Sustained formation of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts in mouse liver by peroxisome proliferators is dependent upon peroxisome proliferator-activated receptor-alpha, but not NADPH oxidase

Reactive oxygen species are thought to be crucial for peroxisome proliferator-induced liver carcinogenesis. Free radicals have been shown to mediate the production of mitogenic cytokines by Kupffer cells and cause DNA damage in rodent liver. Previous in vivo experiments demonstrated that acute administration of the peroxisome proliferator di(2-ethylhexyl) phthalate (DEHP) led to an increase in production of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) radical adducts in liver, an event that was dependent on Kupffer cell NADPH oxidase, but not peroxisome proliferator-activated receptor (PPAR)alpha. Here, we hypothesized that continuous treatment with peroxisome proliferators will cause a sustained formation in POBN radical adducts in liver. Mice were fed diets containing either 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14,643, 0.05% w/w) or DEHP (0.6% w/w) for up to 3 weeks. Liver-derived radical production was assessed in bile samples by measuring POBN radical adducts using electron spin resonance. Our data indicate that WY-14,643 causes a sustained increase in POBN radical adducts in mouse liver and that this effect is greater than that of DEHP. To understand the molecular source of these radical species, NADPH oxidase-deficient (p47phox-null) and PPARalpha-null mice were examined after treatment with WY-14,643. No increase in radicals was observed in PPARalpha-null mice that were treated with WY-14,643 for 3 weeks, while the response in p47phox-nulls was similar to that of wild-type mice. These results show that PPARalpha, not NADPH oxidase, is critical for a sustained increase in POBN radical production caused by peroxisome proliferators in rodent liver. Therefore, peroxisome proliferator-induced POBN radical production in Kupffer cells may be limited to an acute response to these compounds in mouse liver.     

Alterations in the integrity of peroxisomal membranes in livers of mice treated with peroxisome proliferators

Catalase leakage from its particulate compartment within the light mitochondrial fraction of liver was used as an index of the integrity of peroxisomes in untreated mice and in mice treated with the peroxisome proliferators clofibrate(ethyl-p-chlorophenoxyisobutyrate), Wy-14,643(4-chloro-6[2,3-xylidino)-2-pyrimidinylthio]acetic acid) and DEHP(di-(2-ethylhexyl)phthalate).Catalase leakage represented about 2% of the total catalase activity when fractions from untreated mice were incubated at 4°C, increasing to about 5% during 60 min incubation at 37°C. In fractions from livers of mice treated with peroxisome proliferators, catalase leakage was significantly higher, being 7–11% at 4°C and increasing to approximately 20% after 60 min incubation at 37°C. The pattern of release was similar for all proliferators. Parallel data were obtained for catalase latency in these fractions, i.e. following 60 min incubation at 37°C, free (non-latent) catalase activity was 18% in control mice and 65, 67, and 83% in fractions from clofibrate-, Wy-14,643- and DEHP-treated mice, respectively. Differences in catalase leakage from peroxisomes in fractions from untreated mice and clofibrate-treated mice were also apparent following treatments designed to effect membrane permeabilization, as in freeze-thawing, osmotic rupture, and extraction with Triton X-100 and lysophosphatidylcholine.These data are consistent with a significant alteration in the integrity of the membranes of peroxisomes in livers of mice which have been treated with peroxisome proliferators, and furthermore indicate a commonality of effect of these agents.     

Procarbazine genotoxicity in the MutaMouse; strong clastogenicity and organ-specific induction of lacZ mutations.

Procarbazine, a drug used for cancer chemotherapy, is carcinogenic in rodent bioassays. We analyzed the mutagenicity of procarbazine in various organs and the clastogenicity of the drug in hematopoietic cells of the lacZ transgenic MutaMouse. This was part of the second collaborative study of the Mammalian Mutagenesis Study Group of the Japanese Environmental Mutagen Society on the transgenic mouse mutation assay. At 50 mg kg(-1), procarbazine induced micronuclei in hematopoietic cells, but it did not increase the lacZ mutant frequency (MF) in bone marrow. It was also negative in liver, testis, spleen, kidney, and lung. Five daily administrations of 150 mg kg(-1) yielded highly positive responses in the drug's target organs for carcinogenesis (lung, bone marrow, and spleen). Lower positive responses were detected in kidney, which is a minor target organ. Liver showed only a slight increase in lacZ MF and brain showed no increase. The testis MF more than doubled which suggest that procarbazine is mutagenic to germ cells. Thus, we demonstrated that procarbazine has a strong clastogenic effect in hematopoietic cells and is mutagenic in a variety organs after high dose treatment. The induced MF was especially high in procarbazine's target organs for carcinogenesis, which supports the relevance of the transgenic mouse mutation assay for the assessment of potential genotoxins in vivo.     

Effect of peroxisome proliferators on the methylation and protein level of the c-myc protooncogene in B6C3F1 mice liver

Peroxisome proliferators in general are nongenotoxic mouse liver carcinogens for which DNA hypomethylation and altered gene expression are proposed mechanisms. Therefore, the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D), dibutyl phthalate (DBP), gemfibrozil, and Wy-14,643 were evaluated for the ability to alter the methylation and expression of the c-myc protooncogene. Male B6C3F1 mice were administered for 6 days in their diet Wy-14,643 (5-500 ppm), 2,4-D (1,680 ppm), DBP (20,000 ppm), or gemfibrozil (8,000 ppm). All four peroxisome proliferators caused hypomethylation of the c-myc gene in the liver. Wy-14,643 appeared to be the most efficacious with a threshold between 10 and 50 ppm. The level of the c-myc protein was increased by Wy-14,643, but not the other peroxisome proliferators. When female B6C3F1 mice received a two-thirds partially hepatectomy and 16 h later were administered 50 mg/kg Wy-14,643 by gavage, hypomethylation of the gene occurred 24 h later. Hypomethylation was not found in mice that received Wy-14,643 following a sham operation. Hypomethylation of the c-myc gene within 24 h of administering Wy-14,643 after a partial hepatectomy but not after a sham operation supports the hypothesis that the peroxisome proliferators prevent methylation of hemimethylated sites formed by DNA replication.     

Nitrogen-substitution effect on in vivo mutagenicity of chrysene

We have previously reported the in vivo mutagenicity of aza-polycyclic aromatic hydrocarbons (azaPAHs), such as quinoline, benzo[f]quinoline, benzo[h]quinoline, 1,7-phenanthroline and 10-azabenzo[a]pyrene. The 1,10-diazachrysene (1,10-DAC) and 4,10-DAC, nitrogen-substituted analogs of chrysene, were shown to exhibit mutagenicity in Salmonella typhimurium TA100 in the presence of rat liver S9 and human liver microsomes in our previous report, although DACs could not be converted to a bay-region diol epoxide, the ultimate active form of chrysene, because of their nitrogen atoms. In the present study, we tested in vivo mutagenicity of DACs compared with chrysene using the lacZ transgenic mouse (Mutatrade markMouse) to evaluate the effect of the nitrogen substitution. DACs- and chrysene-induced mutation in all of the six organs examined (liver, spleen, lung, kidney, bone marrow and colon). The mutant frequencies obtained with chrysene showed only small differences between the organs examined and ranged from 1.5 to 3 times the spontaneous frequency. The 4,10-DAC was more mutagenic than chrysene in all the organs tested. The highest lacZ mutation frequency was observed in the lung of 4,10-DAC-treated mice and it was 19 and 6 times the spontaneous frequency and the frequency induced by chrysene, respectively. The 1,10-DAC induced lacZ mutation in the lung with a frequency 4.3- and 1.5-fold higher than in the control and chrysene-treated mice, respectively, although the mutant frequencies in the other organs of 1,10-DAC-treated mice were almost equivalent to those of chrysene-treated mice. Not only chrysene but also DACs depressed the G:C to A:T transition and increased the G:C to T:A transversion in the liver and lung. These results suggest that the two types of nitrogen substitutions in the chrysene structure may enhance mutagenicity in the mouse lung, although they showed no difference in the target-organ specificity and the mutation spectrum.     

Epigenetic effects of the continuous exposure to peroxisome proliferator WY-14,643 in mouse liver are dependent upon peroxisome proliferator activated receptor alpha

Peroxisome proliferators are potent rodent liver carcinogens that act via a non-genotoxic mechanism. The mode of action of these agents in rodent liver includes increased cell proliferation, decreased apoptosis, secondary oxidative stress and other events; however, it is not well understood how peroxisome proliferators are triggering the plethora of the molecular signals leading to cancer. Epigenetic changes have been implicated in the mechanism of liver carcinogenesis by a number of environmental agents. Short-term treatment with peroxisome proliferators and other non-genotoxic carcinogens leads to global and locus-specific DNA hypomethylation in mouse liver, events that were suggested to correlate with a burst of cell proliferation. In the current study, we investigated the effects of long-term exposure to a model peroxisome proliferator WY-14,643 on DNA and histone methylation. Male SV129mice were fed a control or WY-14,643-containing (1000ppm) diet for one week, five weeks or five months. Treatment with WY-14,643 led to progressive global hypomethylation of liver DNA as determined by an HpaII-based cytosine extension assay with the maximum effect reaching over 200% at five months. Likewise, trimethylation of histone H4 lysine 20 and H3 lysine 9 was significantly decreased at all time points. The majority of cytosine methylation in mammals resides in repetitive DNA sequences. In view of this, we measured the effect of WY-14,643 on the methylation status of major and minor satellites, as well as in IAP, LINE1 and LINE2 elements in liver DNA. Exposure to WY-14,643 resulted in a gradual loss of cytosine methylation in major and minor satellites, IAP, LINE1 and LINE2 elements. The epigenetic changes correlated with the temporal effects of WY-14,643 on cell proliferation rates in liver, but no sustained effect on c-Myc promoter methylation was observed. Finally, WY-14,643 had no effect on DNA and histone methylation status in Pparalpha-null mice at any of the time points considered in this study. These data indicate the importance of epigenetic alterations in the mechanism of action of peroxisome proliferators and the key role of Pparalpha.     

Specific changes in the protein composition of rat liver in response to the peroxisome proliferators ciprofibrate, Wy-14,643 and di-(2-ethylhexyl)phthalate.

The hypolipidaemic agents ciprofibrate and Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) and the phthalate-ester plasticizer di-(2-ethylhexyl)-phthalate (DEHP), like other peroxisome proliferators, produce a significant hepatomegaly and induce the peroxisomal fatty acid beta-oxidation enzyme system together with profound proliferation of peroxisomes in hepatic parenchymal cells. Changes in the profile of liver proteins in rats following induction of peroxisome proliferation by ciprofibrate, Wy-14,643 and DEHP have been analysed by high-resolution two-dimensional gel electrophoresis. The proteins of whole liver homogenates from normal and peroxisome-proliferator-treated rats were separated by two-dimensional gel electrophoresis using isoelectric focusing for acidic proteins and nonequilibrium pH gradient electrophoresis for basic proteins. In the whole liver homogenates, the quantities of six proteins in acidic gels and six proteins in the basic gels increased following induction of peroxisome proliferation. Peroxisome proliferator administration caused a repression of three acidic proteins in the liver homogenates. By the immunoblot method using polyspecific antiserum against soluble peroxisomal proteins and monospecific antiserum against peroxisome proliferation associated Mr 80000 polypeptide (polypeptide PPA-80), the majority of basic proteins induced by these peroxisome proliferators appeared to be peroxisomal proteins. Polypeptide PPA-80 becomes the most abundant protein in the total liver homogenates of peroxisome-proliferator-treated rats. These results indicate that ciprofibrate, DEHP and Wy-14,643 induce marked changes in the profile of specific hepatic proteins and that some of these changes should serve as a baseline to identify a set of gene products that may assist in defining the specific 'peroxisome proliferator domain'.     

N-Nitrosodi-n-propylamine induces organ specific mutagenesis with specific expression times in lacZ transgenic mice.

The mutagenic and clastogenic effects of N-nitrosodi-n-propylamine (NDPA) in lacZ transgenic mice (MutaMouse) were investigated as a part of the second collaborative study of the transgenic mouse mutation assay by a subgroup of the Mammalian Mutagenesis Study Group, a suborganization of the Environmental Mutagen Society of Japan. Male MutaMouse mice were administered NDPA intraperitoneally at a dose of 250 mg/kg, which is half of the LD(50) of the compound. The clastogenicity of NDPA was examined by the peripheral blood micronucleus test just before and at 24, 48 and 72 h after the treatment. The mutant frequencies in the bone marrow, liver, lung, kidney and urinary bladder were examined by the positive selection method for lacZ kidney. These findings demonstrate that NDPA induces organ-specific mutagenesis with specific expression times, and that the mutagenicity of NDPA in lacZ transgenic mice is consistent with its carcinogenicity.     

Induction of microsomal 1-acylglycerophosphocholine acyltransferase by peroxisome proliferators in rat kidney; co-induction with peroxisomal beta-oxidation

Induction of microsomal 1-acyl-glycerophosphocholine (GPC) acyltransferase in rat tissues by four peroxisome proliferators, clofibric acid, tiadenol, DEHP and PFOA, was examined. Among the nine tissues examined, kidney, liver and intestinal mucosa responded to the challenges by the peroxisome proliferators to induce the enzyme. The treatment of rats with various dose of clofibric acid, tiadenol, DEHP or PFOA resulted in an induction of kidney microsomal 1-acyl-GPC acyltransferase in a dose-dependent manner. Despite the structural dissimilarity of peroxisome proliferators, the induction of microsomal 1-acyl-GPC acyltransferase was highly correlated with the induction of peroxisomal beta-oxidation. The activity of microsomal 1-acyl-GPC acyltransferase was not affected by changes in hormonal (adrenalectomy, diabetes, hyperthyroidism and hypothyroidism) and nutritional (starvation, starvation-refeeding, fat-free-diet feeding and high-fat-diet feeding) states. The induction of renal microsomal 1-acyl-GPC acyltransferase was seen in mice subsequent to the administration of clofibric acid and tiadenol and in guinea pigs subsequent to the administration of tiadenol. These results may indicate that kidney microsomal 1-acyl-GPC acyltransferase is a highly specific parameter responsive to the challenges by peroxisome proliferators and may suggest that the possibility that the inductions by peroxisome proliferators of microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation in kidney are co-regulated.     

Mutagenicity of aristolochic acid in the lambda/lacZ transgenic mouse (MutaMouse)

Aristolochic acid (AA) is found in a plant that causes urothelial carcinomas in patients with Chinese herb nephropathy (CHN). To evaluate the in vivo mutagenicity of AA, we analysed the mutant frequency (MF) in the lacZ and cII gene of 10 organs of the lambda/lacZ transgenic mouse (MutaMouse) after intragastric treatment with AA (15mg/kg per week x 4). Simultaneously, the clastogenicity of AA was evaluated by the peripheral blood micronucleus assay. The nature of the mutations induced by AA was revealed by the sequence analysis of the cII gene, which is also a phenotypically selectable marker in the lambda transgene. MFs in the target organs-forestomach, kidney, and bladder of AA-treated mice were significantly higher than those of control mice (forestomach 33- and 15-fold; kidney 10- and 9-fold; bladder 16- and 31-fold, for the lacZ and cII, respectively). The MFs in non-target organs, except the colon, showed only slight increases. Sequence analysis of cII mutants in target organs revealed that AA induced mainly A:T to T:A transversions whereas G:C to A:T transitions at CpG sites predominated among spontaneous mutations. These results suggested that AA, which is activated by cytochrome P450 and peroxidase to form cyclic nitrenium ions that bind to deoxyadenine, caused the A to T transversions in the target organs of mice.     

Mechanisms of peroxisome proliferator-induced DNA hypomethylation in rat liver

Genomic hypomethylation is a consistent finding in both human and animal tumors and mounting experimental evidence suggests a key role for epigenetic events in tumorigenesis. Furthermore, it has been suggested that early changes in DNA methylation and histone modifications may serve as sensitive predictive markers in animal testing for carcinogenic potency of environmental agents. Alterations in metabolism of methyl donors, disturbances in activity and/or expression of DNA methyltransferases, and presence of DNA single-strand breaks could contribute to the loss of cytosine methylation during carcinogenesis; however, the precise mechanisms of genomic hypomethylation induced by chemical carcinogens remain largely unknown. This study examined the mechanism of DNA hypomethylation during hepatocarcinogenesis induced by peroxisome proliferators WY-14,643 (4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid) and DEHP (di-(2-ethylhexyl)phthalate), agents acting through non-genotoxic mode of action. In the liver of male Fisher 344 rats exposed to WY-14,643 (0.1% (w/w), 5 months), the level of genomic hypomethylation increased by approximately 2-fold, as compared to age-matched controls, while in the DEHP group (1.2% (w/w), 5 months) DNA methylation did not change. Global DNA hypomethylation in livers from WY-14,643 group was accompanied by the accumulation of DNA single-strand breaks, increased cell proliferation, and diminished expression of DNA methyltransferase 1, while the metabolism of methyl donors was not affected. In contrast, none of these parameters changed significantly in rats fed DEHP. Since WY-14,643 is much more potent carcinogen than DEHP, we conclude that the extent of loss of DNA methylation may be related to the carcinogenic potential of the chemical agent, and that accumulation of DNA single-strand breaks coupled to the increase in cell proliferation and altered DNA methyltransferase expression may explain genomic hypomethylation during peroxisome proliferator-induced carcinogenesis.     

Partial purification and immunoreactivity of an 80000-molecular-weight polypeptide associated with peroxisome proliferation in rat liver

Hypolipidaemic drugs and industrial plasticizers such as di-(2-ethylhexyl) phthalate, which cause proliferation of hepatic peroxisomes, also cause an increase in an 80000-mol.wt. polypeptide in the liver of rats and mice. This polypeptide has been designated as PPA-80 (PPA, for peroxisome-proliferation-associated; 80 for 80000mol.wt.). The polypeptide PPA-80 was purified to over 90% purity from livers of rats treated with the peroxisome proliferators Wy-14,643, nafenopin, tibric acid and clofibrate by a single-step preparative sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic procedure. The antibodies raised against the PPA-80 polypeptide isolated from livers of rats treated with Wy-14,643 cross-reacted with polypeptide PPA-80 purified from the livers of rats treated with Wy-14,643, as well as from the livers of rats treated with nafenopin, tibric acid and clofibrate. The anti-(polypeptide PPA-80) antibodies did not cross-react with catalase, a marker enzyme for peroxisomes, or with NADPH–cytochrome P-450 reductase, which has the same approximate mol.wt., 80000. The intensity of immunoprecipitin bands formed with microsomal, large-particle and postnuclear fractions from livers of animals pretreated with peroxisome proliferators was significantly greater compared with equal amounts of protein from corresponding fractions obtained from control animals, suggesting that these agents all enhance the synthesis of the same 80000-mol.wt. polypeptide. Although the polypeptide PPA-80 was increased in the postnuclear, large-particle and microsomal fractions of livers of rats pretreated with peroxisome proliferators, the relative abundance of this peptide in the peroxisome-rich light-mitochondrial fraction and its lack in highly purified mitochondrial fractions suggest the localization of this polypeptide in peroxisomes and/or microsomal fraction. Additional studies are needed to establish unequivocally the subcellular localization of the polypeptide PPA-80 and to ascertain if this polypeptide is identical with the multi-functional protein displaying enoyl-CoA hydratase and β-hydroxyacyl-CoA dehydrogenase activities that was purified by Osumi & Hashimoto [(1979) Biochem. Biophys. Res. Commun. 89, 580–584].     

Changes to the integral membrane protein composition of mouse liver peroxisomes in response to the peroxisome proliferators clofibrate,Wy-14,643 and Di(2-ethyl-hexyl)phthalate

Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes — namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl--p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio] acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate).Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells.These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins.     

Morphological transformation and catalase activity of syrian hamster embryo cells treated with hepatic peroxisome proliferators,TPA and nickel sulphate

The abilities of the hepatic peroxisome proliferators (HPPs) clofibrate, di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)- phthalate (MEHP), 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) and tiadenol to induce morphological transformation and to increase the catalase activity of Syrian hamster embryo (SHE) cells were studied. DEHP, MEHP, clofibrate and tiadenol induced morphological transformation of SHE cells and increased the catalase activity. DEHP was more potent than clofibrate and tiadenol in both inducing catalase and morphological transformation, while MEHP seemed more potent than DEHP in inducing catalase, but not morphological transformation, 2,4,5-T and 2,4-D did not induce morphological transformation, but 2,4,5-T was more potent than clofibrate in increasing the catalase activity. These results show that several HPPs induce morphological transformation of SHE cells and an increase in the catalase activity. There is, however, no direct connection between these two parameters, as seen from the results of 2,4,5-T. The tumor promoter TPA, and the metal salt nickel sulphate, induced morphological transformation of SHE cells without any appreciable increase in the catalase activity. These results further corroborate the dissociation between induction of morphological transformation and the increase in catalase activity.Abbreviations Clofibrate ethyl-2-(p-chlorophenox) isobutyrate - 2,4-D 2,4-dichlorophenoxy acetic acid - DEHP di(2-ethylhexyl)phthalate - HPP hepatic peroxisome proliferator - MEHP mono(2-ethylhexyl)phthalate - SHE Syrian hamster embryo - 2,4,5-T 2,4,5-trichlorophenoxy acetic acid - tiadenol di(hydroxyethylthio)-1,10-decane     

Induction and peroxisomal appearance of gulonolactone oxidase upon clofibrate treatment in mouse liver

Various antihyperlipemic peroxisome proliferators are known to be carcinogenic in rodents but not in human, other primates and guinea pig, which species lost their ability to synthesize ascorbate due to mutations in the gulonolactone oxidase gene. Ascorbate synthesis is accompanied by H2O2 production, consequently its induction can be potentially harmful; therefore, the in vivo effect of the peroxisome proliferator clofibrate was investigated on gulonolactone oxidase expression in mouse liver. Liver weights and peroxisomal protein contents were increased upon clofibrate treatment. Elevated plasma ascorbate concentrations were found in clofibrate-treated mice due to the higher microsomal gulonolactone oxidase activities. Remarkable gulonolactone oxidase activity appeared in the peroxisomal fraction upon the treatment. Increased activity of the enzyme was associated with an elevation of its mRNA level. According to the present results the evolutionary loss of gulonolactone oxidase may contribute to the explanation of the missing carcinogenic effect of peroxisome proliferators in humans.     

The genetic toxicity of the peroxisome proliferator class of rodent hepatocarcinogen

Peroxisome proliferators comprise a structurally diverse class of chemicals. Some of the members of this class show evidence of genetic toxicity (most evidently the in vitro clastogen Wyeth 14,643, WY), while others do not (most evidently methyl clofenapate, MCP). When attempting to understand the mechanism of rodent hepatocarcinogenesis of this class of chemicals the possible role of genetic toxicity should be assessed on a class-wide basis, i.e., if just one peroxisome proliferator is shown to be unequivocally inactive as a genetic toxin, genetic toxicity cannot be implicated in the carcinogenic activity of peroxisome proliferators as a class. In an earlier paper, we established MCP as inactive in a range of in vitro and in vivo genetic toxicity assays. However, the top dose level of MCP that could be tested for induction of chromosome aberrations (clastogenicity) in human lymphocytes and CHO cells was limited by the relative insolubility of the test agent in the assay medium. Methyl clofenapate was not toxic up to a dose that produced precipitate, so cannot be directly compared with WY, which induced aberrations only at toxic dose levels. In the present paper, we have evaluated the clastogenicity of the carcinogenic peroxisome proliferator nafenopin (NAF) at dose levels up to those that are toxic to CHO cells, and found no evidence of chromosome aberration induction. These data isolate further the genetic toxicity of WY from other peroxisome proliferators, and increase confidence in the proposal that genetic toxicity does not play a critical role in the hepatocarcinogenicity of peroxisome proliferators.     

Species differences in hepatic peroxisome proliferation, cell replication and transforming growth factor-beta1 gene expression in the rat, Syrian hamster and guinea pig

The objective of this study was to evaluate species differences in the hepatic effects of three potent rodent peroxisome proliferators, namely methylclofenapate (MCP), ciprofibrate (CIP) and Wy-14,643 (WY), particularly with respect to effects on replicative DNA synthesis and transforming growth factor-beta1 (TGF-beta1) gene expression. Male Sprague-Dawley rats, Syrian hamsters and Dunkin-Hartley guinea pigs were given daily oral doses of 0 (corn oil) and 75 mg/kg MCP for periods of 6 and 21 days. Syrian hamsters and guinea pigs were also treated with 25 mg/kg CIP and 25 mg/kg WY. Relative liver weights were significantly increased in peroxisome proliferator-treated rats and Syrian hamsters, but not in guinea pigs. Hepatic peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities and CYP4A isoform mRNA levels were significantly increased in rats and Syrian hamsters, whereas only minor effects were observed in the guinea pig. Replicative DNA synthesis was studied by implanting 7-day osmotic pumps containing 5-bromo-2'-deoxyuridine during study days -1 to 6 and 14 to 21. Hepatocyte labelling index values were increased by MCP in the rat, but neither MCP, CIP nor WY produced any significant effect on replicative DNA synthesis in the Syrian hamster and guinea pig. MCP treatment increased TGF-beta1 and insulin-like growth factor II/mannose-6-phosphate (IGFII/Man6P) receptor gene expression in the rat. In the Syrian hamster, effects on TGF-beta1 and IGFII/Man6P receptor gene expression were also observed in some instances, whereas TGF-beta1 mRNA levels were essentially unchanged in the guinea pig. These results provide further evidence for marked species differences in response to rodent peroxisome proliferators. While peroxisome proliferators produce a wide spectrum of effects in rat liver, other species such as the Syrian hamster and guinea pig are less responsive and in the case of some endpoints (e.g., cell replication) may be refractory.