Peroxisome-proliferating Agents Research Articles

Expression of cFABP and PPAR in trophoblast cells: effect of PPAR ligands on linoleic acid uptake and differentiation

Throughout gestation, fetal growth depends, in part, on placental transfer of maternal essential fatty acid (EFA) and long-chain polyunsaturated fatty acid. All fatty acid (FA) can cross lipid bilayer by simple diffusion, such as those in the syncytiotrophoblasts, the multinucleated, terminally differentiated trophoblast cells. The trophoblasts differentiation process is accompanied by an increase of human chorionic gonadotropin (hCG) secretion and an inhibition of Human Achaete-Scute Homologue-2 expression (Hash-2). Furthermore, a number of FA-binding proteins (FABPs) have been identified in membrane and cytoplasm of mammalian cells, which are thought to facilitate the transfer of FA across membranes and their intracellular channeling. Thus, the aim of this study was to investigate the implication of cFABPs in linoleic acid (LA) uptake by human trophoblast cells according to differentiation. Moreover, since peroxisome proliferator-activated receptor (PPARs) regulate the expression of cFABP and play an important role in trophoblast cells differentiation, the effects of PPARs ligands are verified on cFABP expression and differentiation. Herein, we reported the increase of the expression of liver and heart FABP (L- and H-FABP) upon differentiation of trophoblast cells, an inhibition of PPARα and β, while PPARγ levels remains unchanged. The nonselective peroxisome-proliferating agents, bezafibrate and LA, impaired trophoblast differentiation, and reduced L- and H-FABP expression. Furthermore, cobalt, a chemical agent known to mimic hypoxia, inhibits trophoblast cells differentiation and diminishes H-, L-FABP and PPARs expression. Finally, both treatments show no influence on LA uptake by trophoblast cells. In conclusion, this study showed that there is no correlation between the expression of H- and L-FABP and LA uptake by trophoblast cells and that bezafibrate and LA greatly impaired trophoblast cells differentiation.

PEX11alpha is required for peroxisome proliferation in response to 4-phenylbutyrate but is dispensable for peroxisome proliferator-activated receptor alpha-mediated peroxisome proliferation.

The PEX11 peroxisomal membrane proteins promote peroxisome division in multiple eukaryotes. As part of our effort to understand the molecular and physiological functions of PEX11 proteins, we disrupted the mouse PEX11alpha gene. Overexpression of PEX11alpha is sufficient to promote peroxisome division, and a class of chemicals known as peroxisome proliferating agents (PPAs) induce the expression of PEX11alpha and promote peroxisome division. These observations led to the hypothesis that PPAs induce peroxisome abundance by enhancing PEX11alpha expression. The phenotypes of PEX11alpha(-/-) mice indicate that this hypothesis remains valid for a novel class of PPAs that act independently of peroxisome proliferator-activated receptor alpha (PPARalpha) but is not valid for the classical PPAs that act as activators of PPARalpha. Furthermore, we find that PEX11alpha(-/-) mice have normal peroxisome abundance and that cells lacking both PEX11alpha and PEX11beta, a second mammalian PEX11 gene, have no greater defect in peroxisome abundance than do cells lacking only PEX11beta. Finally, we report the identification of a third mammalian PEX11 gene, PEX11gamma, and show that it too encodes a peroxisomal protein.

Effects of vehicle, diet and gender on the expression of PMP70- and CYP2K1/2M1-like proteins in the mummichog

The mummichog (Fundulus heteroclitus) has been shown to be responsive to peroxisome proliferating agents (PPAs). Peroxisomes function as important sites for fatty acid β-oxidation. Peroxisome proliferation by PPAs or starvation can lead to changes in the size and number of peroxisomes and the expression of ω-hydroxylases (CYP2K1/2M1 in rainbow trout). Mummichogs were subjected to 96 h fasting or 96 h recovery from fasting. Expression of PMP70- and CYP2K1/2M1-like proteins in vehicle-treated or non-treated controls was compared in both males and females. Fasting and vehicle produced decreases in PMP70- and CYP2K1/2M1-like proteins in both males and females. In reproductive females, decreases due to fasting and vehicle treatment were greater than in female fish that were not gravid. Recovery from fasting resulted in levels of CYP2K1/2M1 near control levels in males while in recovered females, about 2-fold higher levels compared to controls were noted. These results indicate that gender, reproductive status and diet can produce changes in the expressed levels of peroxisomal PMP70 and microsomal CYP2K1/2M1-like proteins in the mummichog.

Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus

Our studies were conducted to explore the role of hepatic fatty acid-binding protein (L-FABP) in fatty acid transport to the nucleus. Purified rat L-FABP facilitated the specific interaction of [(3)H]oleic acid with the nuclei. L-FABP complexed with unlabeled oleic acid decreased the nuclear association of [(3)H]oleic acid:L-FABP; however, oleic acid-saturated bovine serum albumin (BSA) or fatty acid-free L-FABP did not. The peroxisome-proliferating agents LY171883, bezafibrate, and WY-14,643 were also effective competitors when complexed to L-FABP. Nuclease treatment did not affect the nuclear association of [(3)H]oleic acid:L-FABP; however, proteinase treatment of the nuclei abolished the binding. Nuclei incubated with fluorescein-conjugated L-FABP in the presence of oleic acid were highly fluorescent whereas no fluorescence was observed in reactions lacking oleic acid, suggesting that L-FABP itself was binding to the nuclei. The nuclear binding of FABP was concentration dependent, saturable, and competitive. LY189585, a ligand for L-FABP, also facilitated the nuclear binding of fluorescein-conjugated L-FABP, although it was less potent than oleic acid. A structural analog that does not bind L-FABP, LY163443, was relatively inactive in stimulating the nuclear binding. Potential interactions between L-FABP and nuclear proteins were analyzed by Far-Western blotting and identified a 33-kDa protein in the 500 mm NaCl extract of rat hepatocyte nuclei that bound strongly to biotinylated L-FABP. Oleic acid enhanced the interaction of L-FABP with the 33-kDa protein as well as other nuclear proteins. We propose that L-FABP is involved in communicating the state of fatty acid metabolism from the cytosol to the nucleus through an interaction with lipid mediators that are involved in nuclear signal transduction.

Peroxisome proliferating agents, retinoid metabolism, and macrophage activation

Peroxisome proliferating agents (PPAs) are common environmental pollutants that alter hepatic fatty acid metabolism, which often leads to liver disease in PPA-sensitive species. The production of reactive oxygen intermediates (ROIs) and nitric oxide (NO) by activated macrophages is associated with the development of liver pathologies. Retinoids are potent modulators of macrophage activity, and we are testing the hypothesis that PPA-inducible cytochrome P450s metabolize retinoids resulting in altered macrophage function. Preliminary evidence indicates that in vivo exposure of mummichog ( Fundulus heteroclitus) to retinoic acid significantly increases immunostimulated anterior kidney macrophage ROI production, and induces hepatic NO synthase (iNOS, which generates NO). The direct effects of retinoic acid on macrophages were confirmed by in vitro exposure. We have also demonstrated that PPAs can modulate macrophage activity: in vivo exposure of mummichogs to clofibrate suppresses anterior kidney macrophage ROI production, but induces hepatic iNOS. Studies with 2,4-dichlorophenoxyacetic acid (1–21 days exposure) demonstrate both time- and tissue-dependent modulation of macrophage activity. Enhanced iNOS expression was generally associated with induction of PPA-responsive CYP2K1 and/or CYP2M1. The observed PPA-induced macrophage activation could lead to tissue damage in the liver. Moreover, altered macrophage function in general could result in immune system dysfunction.

Induction of lauric acid hydroxylase activity in catfish and bluegill by peroxisome proliferating agents

In mammals, sensitivity to peroxisome proliferation by peroxisome proliferating agents (PPAs) appears to be correlated with inducibility of lauric acid hydroxylase activity. Bluegill and catfish have been shown to respond to PPAs by induction of lauric acid hydroxylase immunoreactive proteins (Haasch, 1996). In this investigation, induction of lauric acid hydroxylase activity was confirmed by HPLC and mass spectral analysis of specific hydroxylation products and possible species-specific differences in metabolism were investigated. Male bluegill, channel catfish and rat, were administered the model PPAs, clofibrate (200 mg kg −1, i.p.), ciprofibrate (100 mg kg −1, i.p.), or olive oil as vehicle control (both sexes of catfish), 48 h prior to hepatic, trunk kidney (catfish only) or kidney (rat) microsome preparation. In general, total metabolism of lauric acid was similar in all species, but female catfish metabolize lauric acid to a greater extent than males. Ciprofibrate treatment produced significant induction of ω- and ω-6 hydroxylation in male catfish kidney. In male bluegill liver, ω-, ω-4 and ω-5 hydroxylations were significantly induced by clofibrate treatment. The data indicate that induction of lauric acid hydroxylase cytochrome(s) P450 occurs in PPA-exposed fish which may be a consideration for environmentally-exposed responsive species.

Expression of PEX11β Mediates Peroxisome Proliferation in the Absence of Extracellular Stimuli

Mammalian cells typically contain hundreds of peroxisomes but can increase peroxisome abundance further in response to extracellular stimuli. We report here the identification and characterization of two novel human peroxisomal membrane proteins, PEX11alpha and PEX11beta. Overexpression of the human PEX11beta gene alone was sufficient to induce peroxisome proliferation, demonstrating that proliferation can occur in the absence of extracellular stimuli and may be mediated by a single gene. Time course studies indicated that PEX11beta induces peroxisome proliferation through a multistep process involving peroxisome elongation and segregation of PEX11beta from other peroxisomal membrane proteins, followed by peroxisome division. Overexpression of PEX11alpha also induced peroxisome proliferation but at a much lower frequency than PEX11beta in our experimental system. The patterns of PEX11alpha and PEX11beta expression were examined in the rat, the animal in which peroxisome proliferation has been examined most extensively. Levels of PEX11beta mRNA were similar in all tissues examined and were unaffected by peroxisome-proliferating agents. Conversely, PEX11alpha mRNA levels varied widely among different tissues, were highest in tissues that are sensitive to peroxisome-proliferating agents, and were induced more than 10-fold in response to the peroxisome proliferators clofibrate and di(2-ethylhexyl) phthalate. Taken together, these data implicate PEX11beta in the constitutive control of peroxisome abundance and suggest that PEX11alpha may regulate peroxisome abundance in response to extracellular stimuli.

Induction of CYP2M1 and CYP2K1 lauric acid hydroxylase activities by peroxisome proliferating agents in certain fish species: Possible implications

In many higher vertebrates, species and gender-specific sensitivity to peroxisome proliferating agents (PPAs) and their toxic effects has been described. Sensitivity may correlate with the induction of lauric acid hydroxylase (LAH) activity. In rainbow trout ( Oncorhynchus mykiss) LAH has been identified with at least two cytochromes P450, CYP2M1 and CYP2K1. Previously, bluegill ( Lepomis macrochirus) and catfish ( Ictalurus punctatus) had been shown to produce CYP2M1 and CYP2K1 immunoreactive protein induction upon treatment with PPAs. The current research extends that observation by demonstration of the associated lauric acid hydroxylase activities after exposure to PPAs. Male bluegill and catfish were treated using a single i.p. injection with olive oil vehicle or ciprofibrate (a model PPA; 100 mg kg −1) or clofibrate (less potent PPA) 48 h prior to microsome preparation. Olive oil-treated female catfish liver and kidney microsomes metabolize lauric acid to a greater extent than male microsomes (83 vs 40% and 77 vs 5%, respectively). Male bluegill liver and male catfish kidney exhibit significant induction of specific LAH after PPA treatment. These observations, taken together with previous work in mammals, may portend possible adverse consequences to sensitive fish species exposed to environmental chemical contamination by PPAs.

Induction of anti-trout lauric acid hydroxylase immunoreactive proteins by peroxisome proliferators in bluegill and catfish

Various chemicals such as phthalate esters, hypolipidemic drugs, solvents like trichloroethylene, and certain herbicides have been identified as peroxisome proliferating agents (PPAs). In many vertebrates, species- and even gender-specific sensitivities to peroxisome proliferation (PP) and toxicity, have been described and may correlate with inducibility of lauric acid hydroxylase activity. Little is known about PP in fish. The rainbow trout does not appear to be inducible for lauric acid hydroxylases (LMC1 and LMC2; CYP2M1 and CYP2K1, respectively) in response to the hypolipidemic drug, clofibrate. Fish other than trout, such as bluegill and catfish, are more acutely sensitive to phthalate ester toxicity. In this study, bluegill and catfish were examined for inducibility of anti-trout LMC1 and LMC2 immunoreactive proteins, 48 h after intra-pentoneal (i.p.) administration of olive oil vehicle, 200 mg/kg clofibrate, or 100 mg/kg ciprofibrate. In control animals, immunoreactive proteins were recognized in a sex-specific manner in both bluegill and catfish liver, and in catfish kidney. In treated animals, male immunoreactive proteins were induced to a greater extent than female, and ciprofibrate was the more potent inducer. The response of bluegill and catfish, therefore, is similar to the response of PPA-sensitive species.

Enantioselective induction of peroxisomal proliferation in CD-1 mice by leukotriene antagonists

The effects of a racemic leukotriene antagonist (MK-0571) and its component enantiomers (L-668,018 and L-668,019) on hepatic peroxisome proliferation were examined in mice, rats, and rhesus monkeys. Administration of racemic MK-0571 to mice resulted in increased liver weights, increased peroxisomal volume density, and a pleiotropic induction of characteristic peroxisomal and nonperoxisomal enzyme activities associated with peroxisomal proliferation. When the individual enantiomers of MK-0571 were administered to mice, a pronounced enantio-selective induction of peroxisome proliferation was observed. Toxicokinetic studies showed that the levels of each enantiomer in the liver or plasma after separate administration were similar. Thus, the enantioselectivity in the induction of peroxisome proliferation could not be explained on the basis of pharmacokinetic differences between the enantiomers. The hepatic peroxisomal response of the rat to MK-0571 was greatly attenuated compared to the mouse. As has been seen with other peroxisome-proliferating agents, MK-0571 had no effect on either peroxisomal volume density or peroxisomal enzyme activity in monkeys. Due to the high degree of enantiomeric discrimination toward the induction of peroxisomal proliferation by these enantiomers, compounds of this type may prove useful as probes to examine the mechanisms by which peroxisomal proliferating agents induce their effects.

Induction of peroxisomes by treatment with perfluorooctanoate does not increase rates of H 2O 2 production in intact liver

Increases in acyl coenzyme A (CoA) oxidase activity due to peroxisome proliferation are postulated to cause oxidative stress via elevated production of H 2O 2, leading to DNA damage. These changes are suspected to be responsible for tumor formation caused by non-genotoxic carcinogens which do not bind to DNA but cause proliferation of peroxisomes. However, the activity of the peroxisomal enzyme acyl CoA oxidase assayed in vitro in the presence of excess fatty acyl CoA substrate may not reflect rates of H 2O 2 generation in intact liver where fatty acid supply is carefully controlled in part by delivery of substrate. The purpose of this work was to determine if rates of hepatic H 2O 2 generation were altered in perfused liver and in vivo following induction of H 2O 2-generating acyl CoA oxidase activity. Injection of the potent peroxisome proliferating agent perfluorooctanoate into rats 5 days prior to sacrifice caused an expected 4-fold increase of H 2O 2-generating acyl CoA oxidase activity measured in hepatic homogenates. In contrast, rates of H 2O 2 generation in perfused liver measured spectrophotometrically (660−640 nm) through a lobe of the liver were not altered by perfluorooctanoate treatment (7.3 ± 1.5 vs. 7.8 ± 0.5 μmol gh in livers from untreated control rats). Similar treatment with perfluorooctanoate also increased in vitro acyl CoA oxidase activity 9-fold in livers from deermice; however, rates of elimination of methanoi, a selective substrate for catalase in rodents whose oxidation is limited by the supply of H 2O 2, were not altered significantly in vivo (control, 110 ± 11 μmol gh vs. perfluorooctanoate, 112 ± 32 μmol gh ). Taken together, these data demonstrate that elevation of H 2O 2 formation by acyl CoA oxidase activity measured in vitro is not necessarily associated with increases in rates of H 2O 2 generation in intact perfused liver or in vivo, most likely due to rate-limitation in intact cells by fatty acid supply. These data do not support the hypothesis that the induction of peroxisomes leads to excessive H 2O 2 production and oxidative stress. It follows that alternative hypotheses to explain carcinogenesis caused by peroxisome-proliferating agents need to be considered.

Effect of the peroxisome proliferator LY171883 on triglyceride accumulation in rats fed a fat-free diet

LY171883 is a leukotriene D 4 antagonist that induces peroxisome proliferation in the rodent liver. Like many peroxisome-proliferating agents, it causes transient lipid accumulation and several other changes in hepatic lipid metabolism. The effect of LY171883 on lipid metabolism was studied further in rats maintained on a fat-free diet. Administration of a fat-free diet for 14 days caused a 5.6-fold increase in liver triglycerides associated with a 3.3-fold increase in fatty acid synthetase. Co-administration of 0.1% LY171883 increased liver triglycerides slightly, whereas 0.3% LY171883 prevented the accumulation of triglycerides. Furthermore, treatment with 0.3% LY171883 reversed the fatty liver in rats pretreated with the fat-free diet for 14 days. Fatty acid synthetase activity increased comparably in all treatment groups, indicating that 0.3% LY171883 did not prevent the lipogenic response to a fat-free diet. In rats treated with 0.3% LY171883, peroxisomal β-oxidation increased 9.5-fold, mitochondrial β-oxidation 4.8-fold, carnitine palmitoyltransferase I 1.9-fold, and plasma ketones 3-fold. In the 0.1% dose group the increases in these parameters were smaller. The data indicate that 0.3% LY171883 sufficiently increased mitochondrial and peroxisomal β-oxidation such that fatty acids generated by lipogenesis were preferentially oxidized rather than esterified to triglycerides. In the 0.1% dose group oxidation was only mildly increased, and the excess fatty acids continued to be esterified.

Peroxisome-associated enzymes and serum lipids in tumour-bearing rats treated with peroxisome-proliferating agents

Xenobiotic induction of liver peroxisomes is associated with hypolipidemia. To test the involvement of the peroxisome proliferation with the hypolipidemia, male rats were inoculated in the groin with five different tumors: an aflatoxin-induced hepatoma, a lasiocarpine-induced hepatoma, an actinomycin-D-induced mesothelioma, a lasiocarpine-induced squamous cell carcinoma, and a methylnitrosourea-induced fibrosarcoma. After the tumours reached a suitable size, the rats were fed diets containing the peroxisome-proliferating hypolipidemic agents tibric acid (2-chloro-5-[3,5-dimethylpiperidinosulfonyl] benzoic acid) or Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid) for 2 weeks. Liver and tumor tissues were then assayed for the peroxisome-associated enzymes, catalase and carnitine acetyltransferase, and correlated with serum levels of triglyceride and cholesterol. The presence of the tumors caused a predictable decrease in liver catalase and a slight elevation of liver carnitine acetyltransferase. Serum cholesterol was elevated slightly, while serum triglyceride levels were elevated, unchanged, or decreased in the tumor-bearing rats maintained on control diet. Inclusion of the xenobiotics in the diet caused increases in liver weight, catalase, and carnitine acetyltransferase. Serum triglycerides were decreased in the three groups which were not already decreased, but a decrease in serum cholesterol was only found in one group after only one of the treatments. The latter finding demonstrates that peroxisomal enzyme induction can be dissociated from the decrease in serum cholesterol. The data were further evaluated by testing for correlations between the changes in these components, comparing changes within groups and between groups. These correlations indicate an inverse biological association between liver catalase and serum cholesterol and between liver carnitine acetyltransferase and serum triglyceride. The latter correlation was inverse only for comparisons between groups, suggesting that carnitine acetyltransferase activity is associate with serum triglycerides only during the perturbational state.