Tumor necrosis factor alpha is not required for WY14,643-induced cell proliferation

It has been proposed that the cytokine tumor necrosis factor alpha (TNFalpha) stimulates peroxisome proliferator-induced hepatic cell proliferation. To test this hypothesis, induction of peroxisome proliferation and hepatocyte proliferation were compared in wild-type C57Bl/6 and TNFalpha knockout mice. Animals were dosed with either vehicle or 100 mg/kg/day WY14,643 by oral gavage for 4 days. Liver to brain weight ratios increased in both wild-type and TNFalpha knockout animals after WY14,643 administration. In addition, WY14,643-treated wild-type C57Bl/6 and TNFalpha knockout mice displayed marked hepatic induction of fatty acyl-CoA oxidase activity (approximately 8-fold) and mRNA content (approximately 5-fold). Electron microscopic examination confirmed increased numbers of peroxisomes in hepatocytes in both mouse models. Moreover, WY14,643 markedly induced hepatic cell proliferation (approximately 15-fold) in both wild-type C57Bl/6 and TNFalpha knockout mice as measured by bromodeoxyuridine incorporation into hepatocyte nuclei. In addition, a 50% decrease in TNFalpha mRNA was observed in wild-type mice after treatment with WY14,643. These results suggest that the hepatocellular proliferation induced after peroxisome proliferator treatment occurs independently of TNFalpha signaling.     

Absence of gamma-glutamyltranspeptidase activity in lung metastasis in rats with hepatocellular carcinomas induced by ciprofibrate, a peroxisome proliferator

The incidence of lung metastasis in rats with hepatocellular carcinomas (HCC) induced by ciprofibrate, a peroxisome proliferator and the expression of gammaglutamyl transeptidase (GGT) in the metastatic lesions was studied. HCC were induced in 75 male F-334 rats by chronic dietary administration of ciprofibrate (0.025% w/w) for 16-22 months. The incidence of lung metastasis was 25% in rats killed between 14 and 16 months which increased to 56% in rats killed between 20 and 22 months. The lung metastases were multifocal and present in both the blood vessels and parenchyma. All the metastatic foci examined for the expression of GGT by histochemical stain were devoid of this enzyme. The results of this study clearly demonstrate the malignant behavior of ciprofibrate-induced liver tumors and the absence of GGT expression in metastatic lesions a phenotypic property that is peculiar to the primary HCC induced by several peroxisome proliferators.     

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.     

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.     

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.     

Peroxisome proliferator-induced hepatocarcinogenesis: histochemical analysis of ciprofibrate-induced preneoplastic and neoplastic lesions for gamma-glutamyl transpeptidase activity

Previous studies revealed that putative preneoplastic and neoplastic lesions induced in the liver by Wy-14,643, a peroxisome proliferator, were gamma-glutamyl transpeptidase (GGT) negative. For ascertainment as to whether phenotypes of foci and carcinomas induced by all peroxisome proliferators are similarly GGT negative, altered areas (AAs), neoplastic nodules (NNs), and hepatocellular carcinomas (HCCs) induced in the livers of male F344 rats by chronic dietary administration of ciprofibrate (0.025% wt/wt in chow; CAS: 52214-84-3) were analyzed histochemically for GGT activity. Eighty-nine percent of AAs, 91% of NNs, and 91% of HCCs were GGT negative. The GGT-negative property of these various hepatic preneoplastic and neoplastic lesions persisted at 8 weeks after the withdrawal of ciprofibrate treatment. The results of this study indicate that the absence of GGT activity is a common feature in hepatic lesions induced by structurally unrelated peroxisome proliferators and is not related to the drug toxicity. The proposal was made that peroxisome proliferators do not derepress the activity of the GGT gene during hepatocarcinogenesis in the rat.     

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.     

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.     

Repeated hepatocyte injury promotes hepatic tumorigenesis in hepatitis C virus transgenic mice

Although hepatitis C virus (HCV) is a well-known causative agent of hepatocellular carcinoma (HCC), the mechanism by which HCV induces HCC remains obscure. To elucidate the role of HCV in hepatocarcinogenesis, a model of hepatocyte injury was established using HCV core transgenic mice, which were developed using C57BL/6 mice transfected with the HCV core gene under control of the serum amyloid P component promoter. After 18–24 months, neither steatosis nor hepatic tumors were found in transgenic mice. The extent of hepatocyte injury and tumorigenesis were then examined in transgenic mice following repeated administration of carbon tetrachloride (CCl₄) using various protocols (20%, 1/week; 10%, 2/week and 20%, 2/week). Serum alanine aminotransferase (ALT) levels did not differ among HCV core transgenic mice and non-transgenic littermates; however, after 40 weeks, hepatic adenomas preferentially developed in transgenic mice receiving 20% CCl₄ once weekly. Moreover, HCC was observed in transgenic mice receiving 2 weekly injections of a 20% solution of CCl₄, and was not observed in the non-transgenic control mice. In conclusion, the HCV core protein did not promote hepatic steatosis or tumor development in the absence of hepatotoxicity. However, the HCV core protein promoted adenoma and HCC development in transgenic mice following repeated CCl₄ administration. These results suggest that hepatotoxicity resulting in an increased rate of hepatocyte regeneration enhances hepatocarcinogenesis in HCV-infected livers. Furthermore, this experimental mouse model provides a valuable method with which to investigate hepatocarcinogenesis.     

Persistence of benzo(a)pyrene metabolite:DNA adducts in lung and liver of mice

The persistence of benzo(a)pyrene (BP) metabolite:DNA adducts has been studied in lung and liver of A/HeJ and C57BL/6J mice after a dose of BP (6 mg/mouse) which induces pulmonary adenomas in A/HeJ mice but not in C57BL/6J mice. BP is not a hepatic carcinogen in either strain. Following p.o. administration of [3H]BP, animals were killed at times ranging from 10 hr to 28 days, and BP metabolite:DNA adducts were analyzed by high-pressure liquid chromatography. The major adduct identified in each tissue was the (+)-7 beta-8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct. A 7 beta, 8 alpha-dihydroxy-9 beta,10 beta,epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, a (-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, and an unidentified adduct were also observed. The disappearance of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP adduct in A/HeJ mice followed first-order kinetics over the time period examined, with a half-life of 18 and 9 days in lung and liver, respectively. The decay of this adduct in C57BL/6J mice was biphasic in both tissues. Our data on cell turnover suggest that there is active removal of adducts in liver, but that normal DNA turnover can account for the partial or possibly total observed disappearance of adducts in lung. These results suggest that the tissue specificity for BP-induced neoplasia in A/HeJ mice may be related to the relative persistence of adducts and high cell turnover rates in lung. In contrast, the results on formation and persistence of adducts and cell turnover do not provide an explanation for the strain difference in susceptibility to BP-induced pulmonary adenomas. It was also shown that the rates of removal of BP metabolite:DNA adducts in A/HeJ mice are not significantly different at a 500-fold lower BP dose.     

Analysis of peroxisome proliferator-induced preneoplastic and neoplastic lesions of rat liver for placental form of glutathione S-transferase and gamma-glutamyltranspeptidase

Structurally unrelated peroxisome proliferators induce altered areas (AA), neoplastic nodules (NN), and hepatocellular carcinomas (HCC) in rats and mice. In this study we have examined several AA, NN, and HCC induced by Wy-14,643 and ciprofibrate in rats for gamma-glutamyltranspeptidase (GGT) and the placental form of glutathione S-transferase (GST-P) by histochemical and immunohistochemical procedures, respectively. In Wy-14,643-treated animals 96-100% of NN and HCC was negative for both GGT and GST-P. Eighty-seven % of the AA was negative for both GGT and GST-P, and only 2% was positive for both the marker enzymes. In ciprofibrate-treated animals 52% and 75% of AA were negative for GST-P and GGT, respectively, and 16% was positive for both the enzymes. However, a large majority of NN and HCC (more than 95%) was devoid of both these marker enzymes. Thus these studies clearly indicate that the hepatic lesions induced by peroxisome proliferators display different phenotypic properties as compared to the lesions induced by commonly used classical liver carcinogens. We conclude that GGT and GST-P are not the ideal markers for identifying AA, NN and HCC induced by peroxisome proliferators.     

Rapid growth of preneoplastic lesions in hepatocarcinogen-Sensitive C3H/HeJ male mice relative to C57BL/6J male mice

We have previously shown that C3H/HeJ male mice are {small tilde}20-fold more susceptible to the induction of liver tumors byN-ethyl-N-nitrosourea (ENU) than are C57BL/6J male mice andthat this difference in sensitivity is largely determined bya single genetic locus (Hcs, hepatocarcinogen sensitivity).In order to determine whether the Hcs locus affects initiationor promotion of hepatocarcinogenesis, we studied the developmentof putatively preneoplastic hepatic lesions that are deficientin glucose-6-phosphatase (G6Pase) in mice treated at 12 daysof age with ENU. In ENU-treated male mice of both strains, thenumber and size of G6Pase-deficient hepatic foci increased overtime between 12 and 24 weeks of age. However, the rate of growthof the lesions was 1.7 times faster for C3H/HeJ male mice (volumedoubling time 2.0 ± 0.1 weeks) than for C57BL/6J mice(3.4 ± 0.4 weeks). Although the number and size of G6Pase-deficientfoci induced by ENU treatment of female C3H/HeJ and C57BL/6Jmice were smaller than for foci in similarly treated male mice,there was no significant difference between the growth ratesof the foci in female C3H/HeJ and C57BL/6J mice. Thus, the phenotypiceffect of the Hcs locus appears to be dependent on promotionof liver tumor induction by the male hormonal environment. Inagreement with studies on the growth rate of the foci in malemice, the [₃H]thymidine labeling index of G6Pase-deficient hepatocytesin C3H/HeJ males (12%) was 1.5-fold higher than in C57BL/6Jmale mice (8.0%) at 20 weeks and 1.2-fold higher at 28 weeks(11% versus 9.5%). The labeling index of histochemically normalhepato cytes in C3H/HeJ male mice (0.38%) was 2.6-fold higherthan in C57BL/6J mice (0.15%) The Hcs locus may affect the promotionphase of hepatocarcinogenesis in male mice by increasing theproliferative rate of both normal and preneoplastic hepatocytes.     

Response of hepatocytes transplanted into syngeneic hosts and heterotransplanted into athymic nude mice to peroxisome proliferators

The development of a transplantation system by which rat hepatocytes can be implanted and remain viable in the dorsal fascia of two-thirds hepatectomized syngeneic hosts provides an opportunity to examine whether such transplanted hepatocytes retain the capacity to recognize and respond to the peroxisome proliferators 2-[4-(2,2- dichlorocyclopropyl )phenoxy]-2- methylpropionic acid (ciprofibrate), a hypolipidemic drug, and di-(2-ethylhexyl)phthalate (DEHP), an industrial plasticizer. Male F344 rats with transplanted rat hepatocytes were fed a control diet or a diet containing either 0.05% ciprofibrate (w/w) or 2% DEHP (v/w). After 4 weeks, the animals were sacrificed, and transplanted hepatocytes revealed a significant increase in the numerical density of peroxisomes in both ciprofibrate- and DEHP-fed rats. The volume density of peroxisomes in transplanted hepatocytes increased 9.2- and 5.3-fold, respectively, in ciprofibrate- and DEHP-fed rats, whereas the volume density of mitochondria remained essentially unchanged. The magnitude of increase in peroxisome volume density in transplanted hepatocytes was comparable to increases in the volume density of these organelles in the liver parenchymal cells of syngeneic hosts. The present study also demonstrates that hepatocytes isolated from cat liver and heterotransplanted into partially hepatectomized athymic nude mice retain their biological integrity and respond to the peroxisome proliferative effect of ciprofibrate. This observation suggests the possibility that hepatocytes obtained from small segments of liver of humans, primates, and other species and heterotransplanted into nude mice might provide a valuable model system for toxicological evaluation of chemicals. These studies suggest that hepatocytes, irrespective of their location in the body, recognize the peroxisome proliferator or its active metabolite(s), which stimulates the expression of peroxisome-specific genes in these cells.     

Peroxisome proliferation and lipid peroxidation in rat liver

Male F344 rats were fed a diet containing the peroxisome proliferators 2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2-methylpropionic acid [ciprofibrate (0.025%)] or [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid [Wy-14643 (0.1%)] for up to 14 months to determine whether hepatic peroxisome proliferation caused by these agents results in the induction of membrane lipid peroxidation in the liver. Peroxidative damage of membrane lipids from whole liver, postnuclear, heavy-particle, microsomal, and nuclear membranes was evaluated by determining the extent of formation of conjugated dienes (ultraviolet absorption, 233 nm). Increased generation of diene conjugates was noted in whole-liver, postnuclear, and heavy-particle membrane lipids of rats fed peroxisome proliferators for 6 months or longer when compared to controls. An additional, more intense absorption profile in the ultraviolet absorption range of approximately 276 nm was noted in the membrane lipids derived from whole liver, postnuclear, and heavy particle pellets, but not in the nuclear and microsomal membrane lipids of livers with peroxisome proliferation. Although the exact chemical nature of this delta 276 nm peak is not clear, it is attributed to the formation of ketone dienes and/or conjugated trienes. The excess lipid peroxidation correlates with the previous observation of accumulation of abundant quantities of lipofuscin in hepatocytes of rats chronically exposed to peroxisome proliferators. The generation of conjugated dienes and ketone dienes and/or trienes together with increased levels of H2O2 generation by peroxisomal enzymes, and decreased levels of hepatic glutathione peroxidase, glutathione reductase, and glutathione-S-transferases, enzymes responsible for the defense against H2O2 damage, suggest the occurrence of membrane lipid peroxidation and oxidative stress in livers of rats treated with carcinogenic peroxisome proliferators.     

Peroxisome proliferators increase ethanol catabolism through utilization of the catalase pathway

We investigated the effects of ciprofibrate, a potent peroxisome proliferator, on ethanol metabolism in mice. The blood alcohol levels of mice fed a liquid diet containing both ciprofibrate and ethanol were markedly depressed compared with mice fed the ethanol-containing diet alone. Ciprofibrate markedly induced enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, hydrogen peroxide, and to a lesser extent catalase in both control and ethanol-diet fed mice. Northern blot analysis indicated no significant upregulation of cytochrome P450IIE1 mRNA by ciprofibrate. Our study suggests that peroxisome proliferators increase ethanol catabolism through hydrogen peroxide production, thus allowing utilization of the catalase pathway. These findings indicate that catalase has the potential to provide a significant pathway for ethanol metabolism under conditions of peroxisome proliferation.     

Genetic control of hepatocarcinogenesis in C57BL/6J and C3H/HeJ inbred mice

Treatment of newborn male C3H/HeJ mice with N, N-dietnyl-nitrosamine(DEN) or N-ethyl-N-nitrosourea (ENU) resulted in the inductionof hepatocellular adenomas and carcinomas with a mean numberof tumors per animal that was 20-to 50-fold higher than thatfor similarly treated C57BL/6J male mice. We used two methodsto study the genetic basis for this difference in susceptibilityto liver tumor induction. Analysis of DEN-induced liver tumormultiplicities as a quantitative genetic trait in segregatingcrosses between C3H/HeJ and C57BL/6J mice indicated that allelicdifferences for at least two loci contributed to the highersensitivity to hepatocarcinogenesis of C3H/HeJ mice relativeto C57BL/6J mice. However, a single locus, which we have denotedHcs (hepato-carcinogen sensitivity), was responsible for 85%of the difference in susceptibility. The C57BL/6J and C3H/HeJalkies at this locus were semi-dominant. This result was confirmedby analysis of hepatocarcinogenesis by ENU in BXH (C57BL/6J? C3H/HeJ) recombinant inbred mice. Four of the nine recombinantinbred strains studied were highly susceptible to the inductionof liver tumors by ENU, three of the strains exhibited the resistantphenotype of the C57BL/6J parent, and two of the strains wereof intermediate sensitivity to hepatocarcinogenesis. Newbornmale C3H/HeJ and C57BL/6J mice did not significantly differin the extent of ethylation of hepatic DNA, or in the relativelevels of N-7-ethylguanine or O⁶-ethylguanine after treatmentwith [1-¹⁴C]DEN.     

Increased incidence of aflatoxin B1-induced liver tumors in hepatitis virus C transgenic mice

Viral hepatitis and aflatoxin B1 (AFB1) exposure are common risk factors for hepatocellular carcinoma (HCC). The incidence of HCC in individuals coexposed to hepatitis C (HCV) or B virus and AFB1 is greater than could be explained by the additive effect; yet, the mechanisms are poorly understood because of the lack of an animal model. Our study investigated the outcomes and mechanisms of combined exposure to HCV and AFB1. We hypothesized that HCV transgenic (HCV-Tg; expressing core, E1, E2 and p7, nucleotides 342-2771) mice will be prone to hepatocarcinogenesis when exposed to AFB1. Neonatal (7 days old) HCV-Tg or C57BL/6J wild-type (WT) mice were exposed to AFB1 (6 μg/g bw) or tricaprylin vehicle (15 μl/g bw), and male offspring were followed for up to 12 months. No liver lesions were observed in vehicle-treated WT or HCV-Tg mice. Tumors (adenomas or carcinomas) and preneoplastic lesions (hyperplasia or foci) were observed in 22.5% (9 of 40) of AFB1-treated WT mice. In AFB1-treated HCV-Tg mice, the incidence of tumorous or pretumorous lesions was significantly elevated (50%, 18 of 36), with the difference largely due to a 2.5-fold increase in the incidence of adenomas (30.5 vs. 12.5%). Although oxidative stress and steatohepatitis were observed in both AFB1-treated groups, molecular changes indicative of the enhanced inflammatory response and altered lipid metabolism were more pronounced in HCV-Tg mice. In summary, HCV proteins core, E1, E2 and p7 are sufficient to reproduce the cocarcinogenic effect of HCV and AFB1, which is a known clinical phenomenon.     

Relationship between hepatic peroxisome proliferation and 8-hydroxydeoxyguanosine formation in liver DNA of rats following long-term exposure to three peroxisome proliferators; di(2-ethylhexyl) phthalate, aluminium clofibrate and simfibrate

To elucidate the relationship between hepatic peroxisome proliferation and oxidative DNA damage induced by hepatocarcinogenic peroxisome proliferators, 3 agents, namely, di(2-ethylhexyl) phthalate (DEHP, aluminium clofibrate and simfibrate were fed at doses of 1.2%, aluminium clofibrate 0.5% and 0.5% in the diet, respectively, to male F-344 rats for up to 1 year. Evidence of hepatic peroxisome proliferation and 8-hydroxydeoxyguanosine (8-OH-dG) formation in liver and kidney DNA were assessed at 1, 2, 3, 6, 9 and 12 months. Peroxisomal beta-oxidation enzyme activities were increased 3- to 8-fold and catalase was elevated to 1.4- to 2.2-fold the control level by DEHP, aluminium clofibrate and simfibrate from months 1 to 12 of the treatment. 8-OH-dG levels in liver DNA of DEHP-, aluminium clofibrate- and simfibrate-fed rats were increased approximately 2-fold after 1 month, the tendency for elevation also being observed in the liver DNA at 2, 3, 9 and 12 months. The results thus clearly demonstrate that persistent peroxisome proliferation in the liver leads to continued specific oxidative DNA damage.