Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: relationship to mitochondrial alterations

Recent studies have indicated a lack of correlation betweenhepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and thecarcinogenicity of peroxisome proliferators (PP) and suggestedthat DNA in intact hepatic nuclei may be insensitive to increasesin 8-OHdG resulting from PP exposure. The possibility that PP-inducedelevations in acyl CoA oxidase (ACO) activity might result inoxidative damage to mitochondrial DNA (mtDNA) was thereforeinvestigated by feeding male F344 rats the hepatocarcinogenicPP Wy-14,643 (Wy, 0.1% in the diet) for 3, 6, 11, or 22 weeks,or clofibric acid (CA, 0.5% in the diet) for 22 weeks. Followingthe respective PP exposures, hepatic peroxisomal acyl CoA oxidaseactivity was determined and DNA isolated from either mitochondriaor unfractionated liver homogenates and analysed for the presenceof 8-OHdG. PP treatment caused an increase in ACO activity (10-to 15-fold) at all time points examined and an increase of 8-OHdG(1.5- to 2-fold) in DNA isolated from unfractionated liver homogenatesfollowing PP treatment for 11 or 22 weeks. No increase of 8-OHdGin mtDNA was detected. However, quantitation of a PCR amplifiedregion from the D-loop of mtDNA demonstrated a 2- to 3-foldincrease in the relative amount of mtDNA in DNA isolated fromunfractionated liver homogenates following 3, 11, and 22 weeksexposure to Wy or CA (22 weeks only). In addition, a slightincrease in the mitochondrial volume density (1.4-fold) wasobserved in electron micrographs of liver samples from ratsexposed to Wy for 22 weeks. These results (i) demonstrate thatPP treatment, at levels which cause an increase in ACO activity,does not cause oxidative damage to mtDNA, and (ii) suggest thatone reason for the observed increase of 8-OHdG in DNA from unfractionatedliver homogenates may be an increase in the amount of mtDNApresent in these samples. Furthermore, these studies provideadditional evidence against a role of oxidative DNA damage,measured as 8-OHdG, in PP-induced rodent hepatocarcinogenesisand suggest that alterations in mitochondria or other effectsmay be more pertinent to PP-related carcinogenesis.     

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.     

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.     

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.     

Significant increase of 8-hydroxydeoxyguanosine in liver DNA of rats following short-term exposure to the peroxisome proliferators di(2-ethylhexyl)phthalate and di(2-ethylhexyl)adipate

8-Hydroxydeoxyguanosine (8-OH-dG) levels were examined in liver and kidney DNA after di(2-ethylhexyl)phthalate (DEHP), di(2-ethylhexyl)adipate (DEHA) and phthalic anhydride administration to male 6-week-old F-344 rats in the diet at concentrations of 1.2, 2.5 and 1.5%, respectively. Significant increases in 8-OH-dG levels were observed only in the liver (target organ of DEHP and DEHA carcinogenesis) DNA after 1 and 2 weeks of treatment with DEHP and DEHA, respectively. The results suggest the involvement of oxidative DNA damage in hepatocarcinogenesis by peroxisome proliferators.     

Improvements in the analytical method for 8-hydroxydeoxyguanosine in nuclear DNA

Modifications at two points in the sequence of 8-hydroxy-2'-deoxyguanosine(8-OH-dG) analysis have contributed to a more accurate and simplifieddetermination of 8-OH-dG in DNA. The first was an improvementin the detection limit for 8-OH-dG in high-performance liquidchromatography analysis and the second was a pronase digestionand ethanol precipitation method (pronase/ethanol method) forDNA isolation which could minimize artificial formation of 8-OH-dG.Since the changes in background current from electrochemicaldetection are regularly periodical, it was possible to reducethis background change by connecting a pressure damper, degassingthe eluent before use and finally subtracting its theoreticalfunction. After this background correction, the detection limitfor 8-OH-dG was improved one order of magnitude, from 20 fmol(5.68 pg) to 1.76 fmol (0.5 pg). Therefore, 0.005 8-OH-dG/10⁵dG can be detected from 50 µg DNA. This improvement willallow the analysis of small samples, tissues from needle biopsies,<5 ml whole blood, etc., and will contribute to the accuracyof 8-OH-dG measurements. The pronasd ethanol method resultedin lower levels of 8-OH-dG than the phenol method in analysesof both rat liver and calf thymus DNA, even after 6 h incubationat 45°C. The level obtained by the pronase/ethanol methodwith butylated hydroxytoluene was approximately equal to orlower than the 8-OH-dG levels reported in normal rat liver.The pronase/ethanol method for DNA isolation can replace thephenol or other methods in 8-OH-dG analysis. This method alsoomits the use of highly toxic organic solvents.     

Aflatoxin B1-induced 8-hydroxydeoxyguanosine formation in rat hepatic DNA

A time- and dose-dependent increase in 8-hydroxydeoxy-guanosine(8-OHdG) was observed in rat hepatic DNA after a single i.p.injection of aflatoxin B₁ (AFB₁). It was also found that pre-treatmentwith selenium or deferoxamine significantly reduced 8-OHdG levelin AFB₁-administered rats. In contrast, no reduction in 8-OHdGconcentration was found in vitamin E-pre-treated rats. Theseresults provide evidence that AFB₁ causes oxidative DNA damagein rat liver, which may involve hydroxyl radicals as the initiatingspecies. It is postulated that AFB₁-induced oxidative DNA damage(8-OHdG formation) may constitute an important pathway in AFB₁hepatocarcinogenesis.     

Induction of altered hepatic foci in rats by the administration of hypolipidemic peroxisome proliferators alone or following a single dose of diethylnitrosamine

The effect of feeding hypolipidemic peroxisome proliferators on the induction of altered hepatic foci (AHF) in Fischer rats was studied in order to determine whether such agents can induce or promote the development of AHF. In the first study, rats were fed ciprofibrate (10 mg/kg/day) for 1 yr. AHF, neoplastic nodules, and hepatocellular carcinomas were induced. The presence of putative gamma-glutamyltranspeptidase (GGT) activity was numerically the most common marker, although it was absent in larger foci and nodules. A deficiency in canalicular ATPase and glucose-6-phosphatase provided the best markers for the larger foci and nodules. In the second study, rats were subjected to partial hepatectomy, and half of the animals were then intubated with diethylnitrosamine (10 mg/kg). One wk later, rats were fed Wy-14,643 at concentrations of 0, 0.05, and 0.1% in the diet for 6 mo. At 6 mo, the number and volume of foci were increased by the feeding of Wy-14,643 after partial hepatectomy alone and were greatly increased when Wy-14,643 was fed after partial hepatectomy/diethylnitrosamine administration. Canalicular adenosine triphosphatase and glucose-6-phosphatase deficiencies were the most common markers of AHF, and AHF of these phenotypes occupied practically all of the focal volume. The larger AHF did not express GGT, and those foci exhibiting GGT were much less common and occupied very little volume. The absence of the GGT protein itself, as opposed to an inhibition of GGT activity, was verified by immunohistochemical staining using an antibody to GGT. These studies show that hypolipidemic peroxisome proliferators can stimulate an increase in AHF following a single dose of diethylnitrosamine and a mitotic stimulus, and they thus can act as promoters in two-stage liver carcinogenesis. GGT is a poor marker for identifying AHF induced by peroxisome proliferators during the early, premalignant phase of hepatocarcinogenesis.     

Effect of the peroxisome proliferators ciprofibrate and perfluorodecanoic acid on hepatic cell proliferation and toxicity in Sprague--Dawley rats

The objective of this study was to compare the effects of perfluorodecanoicacid (PFDA) and ciprofibrate on the induction of hepatic toxicityand on hepatocellular proliferation in rats. In the first study,rats were first subjected to partial hepatectomy and then injectedwith [³H]thymidine (20 µCi/injection) at 23, 24, 25, 47,48 and 49 h afterwards. After a 2 week recovery period, ratswere injected with one of four levels of PFDA (03, 1.0, 3.0or 10 mg/kg/injection) in four i.p. doses every 14 days, orwere fed 0.01% or 0.003% ciprofibrate. Six days after the lastPFDA injection and three days before the animals were killed,an osmotic minipump containing 20 mg/ml 5-bromo-2’-deoxyuridine(BrdU) was implanted s.c for the measurement of DNA synthesis.Peroxisomal fatty acyl-CoA oxidase activity was significantlyenhanced in both PFDA and ciprofibrate-treated groups in a dose-dependentmanner. Hepatotoxicity, measured as the loss of [³H]thymldinefrom hepatic DNA, was not significantly affected by any of thetreatments. Hepatic DNA synthesis was significantly increasedonly in rats receiving the highest dose of PFDA. In order todetermine the time course of ciprofibrate- and PFDA-inducedcell proliferation, we conducted another study with more timepoints. Rats were fed 0.01% ciprofibrate or were injected every14 days with 3 or 10 mg PFDA/kg body weight for 10 days, 24days, 6 weeks, 26 weeks or 54 weeks. Cell proliferation wasquantified as in the first study. Ciprofibrate increased cellproliferation at the early but not the later time points, whereasPFDA increased cell proliferation at most times throughout thestudy. This study demonstrates that PFDA and ciprofibrate donot selectively induce hepatic toxicity and that their effectson cell proliferation do not correlate with their carcinogenicor promoting activities.     

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.     

Elevated 8-hydroxydeoxyguanosine levels in DNA of diethylstilbestrol-treated Syrian hamsters: covalent DNA damage by free radicals generated by redox cycling of diethylstilbestrol

The generation of free radicals by microsome-mediated redox cycling between catechol estrogens or diethylstilbestrol and their corresponding quinones has previously been demonstrated in vitro. However, the reaction of free radicals with DNA has not yet been detected in animals treated with estrogen and is the subject of this investigation. The reaction of guanine bases of DNA with hydroxyl radicals to form 8-hydroxydeoxyguanosine has been used as a monitor of free radical generation in kidney and liver of Syrian hamsters, a species prone to estrogen-induced carcinogenesis. Prior to in vivo measurements, the in vitro hydroxylation of guanine bases of DNA under conditions of redox cycling of estrogen was investigated. In incubations of DNA or deoxyguanosine with hamster kidney microsomes, NADPH, and diethylstilbestrol 4',4"-quinone, the hydroxylation of guanine bases of free deoxyguanosine or of DNA was 50 to 100% higher than in controls. When incubations were carried out in the presence of iron(III) chloride, the hydroxylation of guanine bases was 2.5- or 10-fold higher than control values. There was a 65% increase from control values in levels of 8-hydroxydeoxyguanosine in liver DNA of hamsters treated with 20 mg/kg/day diethylstilbestrol for 3 days and 100 mg/kg on the 4th day. In hamsters treated chronically with diethylstilbestrol implants for 15 days, 8-hydroxydeoxyguanosine levels more than doubled from control values in kidney but not liver DNA. Treatment of hamsters with estradiol for various time periods did not induce any changes in levels of hydroxylated guanine in either kidney or liver. It was concluded that in vitro and in vivo redox cycling of diethylstilbestrol hydroxylated guanine bases in DNA.     

Effect of hypolipidemic peroxisome proliferators on unscheduled DNA synthesis in cultured hepatocytes and on mutagenesis in Salmonella

The peroxisome proliferators Wy-14,643, BR-931, nafenopin and ciprofibrate were tested in the primary hepatocyte culture-unscheduled DNA synthesis assay and in the Ames Salmonella microsome mutagenicity assay. The amount of unscheduled DNA synthesis (UDS) in hepatocytes was determined by quantifying the amount of [3H]thymidine incorporated into DNA in the presence of hydroxyurea after isolation of nuclei from hepatocytes treated with the test agent. Wy-14,643 and BR-931 induced unscheduled DNA synthesis in rat hepatocytes, whereas nafenopin and ciprofibrate had no effect. All of the peroxisome proliferators were negative in the Ames Salmonella assay.     

Patterns of 8-hydroxydeoxyguanosine formation in DNA and indications of oxidative stress in rat and human pleural mesothelial cells after exposure to crocidolite asbestos

Oxidative damage is a proposed mechanism of asbestos-induced carcinogenesis, but the detection of oxidative DNA lesions in target cells of asbestos-induced mesothelioma has not been examined. In studies here, DNA was isolated from both rat pleural mesothelial (RPM) cells and a human mesothelial cell line (MET5A) after exposure in vitro to crocidolite asbestos at various concentrations. DNA was then examined for formation of 8-hydroxydeoxyguanosine (8-OHdG) at 24, 48 and 72 h using HPLC with electrochemical detection. In addition, steady- state mRNA levels of manganese-containing superoxide dismutase (MnSOD) were assessed as an indication of oxidative stress. Whereas RPM cells showed dose-dependent and significant increases in 8-OHdG formation in response to crocidolite asbestos or iron-chelated crocidolite fibers (but not after exposure to glass beads), MET5A cells showed decreases in 8-OHdG. Both cell types exhibited elevations in message levels of MnSOD. In comparison with human MET5A cells, RPM cells exhibited increased cytotoxicity and apoptosis in response to asbestos, as documented by cell viability assays and flow cytometry analysis using propidium iodide. Results in RPM cells indicate that asbestos causes oxidative damage that may result in potentially mutagenic lesions in DNA and/or apoptosis, despite compensatory increases in expression of an antioxidant enzyme.      

Is 8-hydroxydeoxyguanosine a mediator of carcinogenesis by a choline-devoid diet in the rat liver?

The mechanism(s) by which a diet devoid of choline (CD) induceshepatocellular carcinomas in rats remains unknown. Althoughanimals fed this diet develop nuclear lipid peroxidation, suggestingoxidative DNA damage, there is no direct evidence that thisoccurs. In this study, 8-hydroxy-deoxyguanosine (8-OHdG), aDNA adduct generated by reactive oxygen species, was analyzedin the liver of rats fed a CD diet and in controls receivinga choline-sufficient (CS) diet. After partial hepatectomy, theanimals were injected with diethylnitrosamine (DEN, 50 mg/kgbody wt) or with saline and fed a CD or CS diet for 24 weeks.While liver DNA from rats injected either with DEN or salineand fed a CS diet did not show detectable amounts of the nucleotide,those who were fed DEN/CD and saline/CD demonstrated similar,easily measurable levels of 8-OHdG. These results indicate thatthere is a positive association between the continuous administrationof a CD diet and the production of 8-OHdG in liver DNA, andsupport the idea that oxidative DNA damage is involved in carcinogenesisby a CD diet.     

Increased resistance of peroxisome proliferator-induced hepatic lesions to iron overload in rats

Altered areas (AA), neoplastic nodules (NN) and hepatocellular carcinomas (HCC) induced by chronic administration of ciprofibrate and Wy-14,643 were examined for iron accumulation after systemic iron overload. Eighty percent of AA, 90% NN and 100% HCC showed increased resistance to iron accumulation. However, marked heterogeneity was observed in the amount of iron from area to area within the same lesion with some cells containing no iron, while others showing blue reaction to iron. These findings indicate, that putative preneoplastic and neoplastic hepatic lesions induced by peroxisome proliferators exclude iron in a fashion similar to that of hepatic nodules and carcinomas induced by DNA damaging carcinogens.     

Effect of peroxisome proliferators on intrachromosomal and interchromosomal recombination in yeast

Peroxisome proliferators are a class of non-mutagenic hepatocarcinogens, which induce a similar pleiotropic response such as hepatomegaly, proliferation of the peroxisomes in hepatocytes and hepatocarcinogenesis. Peroxisome proliferators are not detectable by the Ames assay and various other short-term tests. Recently a system for intrachromosomal recombination in yeast (DEL) has been shown to be inducible by a variety of non-mutagenic carcinogens. These include many carcinogens that are not detectable by the Ames assay or by various other short-term tests. In the present study the peroxisome proliferators [4-chloro-6-(2,3-xylidino)-2-pyrimidinyl-thio]acetic acid (Wy-14,643); methyl-2-[4-(p-chlorophenyl)phenoxy]2-methyl propionate (methyl clofenopate); 2-methyl-2[p-(1,2,3,4-tetrahydro-1- naphthyl)phenoxy]-propionic acid (nafenopin); 2-[4-(2,2-dichlorocyclopropyl)phenoxy]2-methyl-propionic acid (ciprofibrate); [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)-acetam ide] (BR-931); and ethyl alpha-p-chlorophenoxyisobutyrate (clofibrate) have been tested for their potential to induce DEL as well as interchromosomal recombination in yeast. No evidence for induction of either system has been found in the presence or the absence of the supernatant (S9) from rat liver homogenate. The data support the notion that peroxisome proliferators are truly non-mutagenic carcinogens.     

The peroxisome proliferators are hepatocyte mitogens in chemically- defined media: glucocorticoid-induced PPAR alpha is linked to peroxisome proliferator mitogenesis

Peroxisome proliferator-induced mitogenesis is believed to play a role in hepatocarcinogenesis, but it has not been possible to demonstrate high level induction of DNA synthesis by peroxisome proliferators in cultured hepatocytes. We now show that four structurally dissimilar peroxisome proliferators (methylclofenapate, Wy-14 643, tetradecyl-3- thia acetic acid and clofibrate) cause high level induction of DNA synthesis in primary cultures of rat hepatocytes, routinely 7-9 fold above control, with up to 29% of cells undergoing S-phase. Peroxisome proliferators induce DNA synthesis rapidly, with maximal response 24 h after dosing [compared with 48 h for epidermal growth factor (EGF)]; indeed, peroxisome proliferators were mitogenic in a chemically defined medium, i.e. with no added exogenous growth factors. EGF-treated hepatocytes that had undergone DNA synthesis comprised 23% binucleated cells, whereas hepatocytes induced into S-phase by peroxisome proliferators contained only 3% binucleated cells, demonstrating a distinct response of hepatocytes to peroxisome proliferators and EGF. The presence of a glucocorticoid was essential for peroxisome proliferator-induced DNA synthesis, but not for EGF-induced DNA synthesis, demonstrating that the requirement for glucocorticoids is selective for peroxisome proliferators. Hydrocortisone was shown to induce the expression of peroxisome proliferator activated receptor- alpha (PPAR alpha), and we propose that it is the glucocorticoid- induced expression of PPAR alpha that is essential for peroxisome proliferator mitogenesis. This in vitro system provides a powerful tool for investigating the mechanism and role of peroxisome proliferator- induced mitogenesis in liver growth and carcinogenesis.      

Production of both 8-hydroxydeoxyguanosine in liver DNA and gamma-glutamyltransferase-positive hepatocellular lesions in rats given a choline-deficient, L-amino acid-defined diet

The comparative carcinogenic activities of a choline-deficient, L-amino acid-defined diet (CDAADD) and a purified choline-deficient diet (CDD) for rat liver were studied in terms of both 8-hydroxydeoxyguanosine induction, a marker of DNA damage induced by oxidative stress, and development of gamma-glutamyltransferase (GGT)-positive putative preneoplastic lesions, including foci and hyperplastic nodules. Twelve weeks after the beginning of treatment, DNA damage could be detected in the liver DNA of rats receiving either CDAADD or CDD, the degree being significantly greater in the former case. Similarly, while GGT-positive liver lesions were induced by both CDAADD and CDD, the numbers were higher and the areas of lesions were larger in rats receiving CDAADD than in those given CDD. Histologically, hyperplastic nodules were induced in the livers of animals administered CDAADD whereas only foci were seen in the CDD case. The results thus indicate that oxidative stress might be directly involved in rat liver carcinogenesis by CDD and, to a greater degree, with CDAADD.     

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.