Michelle L. O'Brien

Role of Oxidative Stress in Peroxisome Proliferator-Mediated Carcinogenesis

Abstract In this review, the evidence about the role of oxidative stress in the induction of hepatocellular carcinomas by peroxisome proliferators is examined. The activation of PPAR-α by peroxisome proliferators in rats and mice may produce oxidative stress, due to the induction of enzymes like fatty acyl coenzyme A (CoA) oxidase (AOX) and cytochrome P-450 4A1. The effect of peroxisome proliferators on the antioxidant defense system is reviewed, as is the effect on endpoints resulting from oxidative stress that may be important in carcinogenesis, such as lipid peroxidation, oxidative DNA damage, and transcription factor activation. Peroxisome proliferators clearly inhibit several enzymes in the antioxidant defense system, but studies examining effects on lipid peroxidation and oxidative DNA damage are conflicting. There is a profound species difference in the induction of hepatocellular carcinomas by peroxisome proliferators, with rats and mice being sensitive, whereas species such as nonhuman primates and guinea pigs are not susceptible to the effects of peroxisome proliferators. The possible role of oxidative stress in these species differences is also reviewed. Overall, peroxisome proliferators produce changes in oxidative stress, but whether these changes are important in the carcinogenic process is not clear at this time.

Mechanisms of peroxisome proliferation by perfluorooctanoic acid and endogenous fatty acids.

1. The effects of endogenous fatty acids and perfluorooctanoic acid (PFOA) and its analogs on peroxisomal acyl CoA oxidase (ACO) and microsomal laurate hydroxylase (LH) activities were evaluated in primary cultures of rat hepatocytes and activation of peroxisome proliferator-activated receptor alpha (PPARalpha) in CV-1 cells. The rank order for the stimulation of ACO activity in hepatocytes for selected compounds was PFOA >> octanoic acid>octanedioic acid, perfluorooctanol (inactive). Increases in ACO activity by PFOA, like those of ciprofibrate, were associated with a marked increase in peroxisome number and cytosolic occupancy volume. Maximal effects of ciprofibrate and PFOA on the stimulation of ACO activity were not additive, suggesting that these two compounds share a common pathway of peroxisome proliferation. 2. Saturated monocarboxylic acids of C4 to C18 chain length were inactive, and, among dicarboxylic acids, only small elevations (40-45%) in ACO activity were observed with the long-chain C12 and C16 dioic acids. Of the C18 fatty acids tested, only oleic and linoleic acids, at 1 mM, produced a two- to three-fold elevation in ACO and LH activities. In comparison with endogenous fatty acids, PFOA was more potent and exhibited a different time course and greater magnitude of stimulation of ACO and LH activities in cultured hepatocytes. 3. Addition of mitochondrial beta-oxidation inhibitors (3-mercaptopropionic and 2-bromooctanoic acids) did not alter ACO activity in the presence of octanoic acid or octanedioic acid; nor did they modify the stimulation of ACO activity by PFOA. The carnitine palmitoyltransferase I inhibitor 2-bromopalmitic acid produced a 2.5-fold increase in ACO stimulatory activity and reduced both ciprofibrate- and PFOA-mediated stimulations of ACO activity. 4. Cycloheximide treatment reduced PFOA- and ciprofibrate-induced ACO activities; however, the response to oleic acid was not blocked and increased slightly. 5. In rat and human PPARalpha transactivation assays, the rank order of activation was ciprofibrate > PFOA > oleic acid > or = octanoic acid > octanedioic acid or perfluorooctanol (inactive). PFOA, ciprofibrate and oleic acid were activators of rPPARalpha at concentrations that correlated favorably with the changes in ACO activity in cell culture. Octanoic acid did not increase ACO activity and was a weak activator of PPARalpha. 6. Our findings suggest that fatty acids such as oleic acid (endogenous fatty acids) and PFOA (a stable fatty acid) act through more than one pathway to increase ACO activity in rat hepatocytes. We conclude that the potent effects of PFOA are primarily mediated by a mechanism that includes the activation of liver PPARalpha.

Regulation of protein kinase C δ by estrogen in the MCF-7 human breast cancer cell line☆

Abstract We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) δ in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC δ isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC δ was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC δ expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC δ protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10 −10 –10 −8 M estrogen for 7 days down-regulated specifically PKC δ mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC δ expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor β while the MCF-7 cells expressed predominantly estrogen receptor α and barely detectable levels of estrogen receptor β. These results suggest that the differential ability of estrogen to regulate PKC δ expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Stereoselective effects of chiral clofibric acid analogs on rat peroxisome proliferator-activated receptor ? (rPPAR?) activation and peroxisomal fatty acid ?-oxidation

Enantiomers of a series of substituted analogs of 2-(4-chloronhenoxy)-acetic acid (CPAA) were synthesized and used to examine the influence of steric and structural parameters on peroxisome proliferation. The effects of these compounds were studied on the activation of the peroxisome proliferator-activated receptor α (PPARα) in CV-1 cells using an in vitro co-transfection assay. Selected sets of isomers were tested for their ability to increase peroxisomal fatty acyl-CoA oxidase (ACO) activity in H4IIEC3 (rat Reuber hepatoma) cells. Of the series of 2-substituted analogs studied, the isomers of the n-propyl and phenyl derivatives of CPAA showed a high degree of stereoselectivity [(S)-isomer ≫ (R)-isomer]. In general, the potency of the compound to activate the receptor increased with the size of the 2-alkyl substituent. Among the 4-chlorobenzyloxy- and 4-(4′-chlorophenyl)benzyloxy- analogs studied, 2-[4-(4′-chlorophenyl)-benzyloxy]-propanoic acid exhibited a high degree of stereoselectivity in both the biological systems studied [(R) ≫ (S)]. The congeners of 2-methyl substituted CPAA showed a reverse stereoselectivity [(R) > (S)] as compared to the other 2-substituted analogs [(S) > (R)]. Our results indicate that (1) both structural and steric characteristics of CPAA analogs play an important role in the activation of rPPARα and on stimulation of peroxisomal ACO activities, and (2) clofibric acid and analogs exert their peroxisome proliferative effects by interaction with a specific site on a protein. The enantiomers of the 2-n-propyl and the 2-phenyl CPAA analogs may be useful as mechanistic probes in elucidating the nature of this binding site. Chirality 9:37–47, 1997.

A hypothetical mechanism for fat-induced rodent hepatocarcinogenesis.

The regulation of fat metabolism in higher eukaryotes appears to be intimately associated with the activities of a family of recently identified nuclear receptors called ‘peroxisome proliferator-activated receptors’ (PPAR). In 1990, Issemann and Green1 reported the cloning of a mouse steroid receptor superfamily member that they characterized as a peroxisome proliferator-responsive transcription factor. The steroid receptor family of genes consists of a group of ligand-activated DNA transcription factors that bind regulatory sequences upstream of their target gene(s) resulting in the activation or repression of specific gene transcription.2–4 Subsequently it has been shown that PPAR is a small family of genes with reports of at least α, β and γ isoforms in mouse,5,6 Xenopus,7 rat8 and humans.9,10 An examination of PPAR regulatable promoters suggest this receptor family is intimately involved in fat metabolism including their breakdown,11,12 storage13 and synthesis.14 The complexity of the PPAR activation pathway has been substantially enhanced by the demonstration that PPAR DNA binding is linked to heterodimerization with a member of the retinoid X (RXR) family of receptors,15 and more recent PPAR transcription studies in yeast where it was demonstrated that PPAR activity is contingent upon both RXR and perhaps other mammalian cell-specific factor(s).16 The RXR family of receptors appear to be a point of convergence for several members of the intracellular receptor superfamily of genes17–19 and play a critical role in the transcriptional events associated with these receptors. All of the data accumulated to date clearly implicate PPAR-regulated events in the homeostasis of fats and suggest they may serve as viable targets for drug intervention and regulation of fat metabolism.