Hepatic Peroxisome Proliferation Research Articles

The relationship between liver peroxisome proliferation and adipose tissue atrophy induced by peroxisome proliferator exposure and withdrawal in mice

We have previously demonstrated that severe adipose tissue atrophy occurs upon dietary treatment of mice with potent peroxisome proliferators (PPs). This atrophy occurs subsequent to peroxisome proliferation in the liver and may represent a novel addition to the pleiotropic effects exerted by PPs. In the present study we have characterized the recovery of mice from such atrophy following cessation of exposure. Following termination of treatment with perfluorooctanoic acid (PFOA) for 7 days, the adipose tissue atrophy was rapidly reversed, beginning on 2–5 days of recovery and being complete within 10 days. In contrast, hepatic peroxisome proliferation recovered much more slowly, indicating that these processes are not strictly coordinated. Analysis of lipoprotein lipase and hormone-sensitive lipase activities in adipose tissue revealed that the decrease and increase in these activities, respectively, caused by PFOA were both reversed within 10 days of recovery. Overall, these data provide further support for our previous conclusion that the adipose tissue atrophy induced by PFOA is caused, at least in part, by changes in the activities of lipoprotein lipase and hormone-sensitive lipase. The serum level of cholesterol, which increased after termination of PFOA treatment, returned to normal with a time-course similar to the recovery of adipose tissue weight, although hepatic peroxisome proliferation was still present. The possible relationship between the reduction in serum cholesterol and/or in its availability to peripheral tissues and the associated atrophy of adipose tissues caused by PPs is discussed.

PPARs of the Heart

The peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated transcription factors within the broad nuclear receptor superfamily. The PPAR family includes three members encoded by distinct genes: α, β/δ, and γ (see reviews1,2). PPARα was originally identified as the intracellular receptor for a class of nongenotoxic rodent hepatocarcinogens, which includes the hypolipidemic drug clofibrate, a potent inducer of hepatic peroxisomal proliferation and hypolipidemic agent. The three PPARs are now distinguished by tissue- and developmental-specific patterns of expression and by the distinct, albeit overlapping, nature of ligands capable of activating each receptor. PPARα, which is abundant in tissues with high rates of mitochondrial fatty acid oxidation, such as heart, liver, and kidney, regulates a wide variety of target genes involved in cellular lipid catabolism. In contrast, PPARγ, an adipose-enriched nuclear receptor, directs the expression of genes involved in adipocyte differentiation and fat storage. The function of the ubiquitously expressed PPARβ/δ, is not well understood although some evidence suggests that it exerts actions on the epidermis and activates antiinflammatory programs. Ligand activation of PPARs leads to obligate heterodimerization with the 9- cis retinoic acid-activated receptor, RXR, promoting binding of the complex to cognate DNA response elements within PPAR target gene promoter regions (Figure). A variety of natural and synthetic compounds including fatty acids, eicosanoids, and arachidonic acid derivatives can serve as activators of the PPARs, some in a receptor-specific manner. However, the true endogenous ligands have not been identified. PPAR transcriptional regulatory complex. PPARs bind to sequence-specific target elements (PPREs) as a liganded heterodimer with the retinoid X receptor (RXR). Major functions based on known target genes and examples of relevant tissues (in parentheses) for each member of the PPAR family are shown. The question mark indicates that target genes for PPARβ/δ are largely unidentified. PPARα has been shown to …

ZnCl 2 induces Syrian hamster embryo (SHE) cell transformation

In order to test the hypothesis of a relationship between apoptosis and neoplastic transformation, we studied the transforming potency of zinc, known for its antiapoptotic effects. In this study, zinc chloride (100 μM) was shown to induce morphological transformation (MT) in Syrian hamster embryo (SHE) cells. It was also tested in combination with benzo(a)pyrene (BaP), a positive control for carcinogenicity, or fomesafen, a carcinogenic pesticide with hepatic peroxisomal proliferation properties. A co-exposure of the two carcinogens with 100 μM zinc increased cell transformation in SHE cells. These results were in agreement with the theory of a relationship between the inhibition of apoptosis and induction of cell transformation. The cloning efficiency (CE) of SHE cells seeded at clonal density was raised by zinc, fomesafen and furthermore by the mixture of the two chemicals, which could be explained by the antiapoptotic action of zinc and fomesafen on SHE cells. No change in myc and bax expressions was observed in zinc-treated SHE cells.

Mechanisms of liver steatosis in rats with systemic carnitine deficiency due to treatment with trimethylhydraziniumpropionate

Rats with systemic carnitine deficiency induced by treatment with trimethylhydraziniumpropionate (THP) develop liver steatosis. This study aims to investigate the mechanisms leading to steatosis in THP-induced carnitine deficiency. Rats were treated with THP (20 mg/100 g) for 3 or 6 weeks and were studied after starvation for 24 h. Rats treated with THP had reduced in vivo palmitate metabolism and developed mixed liver steatosis at both time points. The hepatic carnitine pool was reduced in THP-treated rats by 65% to 75% at both time points. Liver mitochondria from THP-treated rats had increased oxidative metabolism of various substrates and of beta-oxidation at 3 weeks, but reduced activities at 6 weeks of THP treatment. Ketogenesis was not affected. The hepatic content of CoA was increased by 23% at 3 weeks and by 40% at 6 weeks in THP treated rats. The cytosolic content of long-chain acyl-CoAs was increased and the mitochondrial content decreased in hepatocytes of THP treated rats, compatible with decreased activity of carnitine palmitoyltransferase I in vivo. THP-treated rats showed hepatic peroxisomal proliferation and increased plasma VLDL triglyceride and phospholipid concentrations at both time points. A reduction in the hepatic carnitine pool is the principle mechanism leading to impaired hepatic fatty acid metabolism and liver steatosis in THP-treated rats. Cytosolic accumulation of long-chain acyl-CoAs is associated with increased plasma VLDL triglyceride, phospholipid concentrations, and peroxisomal proliferation.

Fibrate treatment does not modify the expression of acyl coenzyme A oxidase in human liver

Fibrates induce hepatic peroxisome proliferation and carcinogenesis in rodents by activating peroxisome proliferator-activated receptor alpha (PPAR(alpha)). There is no conclusive evidence that humans are unresponsive to peroxisome proliferation, and concern exists about the long-term safety of fibrate treatment. In a university hospital setting, 48 patients with uncomplicated gallstones and a serum level of low-density lipoprotein cholesterol greater than 130 mg/dL were randomly assigned to open-label treatment with bezafibrate (400 mg/d), fenofibrate (200 mg/d), gemfibrozil (900 mg/d), or placebo for 8 weeks before elective cholecystectomy. Serum samples for lipid determinations were obtained at baseline and before surgery. A liver specimen was obtained at operation, and the relative levels of messenger ribonucleic acid (mRNA) for the wild and truncated forms of PPAR(alpha), acyl coenzyme A oxidase, liver carnitine palmitoyltransferase I, apolipoprotein A-I, and stearoyl coenzyme A desaturase were determined. Fenofibrate, bezafibrate, and gemfibrozil reduced plasma low-density lipoprotein cholesterol levels by 22% (P =.009), 14% (P =.042), and 11% (not significant), respectively. Plasma triglyceride levels decreased significantly (24%-36%; P <.05), whereas high-density lipoprotein cholesterol levels rose nonsignificantly after treatment with the 3 fibrates. Except for a 35% increase of apolipoprotein A-I mRNA after fenofibrate administration (P <.05), none of the individual fibrates induced significant changes in the mRNAs tested, although as a group they increased the mRNA for liver carnitine palmitoyltransferase I by 40%(P =.08; marginally significant). Fibrate administration to humans at pharmacologic doses able to activate PPAR(alpha) and to induce a hypolipidemic effect does not increase the hepatic expression of acyl coenzyme A oxidase, a well-known marker of peroxisome proliferation in rodents.

Mechanisms Involved in Responses to the Peroxisome Proliferator WY-14,643 on Gap Junctional Intercellular Communication in V79 Hamster Fibroblasts

WY-14,643, a potent hepatic peroxisome proliferator, decreased gap junctional intercellular communication when used at low and intermediate concentrations (1 nM to 10 μM) in the V79 Chinese hamster fibroblast cell line. It did not decrease intercellular communication in early passage Syrian hamster embryo fibroblasts. The mechanism of WY-14,643-induced suppression of intercellular communication was studied by preexposure of V79 cells to inhibitors of protein kinase C, mitogen-activated protein kinases, phosphatidylinositol 3-kinase, or mammalian target-of-rapamycin before addition of WY-14,643. Only protein kinase C, particularly the δ isoform, appeared involved in the inhibition of communication by WY-14,643. Also clofibrate-induced suppression of GJIC in V79 cells appeared to involve protein kinase Cδ. Furthermore, WY-14,643 did not cause any activation of the mitogen-activated protein kinases p44/42, p38, or Jun N-terminal kinase. When WY-14,643 was used at a higher concentration (100 μM), intercellular communication was increased both in V79 and Syrian hamster embryo cells. This effect was inhibited by preexposure of V79 cells to brefeldin A. Thus, there may be a pool of connexins in the Golgi complex that could be recruited to the plasma membrane upon exposure to high concentrations of WY-14,643.

Species differences in response to the phthalate plasticizer monoisononylphthalate (MINP) in vitro: a comparison of rat and human hepatocytes

Diisononylphthalate (DINP) is one of the group of dialkyl phthalate esters used widely to impart flexibility to polyvinyl chloride (PVC) products. However, DINP and other phthalates are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent hepatic peroxisome proliferation, induction of DNA synthesis and suppression of apoptosis leading to liver tumours. Despite these adverse effects in rodent liver, humans appear to be nonresponsive to the adverse effects of PPs. Here, we have examined species differences in the response of rat and human hepatocytes to MINP, a principle metabolite of DINP and the proximal peroxisome proliferator. In rat hepatocytes in vitro, MINP caused a concentration-dependent induction of peroxisomal beta-oxidation. Similarly, MINP caused a concentration-dependent suppression of apoptosis and induction of DNA synthesis. In contrast to the pleiotropic response noted in rat hepatocytes, MINP did not cause induction of beta-oxidation, stimulation of DNA synthesis or suppression of apoptosis in human hepatocytes. These data provide evidence for species differences in the hepatic response to the phthalate ester DINP, confirming that human hepatocytes are refractory to the adverse effects noted in rodents.

Profiling of Acyl-CoA Oxidase-Deficient and Peroxisome Proliferator Wy14,643-Treated Mouse Liver Protein by Surface-Enhanced Laser Desorption/Ionization ProteinChip® Biology System

Peroxisome proliferators induce hepatic peroxisome proliferation and hepatocellular carcinomas in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including fatty acids. Mice lacking fatty acyl-CoA oxidase (AOX-/-), the first enzyme of the peroxisomal beta-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation. To investigate proteins involved in peroxisome proliferation, we adopted a novel surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology to compare the protein profiles of control (wild-type), AOX-/-, and wild-type mice treated with peroxisome proliferator, Wy-14,643. The results indicated that the protein profiles of AOX-/- mice were similar to the wild-type mice treated with Wy14,643, but significantly different from the nontreated wild-type mice. Using four different ProteinChip Arrays, a total of 40 protein peaks showed more than twofold changes. Among these differentially expressed peaks, a downregulated peak was identified as the major urinary protein in both AOX-/- and Wyl4,643-treated mice by SELDI. The identification of MUP was further confirmed by two-dimensional electrophoresis and liquid chromatography coupled tandem mass spectrometry (LC-MS-MS). This SELDI method offers several technical advantages for detection of differentially expressed proteins, including ease and speed of screening, no need for chromatographic processing, and small sample size.

Characterization of the adipose tissue atrophy induced by peroxisome proliferators in mice.

In the present study, we characterized the effects of peroxisome proliferators (PP) on adipose tissue in mice. Treatment with potent PP, such as perfluorooctanoic acid (PFOA), 2-methyl-2-(p(1,2,3,4-tetrahydroxy-naphthyl)-phenoxy)propionic acid, (4-chloro-6-(2,3-xylidino)2-pyrimidinylthio) acetic acid, and di(2-ethylhexyl)phthalate, caused dramatic decreases in adipose tissue weight, whereas the moderately potent PP, acetylsalicylic acid, had a relatively weak effect. This decrease in weight reflects a loss of fat from adipocytes rather than a loss of cells, as demonstrated by constant DNA content. The dose-dependency and time-course experiments indicate that peroxisome proliferation occurs simultaneously with or prior to adipose tissue atrophy. Thus, hepatic peroxisome proliferation might result in the increased mobilization of lipids and lipid utilization in liver. The enhanced adipose tissue hormone-sensitive lipase (HSL) activity and down-regulated lipoprotein lipase (LPL) activity observed upon PP treatment might, at least in part, explain the loss of fat via increased FA release from adipocytes and/or decreased FA uptake from the circulation, respectively. In addition, the possible involvement of the increased tumor necrosis factor alpha expression found upon PFOA treatment in reducing the insulin sensitivity of adipose tissue and thereby altering LPL and HSL activities is discussed.

EFFECTS OF THE RAT HEPATIC PEROXISOME PROLIFERATOR, WYETH 14,643, ON THE LACTATING GOAT

ABSTRACTSome peroxisome proliferators have been reported to reduce body weight gain in suckling rats, possibly through a lactational effect. Decreases in milk production or nutritional quality, either as a result of peroxisome proliferator-induced reductions in lipid content or alterations in the hormonal milieu necessary for milk production, could result in pup growth retardation. Wyeth-14,643 (WY) is hypolipidemic agent and a potent inducer of hepatic peroxisome proliferation in rats and mice. As is commonly seen with rodent hepatic peroxisome proliferators, WY produces minimal or no peroxisome induction in guinea pigs or non-human primates. Goats are an excellent model for studying lactation, however, their sensitivity to peroxisome proliferating chemicals is not known. The present study was performed to assess the sensitivity of goats to the hypolipidemic and peroxisome proliferator properties of WY and to determine the effects of WY on milk quantity and quality. Six lactating adult female goats were assigned to either control or treated groups. Goats in the treated group were administered WY (40 mg/kg/day) for 14 consecutive days. The goats were milked twice daily in order to maintain lactation and the quantity of milk collected was recorded. Milk quality was evaluated by determining the content of total fat, protein, and carbohydrate in milk samples collected following 7 and 14 days of treatment. WY administration had no effects on final body weight, liver weight or, gross and histopathological findings. Milk quantity and quality were unaffected by treatment. Serum cholesterol and triglyceride levels were reduced by 25% compared to controls, although only the difference in cholesterol was statistically significant. Hepatic β-oxidation (3 × control) and aromatase (1.5 × control) activities were significantly greater in the treatment group; however, there was no treatment-related effect in the total content of hepatic cytochrome P450. There was no difference in aromatase activity in a pooled ovarian microsome sample. Milk estradiol and prolactin concentrations were not affected by treatment. These findings indicate that goats are weak responders to the hepatic peroxisome proliferator effects of WY. Additionally, the slight serum hypolipidemic effect does not impact milk production or nutritional value.

Perfluorooctanoate, perflourooctanesulfonate, and N-ethyl perfluorooctanesulfonamido ethanol; peroxisome proliferation and mitochondrial biogenesis

Compounds that cause peroxisome proliferation in rats and mice have been reported to interfere with mitochondrial (mt) bioenergetics and possibly biogenesis. The purpose of this investigation was to establish whether proliferation of peroxisomes and mitochondria are necessarily related. Perfluorooctanesulfonate (PFOS) and N-ethyl perfluorooctanesulfonamido ethanol ( N-EtFOSE) were investigated as peroxisome proliferators in comparison to perfluorooctanoic acid (PFOA). Three parameters were chosen to assess peroxisome proliferation, stimulation of lauroyl CoA oxidase activity, reduction of serum cholesterol concentration, and hepatomegaly. mt Biogenesis was assessed through cytochrome oxidase activity, cytochrome content and mitochondrial DNA (mtDNA) copy number. PFOA, PFOS, or N-EtFOSE was administered via a single i.p. injection at 100 mg/kg in male rats, and measurements were made 3 days later. In this model, PFOS and PFOA share similar potencies as peroxisome proliferators, whereas N-EtFOSE showed no activity. mt Endpoints were altered only in the PFOA treatment group, which consisted of a decrease cytochrome oxidase activity in liver tissue and an increase in the mtDNA copy number. None of the perfluorooctanoates significantly altered mt cytochrome content following acute in vivo treatment. These data demonstrate that acute administration of PFOS or PFOA causes hepatic peroxisome proliferation in rats. However, stimulation of mt biogenesis is not a characteristic response of all peroxisome proliferators.

Mouse hepatocyte response to peroxisome proliferators: dependency on hepatic nonparenchymal cells and peroxisome proliferator activated receptor alpha (PPARalpha).

Peroxisome proliferators (PPs) are rodent nongenotoxic hepatocarcinogens that induce peroxisome proliferation and DNA synthesis, and suppress apoptosis in rodent hepatocytes. PPs act through the PP-activated receptor alpha (PPARalpha); tumour necrosis factor alpha (TNFalpha) and hepatic nonparenchymal cells (NPCs), the major source of TNF alpha in the liver, have also been implicated in mediating the rodent hepatic response to PPs. Here we investigate the interaction between PPARalpha and NPCs in regulating the response to PPs. Using normal hepatocyte cultures containing around 20% NPCs, the PP nafenopin (50 microM) induced DNA synthesis and suppressed transforming growth factor beta1-induced apoptosis. However, when the NPCs were removed by differential centrifugation, nafenopin did not induce DNA synthesis or suppress apoptosis in the pure hepatocytes. Reconstitution of the normal hepatocyte cultures by mixing together the pure hepatocytes and the previously separated NPCs in the same proportions as the original cell preparation (17.7+/-8.7% NPCs) restored the response to nafenopin. Interestingly, nafenopin was still able to induce beta-oxidation in the pure hepatocyte cultures, consistent with NPCs being required for PP-induced growth but not for peroxisome proliferation. Next, we evaluated the role of PPARalpha in the hepatocyte dependency upon NPCs. Interestingly, NPCs isolated from PPARalpha-null mice, like those isolated from the wild-type NPCs, restored the hepatocyte response to nafenopin. However, as expected, PPARalpha-null hepatocytes remained non-responsive to PPs, irrespective of the genotype of the added NPCs. These data support a role for NPCs in facilitating a response of hepatocytes to PPs that is ultimately dependent on the presence of PPARalpha in the hepatocyte.

Effects on male rats of di-(2-ethylhexyl) phthalate and di- n-hexylphthalate administered alone or in combination

The effects of phthalate esters of branched chain alcohols, typified by di-(2-ethylhexyl)phthalate (DEHP) differ from those of esters of straight chain alcohols typified by di -n-hexyl phthalate (DnHP). The former induce liver enlargement and proliferation of hepatic peroxisomes, while the latter cause no peroxisome proliferation but cause fat accumulation in the liver. Both classes of phthalate esters are hypolipidaemic and cause thyroid changes associated with an increased rate of thyroglobulin turnover. As phthalate esters are used as mixtures, we have examined the effect of mixtures of the compounds. Groups of five male Wistar albino rats were administered either control diet or diets containing either 10 000 ppm of DEHP, 10 000 ppm of DnHP or 10 000 ppm DEHP plus 10 000 ppm DnHP for 14 days. Rats receiving diets containing DEHP showed the expected increase in relative liver weight, in “peroxisomal” fatty acid oxidation and in CYP4A1. Serum triglyceride and serum cholesterol were also reduced, and the thyroid showed the histological changes mentioned above. Rats consuming diets containing DnHP showed no increase in relative liver weight and no induction of peroxisomal fatty acid oxidation or CYP4A1. However, there was a marked accumulation of fat in the liver. The fall in serum cholesterol was similar to that in rats treated with DEHP, but the fall of serum triglyceride was more pronounced. Thyroidal changes were again observed. In general, changes in rats treated with a mixture of DEHP and DnHP were very similar to those found with rats treated with DEHP alone. The liver was enlarged, and peroxisomal fatty acid oxidation and CYP4A1 were both induced. The amount of fat in the liver was much less than in rats receiving DnHP alone. Thyroid changes were similar to those in rats receiving the individual compounds. The effect on serum cholesterol seemed additive, but the levels of serum triglyceride were intermediate between the groups receiving the single compounds.

Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats.

Wyeth-14,643 (WY) and ammonium perfluorooctanoate (C8) belong to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation in rodents. From previous work, WY, but not C8, has been shown to produce hepatocellular carcinoma in rats, while C8 has been shown to produce Leydig cell adenomas. In addition, based on a review of bioassay data a relationship appears to exist between peroxisome-proliferating compounds and Leydig cell adenoma and pancreatic acinar cell hyperplasia/adenocarcinoma formation. To further investigate the relationship between peroxisome-proliferating compounds and hepatic, Leydig cell, and pancreatic acinar cell tumorigenesis, a 2-year feeding study in male CD rats was initiated to test the hypothesis that peroxisome proliferating compounds induce a tumor triad (liver, Leydig cell, pancreatic acinar cell), and to examine the potential mechanism for the Leydig cell tumors. The study was conducted using 50 ppm WY and 300 ppm C8. The concentration of WY in the diet was decreased to 25 ppm on test day 301 due to increased mortality. In addition to the ad libitum control, a second control was pair-fed to the C8 group. Interim sacrifices were performed at 1- or 3-month intervals. Peroxisome proliferation measured by beta-oxidation activity and cell proliferation were measured in the liver and testis at all time points and in the pancreas beginning at the 9-month time point (cell proliferation only). Serum hormone concentrations (estradiol, testosterone, LH, FSH, and prolactin) were also measured at each time point. Increased relative liver weights and hepatic beta-oxidation activity were observed in both the WY- and C8-treated rats at all time points. In contrast, hepatic cell proliferation was significantly increased only in the WY-treated group. Neither WY nor C8 significantly altered the rate of Leydig cell beta-oxidation or Leydig cell proliferation when compared to the control groups. Moreover, the basal rate of beta-oxidation in Leydig cells was approximately 20 times less than the rate of hepatic beta-oxidation. There were no biologically meaningful differences in serum testosterone, FSH, prolactin, or LH concentrations in the WY- and C8-treated rats when compared to their respective controls. There were, however, significant increases in serum estradiol concentrations in the WY- and C8-treated rats at 1, 3, 6, 9, 15, 18, and 21 months. At 12 months, only the C8-treated rats had elevated serum estradiol concentrations when compared to the pair-fed control. Histopathological evaluation revealed compound-related increases in liver, Leydig cell, and pancreatic acinar cell tumors in both WY- and C8-treated rats. The data support the hypothesis that the peroxisome-proliferating compounds induce the previously described tumor triad. In addition, both C8 and WY produced a sustained increase in serum estradiol concentrations that correlated with the potency of the 2 compounds to induce Leydig cell tumors (i.e., WY caused a more consistent sustained increase in serum estradiol throughout the entire study, and more specifically at the end of the study, than did C8). This study suggests that estradiol may play a role in enhancement of Leydig cell tumors in the rat, and that peroxisome proliferators may induce tumors via a non-LH type mechanism.

Evidence for Peroxisome Proliferator-Activated Receptor (PPAR)α-Independent Peroxisome Proliferation: Effects of PPARγ/δ-Specific Agonists in PPARα-Null Mice

Peroxisome proliferators are a diverse group of compounds that cause hepatic hypertrophy and hyperplasia, increase peroxisome number, and on chronic high-dose administration, lead to rodent liver tumorigenesis. Various lines of evidence have led to the conclusion that these agents induce their pleiotropic effects exclusively via agonism of peroxisome proliferator-activated receptor (PPAR)alpha, a member of the steroid receptor superfamily involved in the regulation of fatty acid metabolism. Recently, agonists of two other members of this receptor family have been identified. PPARgamma is predominantly expressed in adipocytes where it mediates differentiation; PPARdelta is a widely expressed orphan receptor with yet unresolved physiologic functions. In the course of characterizing newer PPAR ligands, we noted that highly selective PPARgamma agonists or dual PPARgamma/PPARdelta agonists, lacking apparent murine PPARalpha agonist activity, cause peroxisome proliferation in CD-1 mice. We therefore made use of PPARalpha knockout mice to investigate whether these effects resulted from agonism of PPARalpha by these agents at very high dose levels or whether PPARgamma (or PPARdelta) agonism alone can result in peroxisome proliferation. We report here that several parameters linked to the hepatic peroxisome proliferation response in mice that were seen with these agents resulted from PPARalpha-independent effects.

Clofibrate-induced in vitro hepatoprotection against acetaminophen is not due to altered glutathione homeostasis.

Prior induction of peroxisome proliferation protects mice against the in vivo hepatotoxicity of acetaminophen and various other bioactivation-dependent toxicants. The mechanisms underlying such chemoresistance are poorly understood, although they have been suggested to involve alterations in glutathione homeostasis. To clarify the role of glutathione in this phenomenon, we isolated hepatocytes from mice in which hepatic peroxisome proliferation had been induced with clofibrate. The cells were incubated with a range of acetaminophen concentrations and the extent of cell killing after up to 8 h was assessed by measuring lactate dehydrogenase leakage from the cells. Hepatocytes from clofibrate-pretreated mice were much less susceptible to acetaminophen than cells from vehicle-treated controls. However, the extent of glutathione depletion during exposure to acetaminophen was similar in both cell types, as were rates of excretion of the product of glutathione-mediated detoxication of acetaminophen's quinoneimine metabolite, 3-glutathionyl-acetaminophen. The glutathione-replenishing ability of clofibrate-pretreated cells after a brief exposure to diethyl maleate also resembled that of control cells. More importantly, prior depletion of glutathione by diethyl maleate did not abolish the resistance of clofibrate-pretreated cells to acetaminophen. Taken together, these findings indicate that although glutathione-dependent pathways may contribute to hepatoprotection during peroxisome proliferation, the resistance phenomenon is not due exclusively to this mechanism.

Sex-dependent regulation of hepatic peroxisome proliferation in mice by trichloroethylene via peroxisome proliferator-activated receptor alpha (PPARalpha).

The mechanism of trichloroethylene-induced liver peroxisome proliferation and the sex difference in response was investigated using wild-type Sv/129 and peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice. Trichloroethylene treatment (0.75 g/kg for 2 weeks by gavage) resulted in liver peroxisome proliferation in wild-type mice, but not in PPARalpha-null mice, suggesting that trichloroethylene-induced peroxisome proliferation is primarily mediated by PPARalpha. No remarkable sex difference was observed in induction of peroxisome proliferation, as measured morphologically, but a markedly higher induction of several enzymes and PPARalpha protein and mRNA was found in males. On the other hand, trichloroethylene induced liver cytochrome P450 2E1, the principal enzyme responsible for metabolizing trichloroethylene to chloral hydrate, only in males, which resulted in similar expression levels in both sexes after the treatment. Trichloroethylene influenced neither the level of catalase, an enzyme involved in the reduction of oxidative stress, nor aldehyde dehydrogenase, the main enzyme catalyzing the conversion to trichloroacetic acid. These results suggest that trichloroethylene treatment causes a male-specific PPARalpha-dependent increase in cellular oxidative stress.

Suppression of mouse hepatocyte apoptosis by peroxisome proliferators: role of PPARα and TNFα

Peroxisome proliferators (PPs) are a diverse group of nongenotoxic chemicals that in rodents cause hepatic peroxisome proliferation, liver enlargement, increased replicative DNA synthesis and suppression of apoptosis. The effects of PPs in vivo can be reproduced in vitro where PPs can induce mouse hepatocyte DNA synthesis and suppress both spontaneous apoptosis and that induced by transforming growth factor β (TGFβ). In vitro, high concentrations (>500 U/ml) of exogenous tumour necrosis factor α (TNFα) [M. Rolfe, N.H. James, R.A. Roberts, TNFα suppresses apoptosis and induces S-phase in rodent hepatocytes: a mediator of the hepatocarcinogenicity of peroxisome proliferators?, Carcinogenesis 18 (1997) 2277–2280] are also able to stimulate hepatocyte DNA synthesis and suppress apoptosis, implicating TNFα in mediating or permitting the liver growth response to PPs. Here, using cultured mouse hepatocytes isolated from PPARα null mice, we have examined the role of the peroxisome proliferator activated receptor α (PPARα) in mediating the suppression of apoptosis caused by PPs. In addition we have investigated further the role of TNFα in mediating the rodent response to PPs. The PP nafenopin (50 μM) was unable to stimulate DNA synthesis measured by bromodeoxyuridine incorporation in these PPARα null mouse hepatocytes (96% of control), unlike epidermal growth factor, a growth factor used as a positive control. In assays of apoptosis using H33258 staining of chromatin condensation, nafenopin was unable to suppress either spontaneous or TGFβ1-induced apoptosis. In contrast, high concentrations of TNFα (>500 U/ml) were able to both stimulate DNA synthesis (204% of control) and suppress apoptosis in PPARα null hepatocytes (40% and 38% of control for spontaneous and TGFβ1-induced apoptosis respectively). However, TNFα could not stimulate β-oxidation of palmitoyl CoA in either PPARα null mouse or B6C3F1 (PPARα wild type) mouse hepatocytes. These data confirm the dependence of the response to PPs on PPARα by demonstrating that PPARα mediates the suppression of hepatocyte apoptosis in response to PPs. In addition, the data provide evidence that high concentrations of TNFα can modulate DNA synthesis and apoptosis in the absence of PPs and PPARα. Thus, in vivo, physiological levels of TNFα may be permissive for a PPARα-dependent growth response to PPs.

Species differences in hepatic peroxisome proliferation, cell replication and transforming growth factor-β1 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-β1 (TGF-β1) 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-β1 and insulin-like growth factor II/mannose-6-phosphate (IGFII/Man6P) receptor gene expression in the rat. In the Syrian hamster, effects on TGF-β1 and IGFII/Man6P receptor gene expression were also observed in some instances, whereas TGF-β1 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.

Species differences in peroxisome proliferation; mechanisms and relevance

Peroxisome proliferators are a class of structurally diverse chemicals, which induce liver carcinogenesis in rodents through interaction and activation of the Peroxisome Proliferator-Activated Receptor α (PPARα). PPARα agonists elicit a powerful pleiotropic response, which include hypolipidaemia. We have examined the response of species that are classically unresponsive to peroxisome proliferators. Whereas hamster responds to PPARα agonists by hepatomegaly and induction of marker genes, the guinea pig does not undergo hepatomegaly or induction of marker genes, such as CYP4A13. Both the hamster and the guinea pig have PPARα, and the guinea pig receptor has been characterised to be fully functional, as demonstrated in reporter gene expression assays. However, the guinea pig PPARα is expressed at low levels in liver, and the currently favoured hypothesis to explain species differences in hepatic peroxisome proliferation invokes the low level of PPARα as the principal determinant of species responsiveness. However, the demonstration that guinea pigs and humans undergo hypolipidaemia induced by PPARα-agonists calls into question the mode of action of PPARα agonists in “non-responsive” species.