or senecionine was given 12 h after PH, very few nodules were initiated. Lasiocarpine pretreatments reduced the initiating activity of diethylnitrosamine and N-nitrosomethylurea in rats subsequently selected with 2-acetylaminofluorene and PH. Resistant nodules selected with lasiocarpine had the typical resistant nodule phenotype (positive for gamma-GT and epoxide hydrolase) and also lacked pyrrolizidine alkaloid-induced megalocytosis. Lasiocarpine treatment also resulted in small regenerative nodular proliferations of hepatocytes that were distinct from resistant nodules because they were negative for gamma-GT and epoxide hydrolase and unrelated to diethylnitrosamine pretreatments. These studies suggest that the hepatocarcinogenicity of pyrrolizidine alkaloids can be better explained by their strong selection (promotion) influence on initiated hepatocytes, rather than by their very weak initiating activity.     

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.     

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.     

Effect of phenobarbital on the gamma-glutamyltranspeptidase activity and the remodeling of nodules induced by the initiation-selection model

The effect of subsequent administration of phenobarbital on the gamma-glutamyltranspeptidase (GGT) activity and on the remodeling of nodules induced by the Solt-Farber procedure was examined in rats. GGT-nodules were initiated by diethylnitrosamine (DENA) followed by selection with 2-acetylaminofluorene (2AAF) in the diet and a partial hepatectomy (PH). Phenobarbital (500 ppm in the drinking water), administered to rats that were previously treated according to the Solt-Farber procedure, (1) increased the persistence of the GGT-nodules, (2) increased the percentage of the liver occupied by GGT positive cells, (3) increased the area of GGT activity per nodule and (4) increased the incidence of eosinophilic lesions. Subsequent treatment with phenobarbital did not alter the incidence of either GGT-nodules or hepatocellular carcinoma. Thus, phenobarbital increased the GGT activity of nodules induced by the Solt-Farber procedure and slowed both the loss of GGT activity by these nodules and their concurrent remodeling, but had no effect on the occurrence of cancer.     

The carcinogenic effect of methapyrilene combined with nitrosodiethylamine given to rats in low doses.

The carcinogenic effects of combinations of methapyrilene hydrochloride (MP), nitrosodiethylamine (NDEA), and phenobarbital (PB) or partial hepatectomy (PH) were examined following sequential treatment of rats. MP is a generally non-genotoxic liver carcinogen of moderate potency, NDEA is a genotoxic liver carcinogen, PB is primarily a liver tumor promoter and PH induces cell proliferation. The dose of each carcinogen was chosen to be below that causing significant liver tumor incidence when given singly. There were 12 protocols involving groups of 28 female rats each. Short treatments with NDEA and MP were followed by 60 weeks of PB promotion or by partial hepatectomy. Each treatment was given separately or in double combination as controls. Several animals of each group were killed at intervals during the experiment for examination of toxic effects and the presence of altered hepatic foci. In only 3 of 12 groups was there a significant incidence of rats with liver neoplasms: the two groups given three treatments: NDEA, MP and PB (86% tumors) or NDEA, MP and PH (33%), and the group receiving NDEA and MP without promotion (46%). The results clearly indicated a co-carcinogenic effect between NDEA and MP. Continuous PB potentiated tumor development, while PH did not. There was no evidence of liver toxicity from any of the treatments, but clear cell foci observed in three groups at weeks 13 and 33 correlated with the later development of liver neoplasms.     

A subnecrogenic dose of diethylnitrosamine is able to initiate hepatocarcinogenesis in the rat when coupled with fasting/refeeding

Caloric restriction causes a generalized decrease in growthrate and has been repeatedly associated with an inhibitory effecton cancer development in several systems. In contrast, exposureto complete fasting followed by refeeding is as metabolic conditionassociated with increased cell turnover in different organs,including the liver. The present study examines whether suchcondition is able to sustain the induction of initiated hepatocytesfollowing a subnecrogenic dose of diethylnitrosamine (DENA).Male Fisher-344 rats were fasted for 4 days and 1 day afterrefeeding they were given a single dose of DENA (20 or 200 mg/kgbody wt, i.p.). Negative and positive control groups were fedad libitum and injected with 20 and 200k mg/kg of DENA, respectively.One week later all animals were subjected to the resistant hepatocytemodel for the selection of hepato-cyte nodules and they werekilled 2 weeks thereafter. Results indicated the presence ofgamma-glutamyltransfer-ase (GGT) positive foci and nodules (38± 7/cm²) in rats regularly fed and given 200 mg/kg ofDENA, while virtually no focal lesions (<1/cm²) were foundin the group receiving 20 mg/kg of DENA and fed throughtoutthe experiment. However, a significant number of GGT positivefoci/nodules (14 ± 7) also developed in rats exposedto fasting and given 20 mg/kg of DENA 24 h after refeeding.No evidence of helpatocellular necrosis was found in the lattergroup follwing DENA administration. No effect of fasting wasobserved when rats received 200 mg/kg of DENA. It is concludedthat fasting/refeeding provides conditions which are able tosustain initiation in rat liver by a subnecrogenic dose of acarcinogen. These findings are in contrast with the commonlyreported inhibitory effect of chronic food restriction on variousstages of carcinogenesis, including initiation.     

Perturbations of endogenous levels of orotic acid and carcinogenesis: effect of an arginine-deficient diet and carbamyl aspartate on hepatocarcinogenesis in the rat and the mouse

Feeding excess orotic acid (OA) In the diet promotes the carcinogenicprocess in different organs Including the liver. A number ofmetabolic and genetic disorders are associated with Increasedsynthesis of endogenous OA and some of these disorders appearto pose an Increased risk of liver cancer development. Thisstudy therefore examines whether excess OA of endogenous originalso exerts a promoting effect on hepatocarcinogenesis in themouse and the rat. Increased endogenous synthesis of OA wasachieved by (i) feeding a diet deficient in arginine (AD) and(ii) feedIng excess dietary carbamylaspartate (CA), a precursorfor the synthesis of OA. A single dose of diethylnitrosamine(DENA) was given i.p. to male Fischer 344 rats (200 mg/kg) orto male DBA/2 mice (90 mg/kg). One week later they were placedon either AD diet or the same diet supplemented with 1.3% arginine(AS) for a total of weeks. Two-thirds partial hepatectomy (PH)was performed at the end of the second week. All animals werethen transferred to a control semisynthetic basal diet for atotal of 20 weeks before they were killed. The results indicatedthat AD diet increased the incidence of hepatic nodules in bothrats (percentage area occupied by nodules was 4.7 ± 0.4in the AD group compared to a control value of 0.7 ±0.5) and mice (4/10 mice had nodules >5 mm diameter in theAD group while none in the AS group had such large nodules).In another experiment male Fischer 344 rats similarly initiatedwIth DENA were exposed to either basal diet or basal diet containing2% CA for 4 weeks coupled with PH performed at the end of thesecond week. This regimen was followed by 20 weeks of feedingbasal diet to both groups. Rats given CA developed larger hepatlcfoci and nodules (0.84 ± 0.56 mm³) compared to the controlgroup, which was fed basal diet throughout the experiment (0.07± 0.03 mm³) Further, both AD diet and dietary CA, likedietary OA, induced an increase In hepatic uridine nucleotides.Taken together, these results suggest that Increased levelsof endogenously synthesized OA, like exo genously supplied excessOA, can induce an Imbalance in hepatic nucleotide pools andcan exert a promoting effect on hepatocarcinogenesis.     

Influence of cell proliferation on initiating activity of pure polychlorinated biphenyls and complex mixtures in resistant hepatocyte in vivo assays for carcinogenicity

The abilities of various pure polychlorinated biphenyls (PCB) and complex mixtures to generate resistant gamma-glutamyltransferase (GGT)-positive hepatocellular nodules was evaluated in F344 rats in which hepatocytes were proliferating. The PCB examined were 2,2',4,4',5,5'-hexachlorobiphenyl (CAS: 35065-27-1), 2,2',4,4'-tetrachlorobiphenyl, 2,2',5,5'-tetrachlorobiphenyl, Aroclor 1254 (CAS: 11097-69-1), and a prepared mixture of pure PCB isomers and congeners similar to those found in human breast milk. The PCB were administered either to male and female suckling rats (weekly for 3 wk) during liver growth or to adult male rats (150-160 g body wt) previously subjected to two-thirds partial hepatectomy (PH). Rats were subsequently given a selection regimen consisting of 3 daily doses (20 mg/kg) of 2-acetylamino-fluorene (2-FAA; CAS: 53-96-3) followed by either PH or necrotizing carbon tetrachloride (CAS: 56-23-5) in adult rats that previously underwent PH. None of the PCB exposures generated GGT-positive nodules after selection, whereas known initiators such as diethylnitrosamine (CAS: 55-18-5), 3-methylcholanthrene (CAS: 56-49-5), benzo[a]pyrene (CAS: 50-32-8), and 2-FAA were active initiators of nodules in suckling or hepatectomized rats. These findings indicate that short-term exposures to these PCB during liver cell proliferation do not show initiating action in an in vivo assay that detects both hepatic and nonhepatic initiating carcinogens.     

The histology and development of hepatic nodules in C3H/He mice following chronic administration of phenobarbitone

Male C3H/He mice were given 0 (control) or 85 mg/kg/day phenobarbitone(PB). Groups of five control and five treated animals were killedat 5, 10, 25, 40 and 55 weeks; additional animals were allowedto live out their natural life span or killed at the terminationof the experiment at 100 weeks. Small nodules of basophiliccells were seen in control animals at 55 weeks. These increasedboth in size and number so that at term nodules were found in17/35 animals examined. Animals given PB showed typical contrilobularhypertrophy. Basophilic nodules were also seen in the treatedanimals and these were similar in form and in number to thoseseen in control animals. The total number of liver nodules seenin animals given PB was, however, greater than the number seenin control animals. The increased nodule burden was a resultof the development of a second nodule type that was first seenin animals killed at 40 weeks. These nodules were formed oflarge epsinophilic cells that had a similar appearence to thehypertrophied cells of the centrilobular region. In contrastthe incidence of carcinoma in PB-treated animals was not increasedover that of the control group.     

Inhibitory Effects of Inhibitors of Arachidonic Acid Metabolism on the Evolution of Rat Liver Preneoplastic Foci into Nodules and Hepatocellular Carcinomas with or without Phenobarbital Exposure

Effects of inhibitors of arachidonic acid (AA) metabolism on the evolution of preneoplastic foci into nodules and of nodules into hepatocellular carcinomas were examined in F344 male rat livers with or without phenobarbital (PB) exposure. p -Bromophenacyl bromide (BPB), acetylsalicylic acid (ASA), and quercetin (QU) were used as inhibitors of phospholipase A₂, cyclooxygenase and lipoxygenase, respectively. Preneoplastic liver foci were induced by initiation with N-nitrosodiethylamine (200 mg/kg, i.p.) followed by selection using the procedure of Cayama et al. For the nodule experiment, starting 1 week after completion of the selection procedure, animals bearing foci were given diets containing 0.05% PB plus 0.75, 1, or 1.5% of one of the inhibitors, 0.05% PB alone, or 0.75, 1 or 1.5% of inhibitor alone, or basal diet for 9 weeks. For the carcinoma experiment, 3 weeks after completion of the selection procedure, animals bearing nodules were given the same diets mentioned above for 29 weeks. BPB, ASA and QU either with or without PB accelerated the remodeling of preneoplastic foci, significantly decreasing the numbers of persistent nodules and hyperplastic nodules. ASA either with or without PB significantly decreased the number of hepatocellular carcinomas per rat. BPB and QU, however, significantly decreased the numbers of hepatocellular carcinomas with but not without PB. The results suggested an involvement of AA metabolism in the process of evolution of preneoplastic foci into nodules and hepatocellular carcinomas in rat liver with or without PB exposure.     

Molecular profiling of genes up-regulated during promotion by phenobarbital treatment in a medium-term rat liver bioassay

In search of genes that are steadily up-regulated during the promotion stage in carcinogenesis, suppression PCR subtractive hybridization and following northern blot screening were performed using a phenobarbital (PB)-promotion model based on a medium-term liver bioassay. Two weeks after a single injection of diethylnitrosamine (DEN; 200 mg/kg body wt, i.p.), rats were given 600 p.p.m. PB in the drinking water for up to 64 weeks. For comparison, animals fed 1 p.p.m. ethinylestradiol (EE) or 3000 p.p.m. butylated hydroxytoluene (BHT) in the diet at promotion stage were also included. Rats were subjected to partial hepatectomy (PH) at week 3. In addition, dose-dependence of PB at week 8 of promotion and responsiveness to representative non-genotoxic carcinogens without DEN initiation were examined. Fragments of a total of 67 different genes were isolated from the up-regulated gene population in the liver at day 10 of PB treatment by subtracting from basal expression of DEN + PH alone. Using northern blot screening for signal-detectable 48 genes, 16 genes showed up-regulation in the livers at week 8 of promotion, common to the PB and EE treatments with the levels being three times or more than the basal expression of unpromoted liver. The majority of these genes were also up-regulated at week 8 by BHT treatment, and were also constitutively expressed in the DEN(-), PH(-) untreated rat livers. Among the up-regulated genes common to the PB and EE promotion, and not responding to the non-genotoxic carcinogens in uninitiated liver, the following six genes showed overexpression in PB-promoted hepatocellular carcinomas at week 64, with the levels three times or more than untreated rat liver: ubiquitously expressed mammalian ABC half transporter, apolipoprotein A4, nuclear receptor binding factor-2, CD81, hypothetical protein (HSPC014) and one unidentified gene. These genes might be candidates for biomarkers in screening of non-genotoxic hepatocarcinogens by analysis in two-stage carcinogenesis models.     

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.     

Comparative tumor-promoting activities of phenobarbital, amobarbital, barbital sodium, and barbituric acid on livers and other organs of male F344/NCr rats following initiation with N-nitrosodiethylamine

Tumor-promoting abilities of four barbiturates, phenobarbital [(PB) CAS: 50-06-6], amobarbital [(AB) CAS: 57-43-2], barbital sodium [(BB) CAS: 144-02-5], and barbituric acid [(BA) CAS: 67-52-7], on the development of neoplasms in livers and other organs of rats following initiation with N-nitrosodiethylamine [(DENA) CAS: 55-18-5] were compared. Four-week-old F344/NCr male rats were given a single ip injection of 75 mg DENA/kg body weight. Beginning 2 weeks later, they were given either tap water (group 1) or drinking water containing 500 ppm of PB (group 2), the sodium salt of BB (group 3), AB (group 4), or BA (group 5) for the remaining experimental period. Control groups (groups 6-10) received an ip injection of saline alone and 2 weeks later were given either tap water or drinking water containing barbiturates as listed above. Animals were sacrificed at either 52 weeks or 78 weeks. None of the barbiturates altered the growth and survival of animals. PB and BB increased liver weights and significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 weeks and hepatocellular foci, hepatocellular adenomas, and trabecular carcinomas at 78 weeks in DENA-treated rats. No such enhancing effects were observed with AB or BA. PB or BB did not significantly enhance the incidence of nonhepatic tumors at 52 weeks. However, at 78 weeks BB significantly enhanced the development of renal tubular adenomas and carcinomas, while PB enhanced the development of thyroid follicular cell neoplasms in DENA-treated rats. These results clearly showed that barbiturates exhibited structure-promoting activity relationships and that their promoting abilities were not restricted to liver alone. Substitution of both hydrogen atoms at the C-5 position of the pyrimidine ring by alkyl or aryl groups appears to be essential but not sufficient for tumor-promoting activity of barbiturates.     

Modulation by indomethacin of diethylnitrosamine-induced early changes in c-myc and c-ras expression and late incidence of preneoplastic lesions in rat liver

We measured the levels of c-myc and c-ras expression before and after diethylnitrosamine (DENA) treatment in the liver of rats previously submitted to partial hepatectomy (PH), in the presence or absence of indomethacin (IMC), given at a dose that reduced by 75% the incidence of preneoplastic foci of altered hepatocytes scored 8 weeks after application of the carcinogen. The time-course evolution of c-myc response to PH was similar in IMC-treated and untreated rats (with a peak at 3-8 h at least as high in IMC-treated animals as in the hepatectomized reference group), whereas the overall c-ras response was significantly reduced by the IMC treatment, resulting in much lower c-ras expression at 18-24 h posthepatectomy. Treatment with DENA 24 h after PH did not significantly modify c-ras expression compared to partially hepatectomized controls. In contrast, DENA treatment resulted in a marked transient increase in c-myc expression that was at least as pronounced, if not the same, in the IMC-treated animals. These results leave open the possibility that increased c-myc expression under DENA influence might play a role in foci induction but exclude that this might be sufficient. They are consistent with a role for c-ras expression in determining the susceptibility of hepatocytes towards the carcinogenic action of DENA.     

The inhibition by methionine and choline of liver carcinoma formation in male C3H mice dosed with diethylnitrosamine and fed phenobarbital

The ability of the dietary methyl donors methionine and choline to inhibit the carcinogenic and tumor-promoting effects of phenobarbital (PB) in the livers of male weanling C3H mice was examined. The mice were fed a commercial rodent diet with or without 0.05% PB. Thirty animals from each set received the diet with either: (1) no dietary supplementation, (2) an additional 1.0% choline chloride, (3) 1.5% DL-methionine or (4) both 1.5% DL-methionine and 1.0% choline chloride. Additional groups of 30 animals with the same eight dietary and PB-treatment regimens described above were given a single initiating dose of 150 mg diethylnitrosamine (DENA)/kg body wt dissolved in saline, or the saline solution only, 1 week prior to the start of PB feeding. The 16 treatment groups were fed their respective diets for 12 months. Statistical trend analysis showed that increasing levels of supplemental methyl donors gave highly significant protection in PB-treated mice (P less than 0.01). The incidence of liver carcinomas in the four dietary groups not receiving PB or DENA varied from 0 to 7%. The PB-treated animals not receiving an initiating dose of DENA developed hepatocellular carcinomas (HCCs) at incidences of 79% in group 1 animals, 74% in group 2 animals, 60% in group 3 animals, and 31% in group 2 animals respectively. Thus, incidence of HCCs in group 4 was significantly lower than in groups 1, 2 or 3 (P less than 0.01). However, the total incidence of liver tumors (adenomas plus carcinomas) was about the same in all DENA or PB-treated groups. Thus, dietary supplementation with methyl donors increased the proportion of animals bearing liver adenomas as their most advanced hepatic lesion in PB-treated mice. In DENA-treated mice fed PB, dietary supplementation with methionine and choline protected against the formation of liver carcinomas (P less than 0.02); however, methionine and choline had no significant effect on liver tumor formation in mice fed the PB-free diets. Methionine and choline supplementation gave significant protection against HCC metastases in the lungs of the tumor-bearing mice in groups initiated with DENA followed by PB promotion. These results support the hypothesis that PB exerts it tumorigenic activity in mice at least in part through a physiological insufficiency of labile methyl groups.     

Enhanced preneoplastic liver lesion development under 'selection pressure' conditions after administration of deoxycholic or lithocholic acid in the initiation phase in rats

The initiating potential of the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), for rat hepatocarcinogenesis was investigated using the development of hyperplastic nodules and/or glutathione S-transferase placental form (GST-P)-positive liver foci as the end point. Five week old male F344 rats were given either basal diet, or diets containing 0.5% DCA or 0.5% LCA for 3 weeks in conjunction with partial hepatectomy performed midway, followed by a selection regimen consisting of 2 weeks feeding of 0.02% 2-acetylaminofluorene diet and a single gastric intubation of carbon tetrachloride. The animals were then placed on either basal diet or a diet containing 0.05% phenobarbital (PB) for 52 weeks. Significantly higher numbers of hyperplastic liver nodules developed in the DCA-treated rats irrespective of PB promotion as compared with the respective control groups. No such increase was evident in the LCA-treated rats. In contrast, both DCA and LCA treatments enhanced the development of GST-P-positive liver foci with or without subsequent PB promotion. Only one hepatocellular carcinoma was diagnosed in a control group animal. The present data indicate that a short period of feeding of DCA and LCA in the initiation stage in conjunction with partial hepatectomy results in enhanced development of preneoplastic liver lesions under selection pressure conditions with or without subsequent PB promotion. They thus confirm and extend our previous finding of enhanced gamma-glutamyltranspeptidase-positive liver foci development in a short-term assay of DCA and LCA, and suggest that these secondary bile acids either possess possible initiating activity or some other priming effect for rat hepatocarcinogenesis.     

Promotion mechanism of phenobarbital and partial hepatectomy in DENA hepatocarcinogenesis cell kinetics effect

Diethylnitrosamine (DENA, 10 mg kg-1 per day) was fed to rats for 2, 4 and 6 weeks. One week after the cessation of DENA, animals were submitted either to partial hepatectomy or to phenobarbital administration. Partial hepatectomy did not promote neoplastic transformation, except after a 6-week DENA treatment. A minimum of phenobarbital was required to reach a significant promoting effect in DENA carcinogenesis. A too-limited treatment was ineffectual but could be compensated for by prolonged DENA administration. The phenobarbital treatment became unnecessary when neoplastic nodules were present. Phenobarbital continuously given after the carcinogen administration promoted neoplastic transformation even after a subcarcinogenic DENA treatment (2 weeks). It accelerated the pathological evolution and increased the tumour incidence. In these conditions, phenobarbital increased the proliferation advantage of preneoplastic cells over normal cells. In the different experimental modalities, the promoting effect was associated with the induction of chronic cell proliferation, the inhibition of the rapid response to the 2/3 partial hepatectomy and the mitotic circadian rhythm normally present during liver regeneration. It is concluded that the promotion mechanism could consist in disturbing the mitotic control in order to maintain, for a long time, a chronic low level of cell proliferation permitting the selective growth of preneoplastic cells and their subsequent transformation.