Evaluation of liver cell proliferation during ciprofibrate-induced hepatocarcinogenesis

To determine if the carcinogenic potential of peroxisome proliferators is dependent upon their ability to induce cell proliferation, we have investigated the extent of cell proliferation in the livers of rats fed ciprofibrate, a peroxisome proliferator. Male rats were maintained on a diet containing ciprofibrate (0.025% w/w) and killed at selected intervals following 1 week of continuous [3H]thymidine labeling. Evaluation of labeling indices demonstrated a significant increase in cell proliferation during the first week but not in rats killed at the end of 5 and 20 weeks of treatment. Increases in hepatocyte nuclear labeling were found at 40 and 70 weeks of ciprofibrate administration which coincided with the appearance in livers of putative preneoplastic and neoplastic lesions. In a short-term feeding study, ciprofibrate and ethoxyquin were fed to rats at a dietary concentration of 0.025% and 0.5%, respectively, either alone or in combination for 7 days. Ciprofibrate and ethoxyquin either alone or in combination produced marked hepatomegaly and a significant increase in DNA synthesis as demonstrated by [3H]thymidine incorporation and autoradiographic studies. DNA synthesis in the group receiving ciprofibrate and ethoxyquin simultaneously, was slightly more than in animals that received either compound alone, suggesting a synergistic effect, although chronic feeding of these agents together resulted in inhibition of liver carcinogenesis (Rao, M. S. et al. (1984) Cancer Res., 44, 1072-1076). The results of this study further suggest that cell proliferation induced by peroxisome proliferators may be less important in carcinogenesis than peroxisome proliferation induced by these compounds.     

Inhibitory effect of antioxidants ethoxyquin and 2(3)-tert-butyl-4-hydroxyanisole on hepatic tumorigenesis in rats fed ciprofibrate, a peroxisome proliferator

The objective of this study was to test the hypothesis that hepatocarcinogenesis by peroxisome proliferators, a novel class of chemical carcinogens, is mediated either directly by carcinogenic H2O2, generated by peroxisomal oxidase(s) or indirectly by free radicals produced from H2O2, and that antioxidants could retard or inhibit neoplasia by scavenging active oxygen (super-oxide radicals O(2), hydrogen peroxide, hydroxyl radicals HO, and singlet oxygen 1O2). Accordingly, the effect of synthetic antioxidants 2(3)-tert-butyl-14-hydroxyanisole and ethoxyquin on the peroxisome proliferator 2-[4-(2,2-dichlorocyclopropyl)phenoxy]2-methyl-propionic acid (ciprofibrate)-induced hepatic tumorigenesis has been examined in male Fischer 344 rats. Rats were fed either a 2(3)-tert-butyl-4-hydroxyanisole (0.5% w/w)- or ethoxyquin (0.5% w/w)-containing diet with or without ciprofibrate (10 mg/kg of body weight) for 60 weeks. Rats fed ciprofibrate (10 mg/kg of body weight) in the diet or fed a diet with no added chemicals served as controls. Results of this study demonstrated that ethoxyquin markedly inhibited the hepatic tumorigenic effect of ciprofibrate, as evidenced by a decreased incidence of tumors, a decreased number of tumors per liver, and a reduced tumor size. 2(3)-tert-Butyl-4-hydroxyanisole also caused a significant decrease in the incidence and number of hepatocellular carcinomas that were larger than 5 mm. The present data suggest that the inhibitory effect of antioxidants on ciprofibrate-induced hepatic tumorigenesis may be due to H2O2 and free radical-scavenging property of ethoxyquin and 2(3)-tert-butyl-4-hydroxyanisole, since these antioxidants do not prevent peroxisome proliferation and induction of H2O2-generating peroxisomal enzymes in livers of rats fed ciprofibrate. Whether the inhibitory effect of antioxidants is exercised on the presumptive H2O2 initiation process and/or on the postinitiation growth phase of foci and nodules in liver is, at present, unknown.     

Short-term exposure to the peroxisome proliferators, perfluorooctanoic acid and perfluorodecanoic acid, causes significant increase of 8-hydroxydeoxyguanosine in liver DNA of rats

To elucidate the relationship between peroxisome proliferation by perfluorinated compounds and oxidative DNA damage, perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), perfluorobutyric acid (PFBA) and perfluorooctane (PFO) were administered to 6-week-old F-344 male rats. After a single intraperitoneal (i.p.) injection of PFOA, PFBA or PFO in corn oil at a dose of 100 mg/kg, significant increases of liver weight and 8-hydroxydeoxyguanosine (8-OH-dG) levels in liver DNA were observed in PFOA-treated rats. Oral administration of powdered diet containing 0.02% PFOA or 0.01% PFDA for 2 weeks resulted in significant increases of liver weight and 8-OH-dG levels in liver DNA in rats given both chemicals. On the other hand, no increase in 8-OH-dG levels in kidney DNA was found in either of the studies. Our results demonstrate that, as with other peroxisome proliferators (phthalic ester plasticizers and hypolipidemic drugs), PFOA and PFDA induced peroxisome proliferation also leads to organ specific oxidative DNA damage.     

Nafenopin-induced rat liver peroxisome proliferation reduces DNA methylation by N-nitrosodimethylamine in vivo

The hypolipidaemic drug nafenopin (NAF) has been shown to enhancethe hepatocarcinogenic effect of N-nitrosodimethylamine (NDMA)and N-nitrosodiethylamine in rats. We have investigated whetherthe NAF-induced peroxisome proliferation in hepatocytes interfereswith NDMA's metabolism and interaction with DNA. Adult maleWistar rats received a single i.p. injection of [¹⁴C]NDMA (2mg/kg) and were killed 4 h later. DNA was isolated from liverand kidney, hydrolysed in 0.1 N HCI and analysed by Sephasorbchromatography. In rats pre-treated with NAF (0.2% in the dietover a period of 3 weeks), the concentration of N7-methylguaninein hepatic DNA (µmol/mol guanine) was 46% below controlvalues. This is probably due to the greater amount of targetDNA, as NAF caused a marked hepatomegaly with a 50% increasein total liver DNA content. Concentrations of N7-methylguaninein kidney DNA were twice as high in NAF-pre-treated animalswhen compared to control rats. This is unlikely to result froma shift in the metabolism of NDMA from liver to other rat tissuessince the time course and extent of the conversion of [¹⁴C]NDMAto ¹⁴CO₂ and ¹⁴C-labelled urinary metabolites were identicalin NAF-treated and control animals. There was no indicationthat NAF inhibits the activity of the hepatic O⁶-alkylguanine-DNAalkyltransferase.     

Supplemental dietary calcium fails to alter the acute effects of 1,2-dimethylhydrazine on O6-methylguanine, O6-alkylguanine-DNA alkyltransferase and cellular proliferation in the rat colon

Prior studies from our laboratory have demonstrated that K-rasG to A mutations were detectable in a high percentage of carcinomaswhich developed in the colons of animals treated with the knowncolonic procarcinogen, 1,2-dimethyl-hydrazine (DMH). Moreover,in this model, the incidence of these mutations was decreasedby a supplemental dietary calcium regimen which concomitantlydecreased the frequency of rats with multiple tumors as wellas tumor size. In an attempt to clarify the possible mechanism(s)involved in this antimutagenic effect of supplemental calcium,two groups of Sprague—Dawley rats were fed semisyntheticdiets containing either 0.87 or 1.80% cakium by weight for 3weeks, s.c. injected with 100 mg/kg of DMH and killed priorto and at various time periods (16-144 h) after injection. Thecolons of animals were analyzed and compared with respect toO⁶-methylguanine content in DNA, O⁶-alkylguanine-DNA alkyltransferaselevels as well as cellular proliferation, as assessed by immunohistochemicalstaining of colonic crypts by bromodeoxyundine. In certain experiments,these parameters were also analyzed in the proximal and distalcolon before and at various times after administration of DMH.The results of these experiments demonstrated that supplementaldietary calcium was not found to influence significantly O⁶-methylguaninelevels, alkyltransferase levels or cellular proliferation inthe entire colon or in either colonic segment before or afterthe acute administration of DMH. DMH did, however, differentiallyalter all three of these biochemical parameters in the colonicsegments (distal > proximal), possibly due to a greater degreeof metabolic activation in the distal colon.     

Formation of 8-hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator

The mechanism by which nongenotoxic peroxisome proliferators induce hepatocellular carcinomas in rats and mice remains intriguing. The available experimental evidence suggests that the proliferation of peroxisomes and induction of peroxisome-associated enzymes results in oxidative stress which then leads to tumorigenesis. However, so far no direct evidence for oxidative DNA damage in livers of peroxisome proliferator-treated animals has been established. In the present study we have examined the DNA obtained from the livers of rats treated with ciprofibrate, a potent peroxisome proliferator, for variable periods of time for 8-hydroxydeoxyguanosine (8-OH-dG), an adduct that results from the damage of DNA caused by hydroxyl radical. Administration of ciprofibrate in diet at a concentration of 0.025% for 16, 28, 36, or 40 weeks resulted in progressive increases in the levels of 8-OH-dG. At 16, 28, and 40 weeks of ciprofibrate treatment, the 8-OH-dG in the liver DNA was significantly increased as compared to controls. This increase in 8-OH-dG levels is attributed to persistent peroxisome proliferation resulting from chronic ciprofibrate treatment as no increase in 8-OH-dG was found in liver DNA of rats that received a single large dose of ciprofibrate. The results of this study clearly demonstrate, for the first time, that persistent proliferation of peroxisomes leads to specific oxidative DNA damage.     

Dose-response studies of MeIQx in rat liver and liver DNA at low doses

2-Amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) is aheterocyclic amine mutagen found in cooked meats and is carcinogenicin mice and rats at high doses (mg/kg body wt). Humans, however,are exposed to low amounts (p.p.b.) in the diet, and the effectscaused by exposure to human equivalent doses of MeIQx have beendifficult to determine accurately. We report on the effect ofMeIQx exposure on liver bioavailability, hepatic DNA bindingand MeIQx persistence in both liver tissue and liver DNA afteracute (24 h), and subchronic (7 day and 42 day) exposures inmale Sprague-Dawley rats. Male Sprague-Dawley rats were administered[2-¹⁴C] MeIQx either by gavage or in the diet for 1, 7 or 42days (1x10^-6mg/kg day up to 3.4x10^-2 mg/kg day dose) and the[2-¹⁴C]MeIQx was measured by accelerator mass spectrometry (AMS).Assessment of the kinetics of hepatic MeIQx DNA adduct formationover 42 days (1.1x10^-4 mg [2-¹⁴C]MeIQx kg daily dose) showsthat steady-state [2-¹⁴C]MeIQx tissue concentrations of 138± 15 pg/g liver and DNA adduct levels of 113 ±10 ag adduct/µg DNA were reached at 14–28 days and28 days respectively. The relationship between administereddose and either hepatic MeIQx DNA adduct levels or MeIQx tissuelevels are linear for the 24 h, 7 day and 42 day exposures.Furthermore, MeIQx adducts persist for at least 14 days afterexposure ceases. These data suggest that bloavailability andDNA adduction by MeIQx increase linearly with increasing dosefor both acute and subchronic exposures. These data also showthat MeIQx DNA adducts are useful in predicting daily exposureand support a linear extrapolation in the risk assessment ofMeIQx. However, the quantitative relationship between DNA adductsand tumor formation will also depend on the specific tissueand the subsequent steps needed for tumor progression.     

Lack of initiating activity of the peroxisome proliferator ciprofibrate in two-stage hepatocarcinogenesis

The peroxisome proliferator ciprofibrate was examined for its ability to initiate hepatocarcinogenesis in rats. Ciprofibrate was fed in the diet at levels of 0%, 0.01% and 0.025% for 2 weeks in order to induce steady-state peroxisome proliferation. Rats were then subjected to partial hepatectomy and then maintained for 6 months on a basal diet or one containing 0.05% phenobarbital. Ciprofibrate treatment did not increase the number or volume of altered hepatic foci (putative preneoplastic lesions). However, ciprofibrate treatment increased liver weights in a dose-dependent manner in rats which did not receive phenobarbital. It is concluded that ciprofibrate-induced peroxisome proliferation is not sufficient to induce initiation, but that a permanent change is produced which results in an increased liver weight.     

Enhancement of methylbenzylnitrosamine-induced esophageal carcinogenesis in zinc-deficient rats: effects on incorporation of [3H]thymidine into DNA of esophageal epithelium and liver

[³H]Thymidine incorporation was measured in esophageal epitheliumand liver DNA of rats fed a control diet ad libitum, a zinc-deficientdiet or a control diet, pair fed to the food intake of zinc-deficientrats. Additional groups of rats on each diet were dosed i.g.(2.5 mg/kg body wt) one or six times with the esophageal carcinogenmethylbenzylnitrosamine (MBN). The zinc-deficient diet significantlyincreased [3H]thymidine incorporation into esophageal epitheliumDNA at 14 days and reached a maximum at 28 days which was approximatelythree times that of the control ad libitum diet rats. Pair feedingsignificantly decreased incorporation into the esophageal epitheliumDNA relative to the control ad libitum diet, by approximately60% after 14 days. None of the diets affected [3H]thymidineincorporation in liver DNA. [³H]Thymidine incorporation wassignificantly inhibited for 48–72 h in the esophagealepithelium and liver DNA of all dietary groups after a singlei.g. dose of MBN before returning to predos-ing levels. Theperiod of inhibition of [3H]thymidine incorporation was lengthenedin all groups after six doses of MBN; the greatest increase(10 days) being noted in the esophageal epithelium DNA of thezinc-deficient rats. This inhibition was followed by a significantincrease above predosing levels in the esophageal epitheliumbut not in the liver. The maximum increase occurred 7 days afterthe completion of MBN dosing in the control ad libitum group,at 14 days in the control pair-fed group and at 36 days in thezinc-deficient group. These findings suggest that the enhancementof MBN-induced esophageal tumors by zinc deficiency is due inpart to the increased proliferation of the target cells witha concomitant greater accessibility of the cellular DNA to thecarcinogen.     

Inhibition of ciprofibrate-induced hepatocarcinogenesis in the rat by dimethylthiourea, a scavenger of hydroxyl radical.

DNA damage caused by oxidative stress is considered to play an important role in peroxisome proliferator-induced hepatocarcinogenesis in rats and mice. In this study, we investigated the effect of dimethylthiourea (DMTU), a known hydroxyl radical scavenger, on ciprofibrate-induced hepatocarcinogenesis. Male F-344 rats were fed a diet containing 0.025% ciprofibrate and given daily intraperitoneal injections of DMTU (5 days a week) at a dose of 50 or 250 mg/kg body weight for 60 weeks at which time the study was terminated. Livers from all animals were analyzed grossly and microscopically for incidence, number and type of tumors. All rats given ciprofibrate alone developed altered areas, neoplastic nodules (NN) and hepatocellular carcinomas (HCC). Combined administration of ciprofibrate and DMTU resulted in inhibition of tumor development. In the group given higher doses of DMTU the incidence of NN was 100% and HCC 0%. The number of tumors per liver also significantly decreased (p<0.001). At lower dose levels DMTU caused significant reduction in the number of tumors per liver (p<0. 05) and a slight reduction (29%) in the incidence of HCC. The inhibitory effect of DMTU on ciprofibrate-induced hepatocarcinogenesis could be explained by hydroxyl radical scavenging properties of DMTU, resulting in decreased free radical induced DNA damage.     

In vivo distribution of a carcinogenic hepatic peroxisome proliferator: whole-body autoradiography of [14C]ciprofibrate in the mouse

The distribution of radiolabel in male mice was studied by whole-bodyautoradiography at intervals after oral administration of [¹⁴C]ciprofibrate,a carcinogenic hepatic peroxisome proliferator. Radioactivitywas rapidly taken up by the liver and to a lesser extent bythe brown fat within 9 h after oral dosing of ciprofibrate.The radioactivity levels in blood, interstitial fluid and fatdecreased during the first 3 days after dosing, but the liverremained densely labeled. Between 3 and 27 days after dosing,liver exhibited a stippled pattern as a result of heavier labelingapparently around the central veins. The relatively low levelsof radiolabel in extra-hepatic tissues observed after oral dosing,together with the prolonged retention in this region of theliver, is consistent with the hepatotropic effects (i.e. hepaticperoxisome proliferation and development of liver tumors) exertedby this compound.     

Response of phenobarbital- and ciprofibrate-exposed hepatocytes to growth factors in type I collagen gels

The response of promoter-exposed hepatocytes to the completehepatic mitogens hepatocyte growth factor (HGF), epidermal growthfactor (EGF) and acidic fibroblast growth factor (aFGF) wasstudied. Male Fisher 344 rats were administered phenobarbitalor ciprofibrate. Hepatocytes were isolated at various time pointsand cultured in type I collagen gels, a 3-dimensional culturesystem that allows stable long-term hepatocyte differentiation.DNA synthetic activity in response to addition of HGF, aFGFor EGF to the cultures was assayed by incorporation of [³H)thymidine.Administration of ciprofibrate to rats caused an immediate decreasedgrowth response in hepatocytes to all three growth factors.Phenobarbital administration caused a gradual decrease in responsivenessto the growth factors: after 10 days, hepatocytes became insensitiveto the mitogenic effects of HGF, EGF and aFGF. However, an earlyincrease in responsiveness to HGF and aFGF occurred in phenobarbital-exposedhepatocytes. The results indicate that phenobarbital and ciprofibratehave similar inhibitory effects on hepatocyte DNA synthesisin culture after long-term in vivo exposure. However, theirearly effects on hepatocyte growth response differ considerably,suggesting that their effects on cellular proliferation occurvia different mechanisms.     

Effect of copper administration on the incorporation of [3H]-thymidine into the liver DNA of rats stimulated by dimethyl-nitrosamine and diethylnitrosamine

The incorporation of [³H]thymidine into liver DNA of rats increased6–8 times 48 h after a single injection of dimethylnitrosamine(DMN, 30 mg/kg) and diethyl-nitrosamine (DEN, 100 mg/kg). Totest the suppressive effect of copper, the incorporation of[³H]thymidine in to liver DNA in the DMN groups or DEN groupspretreated with copper was measured 48 h after the administrationof DMN or DEN. The incorporation of [³H]thymidine into liverDNA of rats stimulated by the injection of DEN was strikinglysuppressed by the injection of cupric acetate (20 mg Ci/kg),but that of rats simultated by the injection of DEN was notsuppressed by the injection of copper. Some other metal salts,silver nitrate (20 mg Ag/kg), nickel acetate (20 mg Ni/kg) andbasic lead acetate (20 mg Pb/kg) did not significantly suppressthe incorporation of [³H]thymidine stimulated by DMN or DEN.The accumulation of copper was much higher in the liver of copper-administeredrats than that of nickel or lead in the liver of nickel-administeredrats or lead-administered rats. The accumulation of silver wascomparatively high in the liver of silver-administered rats.     

Inductions of oxidative DNA damage and mesothelioma by crocidolite, with special reference to the presence of iron inside and outside of asbestos fiber

Inductions of oxidative DNA damage (oh⁸dG) in vitro and peritonealmesothelioma in rats (F344, female) were compared between crocidolite(CR) and de-ironized crocidolite [DCR, washed by HCl and ethylenediaminetetraacetic acid (EDTA)] to verify the hypothesis that reactiveoxygen species contribute to carcinogenesis, focusing on therole of iron present inside or outside of the CR. The yieldof oh⁸dG was 14.6 oh⁸dG/10⁵ in CR and 30.2 in DCR under simpleincubation with DNA. In the incubation systems added severalchemicals and H₂O₂, OCR induced higher levels of oh⁸dG thanCR. Especially, the addition of Fe₂O₃ and H₂O₂ to OCR increasedoh⁸dG in DNA depending on the Fe₂O₃ concentration, however,this tendency was not observed in the same system of CR. Surprisingly,7 out of 10 rats died within 2 days after the injection of 10mg of Fe₂O₃ following the DCR injection (5 mg/rat), showingnecroses of hepatocytes from the surface of each lobe whereCR and Fe₂O₃ particles had been deposited together. There wasno death in other groups of rats. One year after the i.p. injectionof CR (5 mg/rat, single injection), mesotheliomas were foundin all rats administered OCR and Fe (2 mg/rat, once a week,for 35 weeks), in 4 rats of OCR alone (n = 10), in 5 rats ofCR alone (n = 10) and in none of the rats administered Fe₂O₃alone (n = 10). Therefore, present results indicate that theinduction of oxidative DNA damage changed even when the sametype of asbestos was washed by chemical treatment, and Fe₂O₃promoted the development of mesothelioma which was induced byOCR.     

Effects of nickel compounds on incorporation of [3H] thymidine into DNA in rat liver and kidney

In vivo incorporation of [³H] thymidine into DNA was determinedin rats at 28 h after partial hepatectomy. Administration ofnickel carbonyl (Ni(CO)₄) at 2 or 4 h before sacrifice inhibited[³H] thymidine uptake into liver and kidney DNA. For example,in rats killed 4 h after i.v. injection of Ni(CO)₄ (2 mg Ni/100g), [³H]-labelling of liver DNA averaged 54 (SE ± 10)%of controls (p<0.05), and [³H]-labelling of kidney DNA averaged53 (SE ± 6)% of controls (p<0.01). Injection of NiCI₂(2 mg Ni/100 g, i.m.) 4 h before death did not significantlyaffect [³H] thymidine uptake into liver DNA, but did inhibit[³H] thymidine uptake into kidney DNA (65 ± 6%, p<0.02).Binding of ⁶³Ni to DNA in liver and kidney of rats killed 4h after injection of ⁶³Ni(CO)⁴or ⁶³NiCl² ranged from 0.3 to2.2 mol ⁶³Ni/mol of DNA nucleotides. Ultracentrifugation ofDNA on alkaline sucrose gradients did not reveal any differencesbetween sedimentation profiles of hepatic DNA from Ni(CO)⁴-treatedrats versus paired control rats.     

Accumulation and persistence of DNA adducts of the synthetic steroid cyproterone acetate in rat liver

Cyproterone acetate (CPA) is a synthetic steroid which is widelyused in antlandrogenic and gestagenic drugs. We have recentlyshown that CPA induces DNA adducts in cultured rat hepatocytesand in rat liver (1). In the present investigation, we studiedthe persistence and accumulation of CPA-derived DNA adductsin the liver of rats using the 32 technique. To study the persistenceof CPA-DNA adducts, rats were treated with a single oral doseof 10 (female rats) or 100 mg CPA/kg body wt (male rats). FourDNA adducts were detected in the liver of both gender. In femalerats, maximal total DNA adduct levels of 3.40±0.04 adducts/10⁶nucleotides were observed after 1 week. Eleven weeks later,40% of the adducts determined after 1 week were still detectable.In male rats, maximal hepatic DNA adduct levels of     

Effects of dietary sinigrin or indole-3-carbinol on O6-methylguanine-DNA-transmethylase activity and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA methylation and tumorigenicity in F344 rats

The effects of dietary sinigrin and indole-3-carbinol(I3C) onDNA methylation and O⁶-methylguanine-DNA-trans-methylase activity,factors which may be of importance in the induction of tumorigenicityby the tobacco-specific nitrosamine 4-(methymitrosainino)-1-(3-pyridyD-1-butanone(NNK), were investigated. Additionally, the effects of dietarysinigrin on NNK tumorigenicity were assessed in a two-year bioassayin F344 rats. DNA methylation in target tissues of NNK tumorigenesiswas examined in F344 rats administered [³H-CH₃](NNK(0.6mg/kg,four doses)s.c. and fed control or experimental diets for twoweeks. Dietary sinigrin ata concentration of 3 µmol/gdiet decreased 7-methylguanine formation in hepatic DNA, buthad no effect on 7-methylguanine levels of lung or nasal mucosaDNA. Dietary 13C at a concentration of 30µmol/g diet increased7-methylguanine levels in hepatic DNA, but decreased DNA methylationin lung and nasal mucosa. No effects on O⁶-methylguanine-DNA-transmethylaseactivity were observed in tissue extracts derived from the livers,lungs and nasal mucosae of rats fed diets containing sinigrinor 13C. These results suggested that dietary sinigrin mightreduce the incidence of NNK-induced hepatic tumors with no effecton NNK tumorigenesis of the lung and nasal cavity, whereas 13Cmight increase hepatic tumor incidence and reduce NNK tumorigenesisof the lung and nasal cavity. The bioassay results showed thatdietary sinigrin had no effect on NNK tumorigenesis in thesetarget tissues. However, dietary sinigrin plus NNK resultedin a significant incidence of pancreatic tumors, a rare occurrencein F344 rats. While the results from DNA methylation studiesare in agreement with the bioassay data for lung and nasal cavity,the absence of any inhibitory effect of dietary sinigrin onNNK hepatic tumorigenesis indicates that factors other thanDNA methylation and (Amethylguanine repair should be consideredin assessing the effects of dietary compounds on NNK hepatictumorigenesis. The contrary effects on NNK-induced hepatic DNAmethylation by sinigrin and 13C, two major components of cruciferousvegetables, demonstrate the complexities of dietary modulationof carcinogenesis.     

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.     

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.