Biochemical events during initiation of rat hepatocarcinogenesis

Carcinogenesis is a multistep, multistage process that beginswith irreversible, but heritable damage to a single cell. Thepartial hepatectomy/diethylnitrosamine (DEN) model of rat hepatocarcinogenesishas been well characterized and many aspects of the stage ofinitiation are known. Recently, it has been suggested that hepatocytesexpressing the placental isozyme of glutathione S-transferase(PGST) may be one population of initiated cells. Male Fischerrats were subjected to a 70% partial hepatectomy and at thepeak of cell proliferation 24 h later were administered eitherthe solvent trioctanoin, or 10 mg DEN/kg. The rats were administered100 mg bromodeoxyuridine (BrdU)/kg 1 h prior to deathat varioustimes after DEN administration. Since initiation of the carcinogenesisprocess requires the division of cells containing DNA damageto induce mutations, we examined the concentration of alkylatedadducts and the labeling index at various times after DEN administration.In addition, the time course of hepatic PGST expression wasdetermined concurrent with the adduct concentration and labelingindex. During the first day after DEN or solvent administrationto a rat subjected to a 70% partial hepatectomy, a diurnal variationin labeling index was observed. A recovery to post-surgicallabelingindex levels was demonstrated for both the solvent- and DEN-treatedgroups by 7 days. The concentration of threepromutagenic lesionswas maximal at 6 h after DEN administration. The detectablelevel of the O⁶EG adduct was negligible by 24 h after DEN administration,while the two O-alkylpyrimidines, O²ET and O²ET, were retainedfor much longer periods. Single hepatocytes expressing PGSTwere observed by 2 days after DEN administration, while smallfoci of PGST-expressing hepatocytes could be reliably detectedby 2 weeks. Two phases of PGST expression in single hepatocyteswere observed. The first phase was maximal at day 3 and completeby day 6, while the second reached a plateau by day 8 and wasmaintained for the 28 days of the study. The presence of thethree O-alkylation adducts during a time of enhanced cellularproliferation suggests that all three promutagenic adducts maycontribute to the initiation that results in the partial hepatectomy/DENmodel of rat hepatocarcinogenesis.     

Effects of pinocembrin on the initiation and promotion stages of rat hepatocarcinogenesis

Pinocembrin (5, 7-dihydroxyflavanone) is a flavanone extracted from the rhizome of Boesenbergia pandurata. Our previous studies demonstrated that pinocembrin had no toxicity or mutagenicity in rats. We here evaluated its effects on the initiation and promotion stages in diethylnitrosamine-induced rat hepatocarcinogenesis, using short- and medium-term carcinogenicity tests. Micronucleated hepatocytes and liver glutathione-S-transferase placental form foci were used as end point markers. Pinocembrin was neither mutagenic nor carcinogenic in rat liver, and neither inhibited nor prevented micronucleus formation as well as GST-P positive foci formation induced by diethylnitrosamine. Interestingly, pinocembrin slightly increased the number of GST-P positive foci when given prior to diethylnitrosamine injection.     

Proliferation of carcinogen-damaged hepatocytes during cell-cycle-dependent initiation of hepatocarcinogenesis in the rat

Hepatocyte proliferation and damage to DNA were characterized during the initiation phase of carcinogenesis in livers of rats that had received a single administration of the methylating agent methyl(acetoxymethyl)nitrosamine (DMN-OAc). Quiescent non-proliferating hepatocytes in intact livers did not appear to be susceptible to initiation by DMN-OAc, whereas proliferating hepatocytes in the S phase appeared to have greatest risk. To characterize the phenomenology of S-phase-dependent initiation further, the fractions of hepatocytes in the S and M phases of the cell cycle were enumerated at various times after treatment with DMN-OAc. Hepatocytes treated when in G1 experienced a delay of up to 20 h in the onset of S phase and a reduced rate of entry into the S and M cycle phases. Hepatocytes treated when in S phase experienced considerable delay in progression to mitosis due to part to inhibition of DNA replication. Hepatocytes treated when in late S/G2 also demonstrated a delay in progression into mitosis. The levels of 7-methylguanine and O6-methyldeoxyguanosine were quantified in the nuclear DNA of proliferating hepatocytes. The kinetics of removal of these lesions appeared to be first-order (half-life = 24 h). Hepatocyte risk of initiation was modeled by a function which summed over time the product of the fraction of hepatocytes in the S phase and the fraction of residual, unrepaired damage to DNA. For hepatocytes treated when in early G1, the time-weighted frequency of premutagenic DNA damage that was present during DNA replication was estimated to be less than half of that for hepatocytes treated when in early S. The results suggest that cell-cycle-dependent variation in sensitivity to initiation of hepatocarcinogenesis may be, in part, due to efficient removal of potentially carcinogenic lesions from DNA during an extended G1. The apparent high sensitivity of hepatocytes in late S/G2 suggests the contribution of additional factors.     

Phenobarbital as a promoter in the initiation/selection process of experimental rat hepatocarcinogenesis

Supplementary introduction of a phenobarbital (PB) promotionstep after the Solt and Farber procedure dramatically increasesthe number of phenotypically-altered hepatocytes. These hepatocytesoccupy {small tilde}40% of the liver volume after one week ofPB treatment. These areas constitute a relatively uniform cellularpopulation with altered histological pheno-type and with distincthistochemicai markers used by other authors for the detectionof premalignancy. This procedure leads to the appearance ofnumerous hepatocellular carcinomas at approximately the 36 weeksstage. It was suggested that the early hepatocellular alterationsafter the initiation/selection procedure followed by PB mightcorrespond to the hyperplasia of phenotypically-altered epidermalcells at the conversion step of mouse skin tumor promotion.     

Chemopreventive effects of coumaperine from pepper on the initiation stage of chemical hepatocarcinogenesis in the rat.

This study was designed to investigate the chemopreventive action of three natural products, coumaperine, aurapten and an extract from rosemary, against the initiation stage of rat hepato-carcinogenesis. Coumaperine has been isolated from white pepper as a naturally occurring antioxidative agent, but its potential modifying effects on carcinogenesis remain unclear. In experiment 1, a modification of the model developed by Tsuda et al. was applied, with assessment of numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci in male F344 rats. Coumaperine, aurapten and the extract from rosemary were administered i.g. at 100 mg / kg / day once daily for 5 days with initiation by diethylnitrosamine (DEN) on day 4 (20 mg / kg, i.p.). Numbers and areas of GST-P-positive foci in each group given test chemicals tended to be decreased as compared to the vehicle control group values, significance being achieved for number with coumaperine. Experiment 2 was planned to investigate the mechanism of the inhibitory effects of coumaperine. Livers at 8 h after initiation by DEN were examined with coumaperine administered at 100 mg / kg / day once daily for 3 days. Proliferating cell nuclear antigen (PCNA)-positive cells tended to be decreased as compared to the vehicle control, but no effects on apoptosis or cytochrome P-450 (CYP) 2E1 expression were apparent. Our results suggest that coumaperine provides protection against initiation of hepatocarcinogenesis, and that this is related to inhibition of cell proliferation.     

SHORT COMMUNICATION: In vitro colonization ability appears soon after initiation of hepatocarcinogenesis in the rat

Hepatocytes were isolated from rat livers at various intervalsafter initiating hepatocarcinogenesis by a single administrationof methyl(acetoxymethyl)nitrosamine after a partial hepatectomy.Proliferative hepatocyte colonies were regularly observed inprimary cultures derived from initiated livers when these cultureswere incubated in medium containing the liver tumor promoter,phenobarbital. Fewer colonies generally were observed in initiatedcell cultures that were incubated without phenobarbital andin phenobarbital-induced cultures derived from non-initiatedcontrol livers. Because in vitro proliferative activity is acharacteristic of malignant hepatocytes these results suggestthat the feature of in vitro colonization may characterize apopulation of carcinogen-altered hepatocytes.     

Comparison of induction of DNA single-strand breaks and initiation of rat hepatocarcinogenesis by different nitrosamines

The capacities of nitrosamines to induce DNA single-strand breaks (SSB) and to initiate carcinogenesis in rat liver were compared. N-Nitrosodiethanolamine (NDELA), N-nitrosoethylhydroxyethylamine (NEHEA) and N-nitrosodiethylamine (NDEA) were equipotent in inducing DNA SSB when administered by gavage at doses of 0.35 mmol/kg, 0.015 mmol/kg and 0.37 mmol/kg, respectively. Male Wistar rats were injected with these nitrosamines and were then submitted to a selection procedure. Ten rats per group were sacrificed one week after the end of the selection to see the effects of the nitrosamines on the development of preneoplastic lesions. The numbers of gamma-glutamyl transferase (GGT)-positive lesions per cm2 were 0.8, 2.1, 5.2 and 40.1 in rats treated with saline, NDELA, NEHEA and NDEA, respectively. N-Nitrosobis(2,2,2-trifluorethyl)amine (6F-NDEA), a nongenotoxic and noncarcinogenic nitrosamine, induced 0.7 GGT-positive lesions per cm2. Ten rats per group also received 0.05% phenobarbital in their drinking-water and were killed six months after initiation in order to see the effect of the different nitrosamines on the incidence and yield of tumours. Two extrahepatic cancers were found after administration of NEHEA, whereas two hepatocellular carcinomas were detected after injection of NDEA. No cancer developed in the other groups. Although other factors may influence the process, these results indicate that no simple correlation can be established between induction of SSB in DNA and initiation of tumours by nitrosamines in rat liver.     

The role of hepatocyte heterogeneity in the initiation of hepatocarcinogenesis

N-Nitrosodimethylamine (NDMA) is a carcinogen in rat liver whileN-nitrosomethylbenzylamine (NMBzA) produces no liver tumorsbut is a potent esophageal carcinogen in the rat. Both nitrosamines,however, are metabolically activated in the liver and methylatehepatic DNA. The reasons for their different carcinogenic propertiesin rat liver are unclear. Here we show that as expected, NDMAproduces large numbers of putative initiated hepatocytes thatoverexpress the placental form of glutathione S-transferase(GST-P⁺ hepatocytes). Hepatocyte division induced by the hepatotoxiceffect of NDMA occurs principally in the periportal region ofthe liver lobule, while O⁶-methylguanine formation is principallyin the DNA of perivenous cells. These two effects lead to theproduction of GST-P⁺ hepatocytes in roughly equal numbers throughoutthe liver lobule. NMBzA also induces the formation of a small,but significant number of GST-P⁺ hepatocytes. The NMBzA-inducedGST-P⁺ hepatocytes are localized within the perivenous zoneof the liver lobule. Since, unlike NDMA, NMBzA produces no hepatocellularnecrosis and hence does not induce regenerative cell division,these results suggest that NMBzA initiates only those hepatocytesadjacent to the hepatic vein that are spontaneously dividingat the time their DNA becomes methylated by the nitrosamine.We used partial hepatectomy to stimulate cell division in specificregions of the liver lobule. When the peak of DNA methylationproduced by NMBzA in the perivenous cells coincided with a peakof cell division in that region, an increased number of GST-P⁺hepatocytes was induced. Our results suggest that the potencyof initiating agents in the liver depends both on their abilityto form mutagenic lesions in DNA and to induce division in thespecific hepatocytes that contain the modified DNA.     

The role of the stages of initiation and promotion in phenotypic diversity during hepatocarcinogenesis in the rat.

Differences in the distribution of phenotypic alterations in initiated and promoted cell populations reflect both the dose and the action of the specific initiating agent, as well as the mechanism of action of the promoting agent. The use of multiple phenotypic markers to characterize AHF should allow further experimentation on the characteristics of promoting agents in hepatocarcinogenesis, especially their ability to promote separate populations of initiated cells, and on those characteristics of initiated cells that enable certain ones to survive and multiply in the specific environment provided by a promoting agent. Studies of promoting agents with differing mechanisms of action should further allow questions of reversibility, substitution and additivity of the actions of promoting agents to be addressed. The possibility that individual promoting agents do not enhance the growth of the entire population of initiated cells indicates that study of combinations of promoting agents is an important future direction of research. Therefore, information on the characteristics of promoting agents, singly and in combination, is necessary to assess more accurately the contribution of promoting agents to the carcinogenic risk for humans distinct from the effects of initiating agents and complete carcinogens.     

Effect of hepatocarcinogens on the binding of glucocorticoid-receptor complex in rat liver nuclei.

The effects of a number of carcinogens and hepatotoxins on the binding kinetics of the interactions of glucocorticoidcytosol receptor complex with nuclear acceptor sites in rat liver were investigated. Both the apparent sites in rat liver were investigated. Both the apparent concentration of nuclear binding sites and the Kd were significantly diminished following treatment of rats with sublethal doses of the carcinogens aflatoxin B1, diethylnitrosamine, dimethylnitrosamine, thioacetamide, 3'-methyl-4-dimethylaminoazobenzene, 4-dimethylaminoazobenzene, and 3-methylcholanthrene. Treatment with actinomycin D resulted in a slight reduction in the apparent concentration of nuclear acceptor sites but had no effect on the nuclear binding Kd. The hepatotoxic but noncarcinogenic analgesic, acetaminophen, as well as the weakly toxic aflatoxin B1 cognate, aflatoxin B2, were without effect on the kinetics or binding capacity of glucocorticoid-nuclear acceptor site interaction. These experiments suggest that chemically induced alteration of functional glucocorticoid binding sites on chromatin may be involved in the biochemical effects produced in liver by carcinogens of several chemical types. This experimental model may provide a useful approach for further elucidation of early events in carcinogenesis.     

Further characterization of the ability of hepatocarcinogens to lower rat liver aryl sulfotransferase activity

Aryl sulfotransferase (AST) activity in rat liver is thoughtto be a primary pathway in the bio-activation of various hepatocarcinogensto forms which act as ultimate carcinogens in chemical hepatocarcinogenesis.In an effort to understand the significance of rapid and sustaineddecreases in liver AST that accompany dietary administrationof hepatocarcinogens and to further assess its relationshipto carcinogenic processes, we determined the abilities of variousxenobiotics known to be hepatocarcinogens or non-hepatic carcinogensto lower AST activity. We also determined whether the co-administrationof the AST enzyme inhibitor, pentachlorophenol, with hepatocarcinogenswill abrogate the lowering of AST activity caused by hepatocarcinogenswhich do not utilize AST for bio-activation versus hepatocarcinogenswhich can utilize AST. Among carcinogens tested thus far, wehave found the AST activity of liver cytosols to be loweredby the hepatocarcinogens 2-acetylaminofluorene, ethionine, 3'-methyl-4-dimethylaminoazobenzene,thioacetamide, aflatoxin B₁, diethylnftrosamine and benzidine,but not by the non-hepatic carcinogens 2-acetylaminophenanthreneor 3-methylchol-anthrene. Pentachlorophenol reversed activitylosses when co-administered with all carcinogens which lowersAST activity with the exception of ethionine and thioacetamide.We suggest that AST activity lowering is relatively specificfor liver carcinogens and involves two different mechanisms.     

Chronic liver injury promotes hepatocarcinogenesis of the LEC rat

The Long-Evans rat with a cinnamon-like color (LEC) is a mutant rat that spontaneously suffers from chronic liver injury and subsequent hepatocellular carcinoma (HCC) caused by abnormal copper accumulation in the liver. We attempted to elucidate the role of prolonged liver cell injury on LEC rat hepatocarcinogenesis using a copper-deficient diet (CuDD) to inhibit the occurrence of consequent liver injury. The animals were fed the CuDD from the age of 4 weeks until being killed at the age of 10 months. Diethylnitrosamine (DEN) was administered at the age of 8 weeks. Groups fed a basal diet (BD) with or without the administration of DEN were also assigned as control groups. The animals fed the BD manifested liver injury, while those fed the CuDD did not show liver dysfunction until death. The number and volume of glutathione S-transferase placental form (GST-P)-positive preneoplastic lesions in the liver, which were calculated from the data on two- dimensional planes, were examined to clarify the promotive effect of chronic liver injury on the development of HCC. Regarding the size of the lesions, which indicated the intensity of the promotive effect, the lesions in the livers of rats fed the BD with DEN were much larger than those of rats fed the CuDD with DEN. Feeding the LEC rats with CuDD completely suppressed the manifestation of liver injury, and it was clearly shown that prolonged liver injury had a promotive effect on the LEC rat hepatocarcinogenic process.      

Quantitative stereologic study of the effects of varying the time between initiation and promotion on four histochemical markers in rat liver during hepatocarcinogenesis

The effects of varying the interval of time between initiation with diethylnitrosamine (DEN) and promotion by phenobarbital (PB) on the development of altered hepatic foci (AHF) and hepatomas in female Fischer 344 rats was investigated. The intervals between DEN initiation after a 70% partial hepatectomy and a subsequent 6 month period of promotion by feeding of PB were 1 day, 1 week, 1 month, 2 months, 6 months and 11 months. The number and volume percentage occupied by AHF were determined by quantitative stereologic methods on serial frozen sections stained for the markers gamma-glutamyltranspeptidase (GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6Pase) and the placental form of glutathione S-transferase (GST-pi). The number of AHF was greatest when the initiation-promotion interval was only 1 day, and there was a tendency for the number of AHF to decrease as the interval between initiation with DEN and the start of PB promotion was extended. An 11 month delay between initiation and promotion resulted in only 20% fewer AHF than when promotion was begun 1 day after initiation. On the other hand, the volume percentage fraction of AHF did not change when the initiation-promotion interval was increased from 1 day to 2 months. An interval of 6 months roughly doubled the volume percentage fraction, but an interval of 11 months led to a 7- to 8-fold increase in the volume percentage of AHF over that from a 1 day interval. The phenotypic distribution of AHF was significantly lower in relation to certain markers, especially GGT and GST-pi, in those animals only initiated with DEN compared with those initiated with DEN and promoted with PB. When no exogenous promotion was given, there was still a nearly linear increase in both the number and volume percentage occupied by AHF in the liver of rats initiated with DEN. On the other hand, rats subjected to a 1 week interval between DEN initiation and PB promotion exhibited the greatest number of hepatocellular carcinomas 14 months after initiation, compared with other groups. These studies demonstrated a gradually decreasing effectiveness of PB as a promoting agent to stimulate the growth of all AHF initiated by DEN as the interval between initiation and promotion was extended.(ABSTRACT TRUNCATED AT 400 WORDS)     

Promoting effect of oxazepam in rat hepatocarcinogenesis

In order to check whether the benzodiazepine, oxazepam (OZ),has a modulating effect on the development of liver cancer,it was given to rats previously submitted to two different protocolsof hepatocarcinogenesis: the resistant hepatocyte protocol andPitot's model (initiation-promotion). Its effects were comparedwith those of phenobarbital (PB) administered under the sameconditions. As compared with basal diet, a diet containing 0.05%of PB and 0.1% of OZ enhanced, in both models, the developmentof gamma glutamyltransferase-positive lesions in early stages.OZ also had a promoting effect on the development of liver cancersin later stages in both models whereas PB only enhanced it inthe resistant hepatocyte protocol. Thus, like PB, OZ may havea promoting effect on liver cancer in rats.     

Promotion of rat hepatocarcinogenesis by praziquantel.

Praziquantel, the widely used anti-helminthic agent, was investigated for hepatocarcinogenesis-promoting potential using a medium-term liver bioassay system for carcinogens. F344 male rats were given a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and then starting 2 weeks later, received praziquantel in the diet at concentrations of 1.5 or 0.5%, or intragastrically at a dose of 1,500 mg/kg once a week for 6 weeks. Control groups received DEN or praziquantel alone. All rats were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. Development of glutathione S-transferase placental form-positive foci in the liver was significantly increased in terms of both number and area with the 1.5% dose, while only area was affected by the 0.5% dose. The results thus indicate that praziquantel at high dose has promoting potential in rat hepatocytic tumorigenesis.