The role of reactive oxygen species and subsequent DNA-damage response in the emergence of resistance towards resveratrol in colon cancer models.

In spite of the novel strategies to treat colon cancer, mortality rates associated with this disease remain consistently high. Tumour recurrence has been linked to the induction of resistance towards chemotherapy that involves cellular events that enable cancer cells to escape cell death. Treatment of colon cancer mainly implicates direct or indirect DNA-damaging agents and increased repair or tolerances towards subsequent lesions contribute to generate resistant populations. Resveratrol (RSV), a potent chemosensitising polyphenol, might share common properties with chemotherapeutic drugs through its indirect DNA-damaging effects reported in vitro. In this study, we investigated how RSV exerts its anticancer effects in models of colon cancer with a particular emphasis on the DNA-damage response (DDR; PIKKs-Chks-p53 signalling cascade) and its cellular consequences. We showed in vitro and in vivo that colon cancer models could progressively escape the repeated pharmacological treatments with RSV. We observed for the first time that this response was correlated with transient activation of the DDR, of apoptosis and senescence. In vitro, a single treatment with RSV induced a DDR correlated with S-phase delay and apoptosis, but prolonged treatments led to transient micronucleations and senescence phenotypes associated with polyploidisation. Ultimately, stable resistant populations towards RSV displaying higher degrees of ploidy and macronucleation as compared to parental cells emerged. We linked these transient effects and resistance emergence to the abilities of these cells to progressively escape RSV-induced DNA damage. Finally, we demonstrated that this DNA damage was triggered by an overproduction of reactive oxygen species (ROS) against which cancer cells could adapt under prolonged exposure to RSV. This study provides a pre-clinical analysis of the long-term effects of RSV and highlights ROS as main agents in RSV's indirect DNA-damaging properties and consequences in terms of anticancer response and potent resistance emergence.

Resveratrol, a phytochemical inducer of multiple cell death pathways: apoptosis, autophagy and mitotic catastrophe.

Cancers are the largest cause of mortality and morbidity in industrialized countries. In the field of the medicinal chemistry of natural products, numerous studies have reported interesting properties of trans-resveratrol as a chemopreventing agent against cancers, inflammation, and viral infection. Tumor growth inhibition has been linked to the ability of resveratrol to arrest cell cycle progression and to trigger cell death. This review focuses on the pathways that mediate resveratrol-induced cell death. Resveratrol impacts on the mitochondrial functions (respiratory chain, oncoproteins, gene expression, etc), in which p53 protein can be involved and its acetylated or phosphorylated forms. This polyphenol also affects death receptor distribution in ceramide-enriched membrane platforms which serve to trap and cluster receptor molecules, and facilitates the formation of a death-inducing signaling complex in the cell. To induce apoptosis, resveratrol also activates the ceramide / sphingomyelin pathway, which promotes ceramide generation and the downstream activation of kinase cascades. Resveratrol can activate alternative pathways to cell death such as those leading to autophagy, senescence or mitotic catastrophe. Furthermore, numerous attempts have been made using resveratrol analogs to improve the molecule's ability to block cell proliferation and induce cell death. Moreover, structural modification of natural phenolics is expected to produce analogs that may be useful tools to study the structure-activity relationships. Lastly, in various cancer types, resveratrol behaves as a chemosensitizer that lowers the threshold of cell death induction by classical anticancer agents and counteracts tumor cell chemoresistance.

Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol.

BACKGROUND AND AIMS: Oxysterols are assumed to play important roles in age-related macular degeneration, a major cause of blindness. So we characterized the cytotoxic, oxidative, inflammatory, and angiogenic activities of oxysterols (7ß-hydroxycholesterol (7ß-OH), 7-ketocholesterol (7KC), 25-hydroxycholesterol (25-OH)) in human retinal ARPE-19 cells, and evaluated the protective effects of resveratrol (Rsv: 1 µM), a polyphenol from red wine. METHODS: ARPE-19 cells were treated with 7ß-OH, 7KC, or 25-OH (5-40 µg/mL; 24-48 h) without or with Rsv. Cell viability was determined using trypan blue and the MTT assay. Cell death was characterized by electron microscopy and in situ detection of activated caspases with fluorochrome-labeled inhibitors of caspases. Reactive oxygen species (ROS) production was measured with hydroethidine. ELISA methods and a cytometric bead assay were used to quantify cytokines involved in inflammation (IL-8, IL-1ß, IL-6, IL-10, IL-12p70, TNF-α, MCP-1) and VEGF. RESULTS: 7ß-OH and 7KC triggered a caspase-independent cell death process associated with the presence of multilamellar cytoplasmic structures evocating phospholipidosis, increased ROS production, and IL-8 secretion. 7ß-OH enhanced VEGF secretion. No cytotoxic effects were identified with 25-OH, which highly stimulated ROS production, MCP-1, and VEGF secretion. With oxysterols, no IL-10, TNF-α, and IL-12p70 secretion were detected. 25-OH induced IL-8 secretion through the MEK/ERK½ signaling pathway, and Rsv showed cytoprotective activities and inhibited VEGF secretion. CONCLUSION: 7ß-OH, 7KC, and 25-OH have cytotoxic, oxidative, inflammatory, and/or angiogenic activities on ARPE-19 cells. As Rsv has some protective effects against oxysterol-induced cell death and VEGF secretion it could be valuable in ARMD treatment.

The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPARα and HNF4α in the Liver.

PPARα and HNF4α are nuclear receptors that control gene transcription by direct binding to specific nucleotide sequences. Using transgenic mice deficient for either PPARα or HNF4α, we show that the expression of the peroxisomal 3-keto-acyl-CoA thiolase B (Thb) is under the dependence of these two transcription factors. Transactivation and gel shift experiments identified a novel PPAR response element within intron 3 of the Thb gene, by which PPARα but not HNF4α transactivates. Intriguingly, we found that HNF4α enhanced PPARα/RXRα transactivation from TB PPRE3 in a DNA-binding independent manner. Coimmunoprecipitation assays supported the hypothesis that HNF4α was physically interacting with RXRα. RT-PCR performed with RNA from liver-specific HNF4α-null mice confirmed the involvement of HNF4α in the PPARα-regulated induction of Thb by Wy14,643. Overall, we conclude that HNF4α enhances the PPARα-mediated activation of Thb gene expression in part through interaction with the obligate PPARα partner, RXRα.

Human hepatic cell uptake of resveratrol: involvement of both passive diffusion and carrier-mediated process.

This work reports significant advances on the transport in hepatic cells of resveratrol, a natural polyphenol with potent protective properties. First, we describe a new simple technique to qualitatively follow resveratrol cell uptake and intracellular distribution, based on resveratrol fluorescent properties. Second, the time-course study and the quantification of (3)H-labelled resveratrol uptake have been performed using human hepatic derived cells (HepG2 tumor cells) and hepatocytes. The temperature-dependence of the kinetics of uptake as well as the cis-inhibition experiments agree with the involvement of a carrier-mediated transport in addition to passive diffusion. The decrease of passive uptake resulting from resveratrol binding to serum proteins brings to light a mediated mechanism in physiological situation.

Genetic-dependency of peroxisomal cell functions - emerging aspects.

This paper reviews aspects concerning the genetic regulation of the expression of the well studied peroxisomal genes including those of fatty acid beta-oxidation enzymes; acyl-CoA oxidase, multifunctional enzyme and thiolase from different tissues and species. An important statement is PPARalpha, which is now long known to be in rodents the key nuclear receptor orchestrating liver peroxisome proliferation and enhanced peroxisomal beta-oxidation, does not appear to control so strongly in man the expression of genes involved in peroxisomal fatty acid beta-oxidation related enzymes. In this respect, the present review strengthens among others the emerging concept that, in the humans, the main genes whose expression is up-regulated by PPARalpha are mitochondrial and less peroxisomal genes. A special emphasis is also made on the animal cold adaptation and on need for sustained study of peroxisomal enzymes and genes; challenging that some essential roles of peroxisomes in cell function and regulation still remain to be discovered.

Peroxisome-proliferator-activated receptors as physiological sensors of fatty acid metabolism: molecular regulation in peroxisomes.

The enzymes required for the beta-oxidation of fatty acyl-CoA are present in peroxisomes and mitochondria. Administration of hypolipidaemic compounds such as clofibrate to rodents leads to an increase in the volume and density of peroxisomes in liver cells. These proliferators also induce simultaneously the expression of genes encoding acyl-CoA oxidase, enoyl-CoA hydratase-hydroxyacyl-CoA dehydrogenase (multifunctional enzyme) and thiolase (3-ketoacyl-CoA thiolase). All these enzymes are responsible for long-chain and very-long-chain fatty acid beta-oxidation in peroxisomes. Similar results were observed when rat hepatocytes, or liver-derived cell lines, were cultured with a peroxisome proliferator. The increased expression of these genes is due to the stimulation of their transcription rate. These results show that the peroxisome proliferators act on the hepatic cells and regulate the transcription through various cellular components and pathways, including peroxisome-proliferator-activated receptor alpha (PPARalpha). After activation by specific ligands, either fibrates or fatty acid derivatives, PPARalpha binds to a DNA response element: peroxisome-proliferator-responsive element (PPRE), which is a direct repeat of the following consensus sequence: TGACCTXTGACCT, found in the promoter region of the target genes. PPARalpha is expressed mainly in liver, intestine and kidney. PPARalpha is a transcriptional factor, which requires other nuclear proteins for function including retinoic acid X receptor (RXRalpha) and other regulatory proteins. From our results and others we suggest the role of PPARalpha in the regulation of the peroxisomal fatty acid beta-oxidation. In this regard, we showed that although PPARalpha binds to thiolase B gene promoter at -681 to -669, a better response is observed with hepatic nuclear factor 4 ("HNf-4"). Moreover, rat liver PPARalpha regulatory activity is dependent on its phosphorylated state. In contrast, a protein-kinase-C-mediated signal transduction pathway seems to be modified by peroxisome proliferators, leading to an increase in the phosphorylation level of specific proteins, some of which have been shown to be involved in the phosphoinositide metabolism.

Identification of novel peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in mouse liver using cDNA microarray analysis.

Peroxisome proliferators, which function as peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists, are a group of structurally diverse nongenotoxic hepatocarcinogens including the fibrate class of hypolipidemic drugs that induce peroxisome proliferation in liver parenchymal cells. Sustained activation of PPARalpha by these agents leads to the development of liver tumors in rats and mice. To understand the molecular mechanisms responsible for the pleiotropic effects of these agents, we have utilized the cDNA microarray to generate a molecular portrait of gene expression in the liver of mice treated for 2 weeks with Wy-14,643, a potent peroxisome proliferator. PPARalpha activation resulted in the stimulation of expression (fourfold or greater) of 36 genes and decreased the expression (fourfold or more decrease) of 671 genes. Enhanced expression of several genes involved in lipid and glucose metabolism and many other genes associated with peroxisome biogenesis, cell surface function, transcription, cell cycle, and apoptosis has been observed. These include: CYP2B9, CYP2B10, monoglyceride lipase, pyruvate dehydrogenase-kinase-4, cell death-inducing DNA-fragmentation factor-alpha, peroxisomal biogenesis factor 11beta, as well as several cell recognition surface proteins including annexin A2, CD24, CD39, lymphocyte antigen 6, and retinoic acid early transcript-gamma, among others. Northern blotting of total RNA extracted from the livers of PPARalpha-/- mice and from mice lacking both PPARalpha and peroxisomal fatty acyl-CoA oxidase (AOX), that were fed control and Wy-14,643-containing diets for 2 weeks, as well as time course of induction following a single dose of Wy-14,643, revealed that upregulation of genes identified by microarray procedure is dependent upon peroxisome proliferation vis-à-vis PPARalpha. However, cell death-inducing DNA-fragmentation factor-alpha mRNA, which is increased in the livers of wild-type mice treated with peroxisome proliferators, was not enhanced in AOX-/- mice with spontaneous peroxisome proliferation. These observations indicate that the activation of PPARalpha leads to increased and decreased expression of many genes not associated with peroxisomes, and that delayed onset of enhanced expression of some genes may be the result of metabolic events occurring secondary to PPARalpha activation and alterations in lipid metabolism.

Ciprofibrate stimulates protein kinase C-dependent phosphorylation of an 85 kDa protein in rat Fao hepatic derived cells.

The effect of ciprofibrate on early events of signal transduction was previously studied in Fao cells. Protein kinase C (PKC) assays performed on permeabilized cells showed a more than two-fold increase in PKC activity in cells treated for 24 h with 500 microM ciprofibrate. To show the subsequent effect of this increase on protein phosphorylation, the in vitro phosphorylation on particulate fractions obtained from Fao cells was studied. Among several modifications, the phosphorylation of protein(s) with an apparent molecular mass of 85 kDa was investigated. This modification appeared in the first 24 h of treatment with 500 microM ciprofibrate. It was shown to occur on Ser/Thr residue(s). It was calcium but not calmodulin-dependent. The phosphorylation level of this/these protein(s) was reduced with kinase inhibitors and especially with 300 nM GF-109203X, a specific inhibitor of PKC. All these results suggest that the phosphorylation of the 85 kDa protein(s) is due to a PKC or to another Ser/Thr kinase activated via a PKC pathway. A possible biochemical candidate for 85 kDa protein seems to be the beta isoform of phosphatidylinositol 3-kinase regulatory subunit.

Regulation of the peroxisomal beta-oxidation-dependent pathway by peroxisome proliferator-activated receptor alpha and kinases.

The first PPAR (peroxisome proliferator-activated receptor) was cloned in 1990 by Issemann and Green (Nature 347:645-650). This nuclear receptor was so named since it is activated by peroxisome proliferators including several drugs of the fibrate family, plasticizers, and herbicides. This receptor belongs to the steroid receptor superfamily. After activation by a specific ligand, it binds to a DNA response element, PPRE (peroxisome proliferator response element), which is a DR-1 direct repeat of the consensus sequence TGACCT x TGACCT. This mechanism leads to the transcriptional activation of target genes (Motojima et al., J Biol Chem 273:16710-16714, 1998). After the first discovery, several isoforms were characterized in most of the vertebrates investigated. PPAR alpha, activated by hypolipidemic agents of the fibrate family or by leukotrienes; regulates lipid metabolism as well as the detoxifying enzyme-encoding genes. PPAR beta/delta, which is not very well known yet, appears to be more specifically activated by fatty acids. PPAR gamma (subisoforms 1, 2, 3) is activated by the prostaglandin PGJ2 or by antidiabetic thiazolidinediones (Vamecq and Latruffe, Lancet 354:411-418, 1999). This latter isoform is involved in adipogenesis. The level of PPAR expression is largely dependent on the tissue type. PPAR alpha is mainly expressed in liver and kidney, while PPAR beta/delta is almost constitutively expressed. In contrast, PPAR gamma is largely expressed in white adipose tissue. PPAR is a transcriptional factor that requires other nuclear proteins in order to function, i.e. RXRalpha (9-cis-retinoic acid receptor alpha) in all cases in addition to other regulatory proteins. Peroxisomes are specific organelles for very long-chain and polyunsaturated fatty acid catabolism. From our results and those of others, the inventory of the role of PPAR alpha in the regulation of peroxisomal fatty acid beta-oxidation is presented. In relation to this, we showed that PPAR alpha activates peroxisomal beta-oxidation-encoding genes such as acyl-CoA oxidase, multifunctional protein, and thiolase (Bardot et al., FEBS Lett 360:183-186, 1995). Moreover, rat liver PPAR alpha regulatory activity is dependent on its phosphorylated state (Passilly et al., Biochem Pharmacol 58:1001-1008, 1999). On the other hand, some signal transduction pathways such as protein kinase C are modified by peroxisome proliferators that increase the phosphorylation level of some specific proteins (Passilly et al. Eur J Biochem 230:316-321, 1995). From all these findings, PPAR alpha and kinases appear to play an important role in lipid homeostasis.

Peroxisomes: biochemistry, molecular biology and genetic diseases- a video programme for teaching students.

Peroxisome proliferation in rodent liver is a good biochemical marker of toxicology for several classes of xenobiotics, including fibrates; phthalates and adipates; or chlorophenoxy-acetate, a herbicide. Research in peroxisomes provides a good example of the integration of fields related to basic sciences and biomedical and industrial health. A 25min video programme illustrates techniques involved in the characterization of purified peroxisomes (Fig. 1) and membranes, immunoblotting, measurement of proliferation markers, mRNA analysis at the post-transcriptional level, DNA techniques, cell cultures as biological models and computer analysis. It is aimed at undergraduates and non-biochemist advanced students in biology.

Inhibitory effect of resveratrol on the proliferation of human and rat hepatic derived cell lines.

Resveratrol is a polyphenolic compound especially produced by grapevine and consequently found in wine. Based on epidemiological studies resveratrol may act as a cancer chemopreventive compound. The ability of resveratrol to inhibit cell proliferation was studied in rat hepatoma Fao cell line and human hepatoblastoma HepG2 cell line. The results show that resveratrol strongly inhibits cell proliferation at the micromolar range in a time- and dose-dependent manner. Concentrations higher than 50 microM become toxic. Fao cells are more sensitive than HepG2 cells. Interestingly, the presence of ethanol lowers the threshold of resveratrol effect. Resveratrol appears to prevent or to delay the entry to mitosis since no inhibition of [3H]thymidine incorporation is observed, while there is an increase of cell number in S and G2/M phases. In conclusion, resveratrol shows a strong inhibition of hepatic cell proliferation where alcohol may act as an enhancing agent.

The peroxisome proliferator response element (PPRE) present at positions -681/-669 in the rat liver 3-ketoacyl-CoA thiolase B gene functionally interacts differently with PPARalpha and HNF-4.

Although previous data showed that the putative thiolase B PPRE located at -681/-669 bind the PPARalpha-RXRalpha heterodimer in vitro (Kliewer et al. (1992) Nature 358, 771-774), there is no evidence about the functional role of this element. By gel mobility-shift assay, we found an interaction of this PPRE with not only PPARalpha but also with HNF-4. By transfection of cells with the putative PPRE-driven luciferase reporter vector and PPARalpha, we found no significant activation of the luciferase gene expression, in contrast to the case with reporter expression driven by the PPRE of the peroxisomal bifunctional enzyme. On the other hand, HNF-4 activated the luciferase gene expression driven by the putative thiolase PPRE. We suggest that the thiolase B gene induction by peroxisome proliferators employs either another PPRE or this one in combination with other gene regulatory element(s) to lead to the strong gene expression observed in the presence of peroxisome proliferators.

Evolutionary aspects of peroxisomes as cell organelles, and of genes encoding peroxisomal proteins.

Peroxisomes are present in most eukaryotic cell types, and have different enzymatic content and metabolic functions throughout the life scale. The endosymbiotic origin of these DNA-devoid organelles is supported by evolutionary data concerning genes encoding not only most peroxisomal proteins, but also several transcriptional factors regulating their expression such as peroxisome proliferator-activated receptors.

Properties of a fluorescent bezafibrate derivative (DNS-X). A new tool to study peroxisome proliferation and fatty acid beta-oxidation.

The first peroxisome proliferator-activated receptor (PPAR) was cloned in 1990 by Issemann and Green. Many studies have reported the importance of this receptor in the control of gene expression of enzymes involved in lipid metabolic pathways including mitochondrial and peroxisomal fatty acid beta-oxidation, lipoprotein structure [apolipoprotein (apo) A2, apo CIII], and fatty acid synthase. By using radiolabeled molecules, it was shown that peroxisome proliferators bind and activate PPAR. As an alternative method, we developed a fluorescent dansyl (1-dimethylaminonaphthalene-5-sulfonyl) derivative peroxisome proliferator from bezafibrate (DNS-X), a hypolipidemic agent that exhibits an in vitro peroxisome proliferative activity on rat Fao-hepatic derived cultured cells. However, until now, the effect of this new compound on the liver of animals and subcellular localization was unknown. In addition to in vivo rat studies, we present a more efficient large-scale technique of DNS-X purification. Treating rats (DNS-X in the diet at 0.3% w/w) for 6 d leads to a hepatomegaly and a marked increase in liver peroxisomal palmitoyl-CoA oxidase activity. We also developed a method to localize and quantify DNS-X in tissues or cell compartment organelles. The primarily cytosolic distribution of DNS-X was confirmed by direct visualization using fluorescence microscopy of cultured Fao cells. Finally, transfection assay demonstrated that DNS-X enhanced the PPAR alpha activity as well as other peroxisome proliferators do.

Relationship between signal transduction and PPAR alpha-regulated genes of lipid metabolism in rat hepatic-derived Fao cells.

The goal of this study was to characterize phosphorylated proteins and to evaluate the changes in their phosphorylation level under the influence of a peroxisome proliferator (PP) with hypolipidemic activity of the fibrate family. The incubation of rat hepatic derived Fao cells with ciprofibrate leads to an overphosphorylation of proteins, especially one of 85 kDa, indicating that kinase (or phosphatase) activities are modified. Moreover, immunoprecipitation of 32P-labeled cell lysates shows that the nuclear receptor, PP-activated receptor, alpha isoform, can exist in a phosphorylated form, and its phosphorylation is increased by ciprofibrate. This study shows that PP acts at different steps of cell signaling. These steps can modulate gene expression of enzymes involved in fatty acid metabolism and lipid homeostasis, as well as in detoxication processes.

Are physiological clonal expansion and maturation pathways a basis for discrepant behaviour of colon tumoral cell growth in response to the thiazolidinedione PPARgamma agonists?

Recently, anti-diabetic thiazolidinediones, pharmacological agonist ligands of peroxisome proliferator-activated receptor gamma (PPARgamma), have been shown to induce either protective or permissive effects towards colon epithelium tumoral cell growth. Several attractive explanations have been proposed but no final answer to these is currently provided. It is not the purpose of the authors to bring this final answer but to offer another attractive hypothesis which might help our approach to explore further this exciting field of medical research.

Phosphorylation of peroxisome proliferator-activated receptor alpha in rat Fao cells and stimulation by ciprofibrate.

The basic mechanism(s) by which peroxisome proliferators activate peroxisome proliferator-activated receptors (PPARs) is (are) not yet fully understood. Given the diversity of peroxisome proliferators, several hypotheses of activation have been proposed. Among them is the notion that peroxisome proliferators could activate PPARs by changing their phosphorylation status. In fact, it is well known that several members of the nuclear hormone receptor superfamily are regulated by phosphorylation. In this report, we show that the rat Fao hepatic-derived cell line, known to respond to peroxisome proliferators, exhibited a high content of PPARalpha. Alkaline phosphatase treatment of Fao cell lysate as well as immunoprecipitation of PPARalpha from cells prelabeled with [32P] orthophosphate clearly showed that PPARalpha is indeed a phosphoprotein in vivo. Moreover, treatment of rat Fao cells with ciprofibrate, a peroxisome proliferator, increased the phosphorylation level of the PPARalpha. In addition, treatment of Fao cells with phosphatase inhibitors (okadaic acid and sodium orthovanadate) decreased the activity of ciprofibrate-induced peroxisomal acyl-coenzyme A oxidase, an enzyme encoded by a PPARalpha target gene. Our results suggest that the gene expression controlled by peroxisome proliferators could be mediated in part by a modulation of the PPARalpha effect via a modification of the phosphorylation level of this receptor.

Medical significance of peroxisome proliferator-activated receptors.

Peroxisome proliferator-activated receptors (PPAR) were discovered in 1990, ending 25 years of uncertainty about the molecular mechanisms of peroxisome proliferation. Subsequently, PPARs have improved our understanding of adipocyte differentiation. But there is more to PPARs than solving a puzzle about an organelle (the peroxisome) long considered an oddity, and their medical significance goes beyond obesity too. Enhanced PPAR type alpha expression protects against cardiovascular disorders though the role of enhanced PPARgamma expression seems less favourable. PPAR mechanisms, mainly via induction of more differentiated cell phenotypes, protect against some cancers. The differentiation of many cell types (hepatocyte, fibroblast, adipocyte, keratinocyte, myocyte, and monocyte/macrophage) involves PPARs, and these nuclear receptors are now attracting the attention of many medical specialties and the pharmaceutical industry.

Carcinogenic aspect of xenobiotic molecules belonging to the peroxisome proliferator family.

It is known that a short-term exposure of rat, mice or incubation of hepatic cells with fibrate molecules leads to increase in peroxisome number and cell hyperplasia. Further, long-term incubation of cells (at least a year) show transformed characteristics with foci and nodules. To explain the hepatocarcinogenic effect of peroxisome proliferators in rodents we studied the effect of peroxisome proliferators on rat liver oncogenes expression. Earlier, we reported an increase in liver and kidney mRNA level of c-myc and N-myc. Since several metabolic genes are activated by PPAR (peroxisome proliferators activated receptor) through a PPRE (peroxisome proliferator response element), we suggest the involvment of PPAR in oncogene activation, because of the presence of PPRE in the N-myc 5'-upstream region. We showed by flow cytometric analysis that ciprofibrate increased the size of rat Fao derived cell line and the activity of palmitoyl CoA oxidase, a peroxisome proliferation enzyme marker for studying peroxisome proliferation was increased. The above effects which can contribute to hepatocarcinogenesis seem to be restricted to rat and mice, which show strong response to peroxisome proliferators. Indeed, no changes are observed in weak responsive species such as humans (using hepatic derived cell lines) and guinea pig. These data provide arguments for the non-carcinogenic effect of this xenobiotic class in human especially when sensitive, or normal individuals are exposed either to hypolipidaemic agents of the fibrate family.