Long-term effects of hypolipidemic peroxisome proliferator administration on hepatic hydrogen peroxide metabolism in rats

The effects of prolonged dietary administration of peroxisome proliferators, such as clofibrate, bezafibrate and di(2-ethylhexyl)phthalate (DEHP), on hepatic hydrogen peroxide (H2O2) level and on hepatic activities of the enzymes relating to H2O2 metabolism were examined. Male rats were treated for 79 weeks with the above three peroxisome proliferators. The activities of the peroxisomal beta-oxidation and catalase were increased 8- to 20-fold and 2- to 3-fold, respectively, after 2 or 4 weeks of treatment with these peroxisome proliferators. However at 79 weeks the peroxisomal beta-oxidation activity was 3-8 times that of control. The level of catalase activity was kept at approximately 2-fold even after prolonged treatment of peroxisome proliferators. Although the activities of glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST) were decreased 50-60% at 4-12 weeks by the treatment with peroxisome proliferators, from 20 to 79 weeks those activities approached control levels in the case of clofibrate and bezafibrate but not DEHP-fed rats; GSH-Px and GST activities were kept at approximately 40% those of control. However hepatic capacities of H2O2-degrading enzymes, catalase and GSH-Px, apparently exceeded the H2O2-generating levels obtained on the basis of peroxisomal beta-oxidation activities in the livers of control and treated rats throughout the experimental period. The hepatic H2O2 levels increased only slightly but this increase did not correspond to changes in peroxisomal beta-oxidation. Our results suggest that a large part of H2O2 produced by peroxisomal beta-oxidation could be rapidly scavenged by catalase and GSH-Px in the liver of rats treated with peroxisome proliferators.     

Lack of expression of glutathione-S-transferase P, gamma-glutamyl transpeptidase, and alpha-fetoprotein messenger RNAs in liver tumors induced by peroxisome proliferators

Many structurally unrelated nonmutagenic peroxisome proliferators induce altered areas, neoplastic nodules, and hepatocellular carcinomas in rats. Unlike the lesions induced by genotoxic hepatocarcinogens, these lesions do not stain positively for the phenotypic markers gamma-glutamyl transpeptidase (GGT) and glutathione-S-transferase P (GST-P). To ascertain whether the absence of immunocytochemically detectable GST-P and GGT proteins in peroxisome proliferator-induced neoplastic lesions is due to the absence of specific mRNAs, we analyzed the total RNA isolated from hepatocellular carcinomas induced by three different peroxisome proliferators (ciprofibrate, Wy-14643, and BR-931) and the genotoxic carcinogens, 2-acetylaminofluorene and aflatoxin B1 (AFB), for the presence of GST-P, GGT, and alpha-fetoprotein (AFP) mRNAs. Northern and dot blot analysis of total RNA isolated from liver tumors induced by three different peroxisome proliferators revealed no detectable GST-P, GGT, and AFP mRNAs. GST-P mRNA was also not detected in a transplantable hepatocellular carcinoma established from a liver tumor induced by ciprofibrate. In contrast, GST-P mRNA levels were high in primary liver tumors induced by both 2-acetylaminofluorene and AFB and the two transplantable hepatocellular carcinomas established from such tumors. By immunoblot method, GST-P protein was found to be abundant in both primary and transplantable liver tumors induced by genotoxic carcinogens but not in those derived from peroxisome proliferator treatment. The GGT and AFP mRNAs were also not found in all 18 liver tumors induced by peroxisome proliferators that were analyzed and also in the ciprofibrate-derived transplantable liver tumor. The expression of GGT and AFP genes in liver tumors induced by 2-acetylaminofluorene and AFB was variable. These studies with peroxisome proliferators show that the GST-P and GGT gene derepression is not essential for the hepatocarcinogenesis or successful tumor transplantation. Further characterization of the molecular basis for the differential expression, particularly of the GST-P gene in liver tumors, may help identification of the critical event(s) in hepatocarcinogenesis by genotoxic carcinogens and nongenotoxic peroxisome proliferators.     

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.     

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.     

Induction of peroxisome proliferation and hepatic tumours in C57BL/6N mice by ciprofibrate, a hypolipidaemic compound

The hepatic effects of ciprofibrate, a potent peroxisome proliferator, were evaluated in male C57BL/6N mice, a mouse strain with very low incidence of spontaneous liver tumour development. Dietary feeding of ciprofibrate (0.0125% or 0.025% w/w) for 2 weeks resulted in a marked proliferation of peroxisomes (9-fold increase) and several-fold increase (8- to 10-fold) in the activity of peroxisomal beta-oxidation enzymes. Feeding ciprofibrate at 0.025% concentration for 15 months followed by a 0.0125% for 6 months led to the development of hepatic adenomas in 8/14 (57%) and hepatocellular carcinomas (HCC) in 3/14 (21%) mice. In mice given 0.0125% ciprofibrate for 18 months 5 of 8 (62%) and 3 of 8 (37%) developed adenomas and HCC respectively. Similar to the findings observed in rats, both the adenomas and HCC were negative for gamma-glutamyltranspeptidase. These results in C57BL/6N mice of hepatocarcinogenic effect of ciprofibrate, a non-genotoxic chemical, indicate that peroxisome proliferation can be used as a reliable parameter to evaluate the carcinogenicity of hypolipidaemic compounds.     

Expression of peroxisome proliferator-activated receptor alpha,and PPARalpha regulated genes in spontaneously developed hepatocellular carcinomas in fatty acyl-CoA oxidase null mice

Fatty acyl-CoA oxidase null mice (AOX-/-) develop hepatocellular carcinomas in 100% of animals between 10 and 15 months. We evaluated spontaneously developed HCC in AOX-/- mice for PPARalpha, PPARalpha regulated genes and peroxisome volume density and compared with adjacent non-neoplastic liver and liver in wild-type (AOX+/+) and heterozygous (AOX+/-) mice. The level of PPARalpha mRNA was 2.5-fold higher in HCC compared to the adjacent liver. mRNAs of PPARalpha regulated genes such as peroxisomal bifunctional enzyme, thiolase, cytochrome P450 CYP4A1 and CYP4A3 were similar in HCC and adjacent liver and increased by 7- to 22-fold compared with wild-type and heterozygous mice. Immunoblot analysis of HCC showed high amounts of PPARalpha, peroxisomal bifunctional enzyme and thiolase. Electron microscopic examination revealed 3.8 and 8.3-fold increase in the volume density of peroxisomes in HCC and adjacent liver, respectively, compared to the volume density in wild-type mice. These results demonstrate that spontaneously developed HCC in AOX-/- mice display a similar type of pleiotropic responses to high levels of PPARalpha ligands as the non-neoplastic liver. The changes observed in HCC and adjacent liver in AOX-/- mice were identical to those observed in rats and mice exposed to peroxisome proliferators.     

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 proliferation and modulation of rat liver carcinogenesis by 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, perfluorooctanoic acid and nafenopin

Using an initiation--selection--promotion protocol for induction of liver tumors in Wistar rats, the modulating action of various peroxisome proliferators on neoplasia as well as on selected biochemical parameters was studied. After treatment with diethylnitrosamine (DEN), the animals were subsequently subjected to a selection procedure involving feeding of 2-acetylaminofluorene (2-AAF), and in the middle of the 2-AAF treatment, a single necrogenic dose of carbon tetrachloride. Following a recovery period, the rats were fed a diet containing 0.1% nafenopin (NAF), 0.015% perfluorooctanoic acid (PFOA), 0.05% 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05% 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) or 0.05% phenobarbital (PB) as a positive control. When the animals were killed, 7 months after initiation, the incidence of hepatocellular carcinoma was 83, 33 and 16% in the animals treated with NAF, PFOA or 2,4,5-T respectively. No cancers were observed in controls, or in the 2,4,-D groups. In comparison with controls, NAF and PFOA caused a 60-and 24-fold increase inthe peroxisomal beta-oxidation of fatty acids respectively, but only about a 2-fold increase in the catalase activity, 2,4-D and/or 2,4,5-T were much less active in this respect, giving approximately a doubling in the rate of fatty acid oxidation. The specific activity of D-amino acid and glycolate oxidases were significantly depressed, whereas the urate oxidase levels were apparently unaffected by the NAF and PFOA treatment. The results suggest that the selective induction of peroxisomal fatty acid oxidation is consistent with the hypothesis that imbalance between H2O2 overproduction and its destruction could play a role in the modulation of hepatocarcinogenesis by peroxisome proliferators.     

Expression of peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase enzyme and its mRNA in peroxisome proliferator-induced liver tumors

We have examined ciprofibrate and dehydroepiandrosterone (DHEA)-inducedhepatic lesions for the peroxisomal ß-oxidation systemenzyme peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase(PBE) and its mRNA using SDS-polyacrylamide gel electrophoresis,antibodies and cDNA probe. All 12 neoplastic nodules and ninehepatocellular carcinomas (HCCs) that were analyzed for PBEmRNA by in situ hybridization showed an intense signal comparableto the adjacent non-neoplastic liver. SDS-polyacrylamide gelelectrophoresis of postnuclear fractions of six HCC and adjacentliver tissue showed a marked increase in an 80 kDa polypeptide.Immunoblot and Northern blot analysis showed a marked increasein PBE enzyme and PBE mRNA respectively in HCC and adjacentnon-neoplastic liver tissue. In control livers (animals nottreated with peroxisome proliferators), the levels of PBE enzymeand mRNA were very low or undetectable. The results of thisstudy clearly indicate that peroxisomal proliferator (PP)-inducedliver lesions express peroxisomal enzymes to the same extentas adjacent liver and that these enzymes are not useful markersfor identification of PP-induced lesions.     

Hepatic zonation of the induction of cytochrome P450 IVA, peroxisomal lipid beta-oxidation enzymes and peroxisome proliferation in rats treated with dehydroepiandrosterone (DHEA). Evidence of distinct zonal and sex-specific differences

Dehydroepiandrosterone (DHEA) is an intermediate product in the synthesis of male and female sex hormones in the adrenal cortex of man. In livers of rats and mice DHEA increases the levels of cytochrome P450 IVA and peroxisomal beta-oxidation enzymes associated with peroxisome proliferation. Prolonged treatment of rats with DHEA induces liver tumors that are more frequent in females arising mainly in the periportal regions of the liver lobule (Metzger et al., Toxicol. Pathol. 23, 591-605, 1995). Because of paucity of information on hepatic zonation of peroxisomal response to DHEA and controversial reports on gender-specific differences of its effects the present study was undertaken using qualitative immunohistochemical and quantitative immunoelectron microscopical techniques in addition to Western blotting. Rats were treated for 24 weeks with 0.6% DHEA supplied with diet. Immunoblot analysis revealed marked induction of peroxisomal beta- oxidation enzymes, which by quantitative analysis was equally strong in male and female animals, whilst catalase and urate-oxidase were not increased. Cytochrome P450 IVA, in contrast, was induced significantly stronger in male than in female rats. Immunohistochemistry confirmed the induction of cytochrome P450 IVA showing a marked lobular gradient in female animals with strong induction in pericentral and almost no induction in periportal regions of the liver lobule. In male animals cytochrome P450 IVA was expressed more uniformly across the liver lobule. A similar sex specific zone-dependent response was observed for peroxisomes. DHEA induced in females a significant zonal gradient with marked peroxisome proliferation and a strong induction of peroxisomal hydratase/dehydrogenase in pericentral hepatocytes and a much smaller response in periportal regions. Livers of male animals, in contrast, showed a uniform peroxisomal proliferation to DHEA with only slight zonal differences. The striking homologies of the induction patterns of cytochrome P450 IVA and the peroxisome proliferation in both sexes support the notion of a functional relationship. In view of the almost exclusive periportal localization of DHEA-induced tumors in female rats in contrast to the pericentral localization of the peroxisomal proliferation shown by this study, it seems likely that other factors in addition to peroxisome proliferation may contribute to the hepatocarcinogenic effect of DHEA.      

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.     

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.     

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.     

Evidence that reduction of hepatocyte growth factor (HGF) is not required for peroxisome proliferator-induced hepatocyte proliferation

The mechanisms underlying peroxisome proliferator-induced hepatocarcinogenesis are not understood. Because of the uncertainty of human cancer risk associated with peroxisome proliferators, delineating the mechanisms of carcinogenesis by these agents is of great interest. Alterations in liver growth factors were postulated to contribute to the carcinogenic effect of peroxisome proliferators. Administration of these compounds to rodents results in down-regulation of hepatocyte growth factor (HGF) and supplementing culture medium with HGF is reported to suppress cell proliferation of preneoplastic and neoplastic cells from WY-14,643-treated livers. Combined, these observations suggest that reduced levels of hepatic HGF contribute to the mechanisms underlying peroxisome proliferator-induced hepatocarcinogenesis. To determine if HGF can prevent the effects of peroxisome proliferators in liver, the short-term influence of WY-14,643 in two different lines of HGF transgenic mice was examined. Mice were fed either a control diet or one containing 0.1% WY-14-643 for one week. Hepatomegaly was found in both HGF transgenic mouse lines fed WY-14,643 compared with controls. Additionally, hepatic expression of typical mRNA markers of peroxisome proliferation including those encoding peroxisomal fatty acid metabolizing enzymes and cell cycle control proteins were all significantly elevated in HGF transgenic mice fed WY-14,643 compared with controls. Down-regulation of HGF was found to be dependent on PPARalpha since lower levels of HGF mRNA and protein were observed in wild-type mice fed WY-14,643 for 1 week and not in similarly treated PPARalpha-null mice. These results demonstrate that the early increase in hepatic mRNAs associated with peroxisome and cell proliferation induced by WY-14,643 treatment can not be prevented by overexpression of HGF in vivo.     

Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) in rats

The mechanism of hepatocarcinogenesis caused by peroxisome proliferators (PP) is poorly understood, making it difficult to predict the carcinogenicity of PP to rodents or other species. It has been suggested that the carcinogenic potential of individual PP in rodents is correlated with the degree of PP-induced hepatic peroxisome proliferation. To evaluate this possible correlation, di(2-ethylhexyl)phthalate (DEHP) at 1.2% and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) at 0.1% were fed to male F-344 rats for up to 365 days and hepatocytic peroxisome proliferation and DNA replication were measured. All rats fed Wy-14,643 for 365 days had numerous grossly visible nodules in comparison to none in the livers of DEHP-fed or control rats. Despite this difference in the induction of tumors, both DEHP and Wy-14,643 increased the peroxisomal volume density 4- to 6-fold from 8 to 365 days of treatment. Peroxisomal beta-oxidation enzyme activities were increased 8-fold by both DEHP and Wy-14,643 after 18 days. At later time points (77 to 365 days), these enzyme activities were about 25% higher in livers of Wy-14,643- than DEHP-fed rats. DEHP or Wy-14,643 increased absolute liver weights 50 to 75% above controls after 18 to 365 days of feeding. Labeling of hepatocyte nuclei with a single injection of tritiated thymidine revealed a rapid burst in replicative DNA synthesis in both DEHP and Wy-14,643-fed rats, with a return to control levels by 4 days. Additional rats were implanted with 7-day osmotic pumps containing tritiated thymidine. With this more extended method of labeling a 5- to 10-fold increase in replicative DNA synthesis was observed in rats receiving Wy-14,643 for 39 to 365 days as compared to DEHP-fed rats or controls. In conclusion, when performed under conditions similar to the tumorigenicity studies, the degree of peroxisome proliferation correlated poorly with the relative hepatocarcinogenicity of DEHP and Wy-14,643. However, a strong correlation was observed between the relative hepatocarcinogenicity of DEHP and Wy-14,643 and the ability to induce a persistent increase in replicative DNA synthesis. These data emphasize the possible importance of cell replication in the mechanism of PP-induced hepatocarcinogenesis.     

In vitro steady-state levels of hydrogen peroxide after exposure of male F344 rats and female B6C3F1 mice to hepatic peroxisome proliferators

The hypothesis that hepatocarcinogenesis resulting from treatmentof rats and mice with peroxisome proliferators is linked toincreased cellular levels of hydrogen peroxide from peroxisomalß-oxidation was investigated. Male F344 rats and femaleB6C3F2₁ mice were treated for 14 days with di(2-ethylhexyl)phthalate(DEHP) or di(2-ethylhexyl)adipate (DEHA), industrial plasticizers,or nafenopin, a hypolipi-demic drug. Activities of enzymes responsiblefor the production [peroxisomal palmitoyl CoA oxidase (PCO)]and degradation [catalase (Cat) and glutathione peroxidase (GSHPx)]of H₂O₂ were assayed in liver homogenates prepared from treatedanimals. The activities of the peroxisomal enzymes PCO and Catwere enhanced 5- to 25-fold and 1.5-to 3-fold respectively bytreatment with the peroxisome proliferators. The activity ofGSHP_x, a cytoplasmk enzyme, was decreased 40–60% in liverhomogenates prepared from treated pnimalx compared to controlanimals. A kinetic treatment of the rates of formation of hydrogenperoxide by PCO, and of degradation of hydrogen peroxide bycatalase was used to estimate steady-state hydrogen peroxideconcentrations ([H₂O₂]) during peroxisomal oxidation of palmitoylCoA. Increases in peroxisomal steady-state [H₂O₂] for the F344rat liver homogenates correlated well with the carcinogenicpotential of these chemicals, determined in previous carcinogenicitystudies. Increases in the steady-state [H₂O2] were also calculatedfor liver homogenates prepared from mice treated with thesecompounds. Decreases in liver lipid peroxidation were observedafter treatment with each chemical in both species. The resultsof these studies are consistent with an involvement of increasedperoxisomal hydrogen peroxide in the hepatocarcinogenesis ofthese compounds.     

Differential susceptibility of mice humanized for peroxisome proliferator-activated receptor alpha to Wy-14,643-induced liver tumorigenesis

Peroxisome proliferators, such as lipid-lowering fibrate drugs, are agonists for the peroxisome proliferator-activated receptor alpha (PPARalpha). Sustained activation of PPARalpha leads to the development of liver tumors in rodents. Paradoxically, humans appear to be resistant to the induction of peroxisome proliferation and development of liver tumors by peroxisome proliferators. To examine the species differences in response to peroxisome proliferators, a PPARalpha humanized mouse (hPPARalpha) was generated, in which the human PPARalpha was expressed in liver under control of the Tet-OFF system. To evaluate the susceptibility of hPPARalpha mice to peroxisome proliferator-induced hepatocarcinogenesis, a long-term feeding study of Wy-14,643 was carried out. hPPARalpha and wild-type (mPPARalpha) mice were fed either a control diet or one containing 0.1% Wy-14,643 for 44 and 38 weeks, respectively. Gene expression analysis for peroxisomal and mitochondrial fatty acid metabolizing enzymes revealed that both hPPARalpha and mPPARalpha were functional. However, the incidence of liver tumors including hepatocellular carcinoma was 71% in Wy-14,643-treated mPPARalpha mice, and 5% in Wy-14,643-treated hPPARalpha mice. Upregulation of cell cycle regulated genes such as cd1 and Cdks were observed in non-tumorous liver tissue of Wy-14,643-treated mPPARalpha mice, whereas p53 gene expression was increased only in the livers of Wy-14,643-treated hPPARalpha mice. These findings suggest that structural differences between human and mouse PPARalpha are responsible for the differential susceptibility to the peroxisome proliferator-induced hepatocarcinogenesis. This mouse model will be useful for human cancer risk assessment of PPARalpha ligands.     

Hepatocarcinogenicity of dehydroepiandrosterone in the rat.

Dehydroepiandrosterone, a major secretory steroid hormone of the human adrenal gland, possesses mitoinhibitory and anticarcinogenic properties. It also induces peroxisome proliferation in the livers of rats and mice. Because peroxisome proliferators exhibit hepatocarcinogenic potential, it is necessary to examine the long term hepatic effects of dehydroepiandrosterone since this hormone is contemplated for use as a potential cancer chemopreventive agent in humans. Dehydroepiandrosterone was administered in the diet at a concentration of 0.45% to F-344 rats for up to 84 weeks. At the termination of the experiment, 14 of 16 rats developed hepatocellular carcinomas. Liver tumors induced by dehydroepiandrosterone lacked gamma-glutamyl transpeptidase and glutathione S-transferase (placental form); these phenotypic properties are identical to the features exhibited by liver tumors induced by other peroxisome proliferators. Dehydroepiandrosterone was also shown to markedly inhibit liver cell [3H]thymidine labeling indices, suggesting that cell proliferation is not a critical feature in liver tumor development with this agent. These results show that although dehydroepiandrosterone exerts anticarcinogenic effects in a variety of tissues, the peroxisome-proliferative property makes it a hepatocarcinogen.     

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.