The peroxisome proliferator nafenopin does not suppress hepatocyte apoptosis in guinea-pig liver in vivo nor in human hepatocytes in vitro

In rats and mice, nafenopin is a nongenotoxic hepatocarcinogen, which induces hepatic DNA synthesis and enzyme induction both in vivo and in hepatocyte cultures in vitro. However, humans and guinea-pigs are considered to be non-responsive to the liver growth effects of peroxisome proliferators (PPs). The ability to stimulate cell replication coupled with the ability to suppress apoptosis is thought to underpin the carcinogenicity of nongenotoxic carcinogens such as PPs. Previous studies in this laboratory have shown that in rats in vivo and in vitro nafenopin suppressed spontaneous hepatocyte apoptosis and that induced by the physiological negative growth regulator transforming growth factors β1 (TGFβ1). In addition nafenopin suppressed apoptosis in cultured hepatocytes from guinea-pig and hamster. The effects of PPs on apoptosis in human hepatocyte cultures is not known. To correlate these previous in vitro findings to the known species differences in hepatocarcinogenicity of PPs we have investigated the effects of nafenopin on guinea-pig liver growth in vivo. Also, we have examined the effects of nafenopin on apoptosis in cultures of human hepatocytes, a valuable model for human risk assessment. Nafenopin did not inhibit either spontaneous or TGFβ1 induced apoptosis in human hepatocytes in vitro. Administration of nafenopin to guinea-pigs in vivo produced none of the changes seen previously in responsive species, such as rats and mice. There was no change in liver/body weight ratio, peroxisomal volume of hepatocytes or DNA synthesis as determined by incorporation of bromodeoxyuridine and there was no suppression of apoptosis. The lack of response to nafenopin in guinea-pigs in vivo and human hepatocytes in vitro provides further evidence that these species may be refractory to the liver growth effects of PPs despite the ability of guinea-pigs and humans to respond to PPs by alterations in lipid metabolism. The data presented add to our overall understanding of species differences in response to the PP class of rodent nongenotoxic carcinogens. Received: 9 June 1998 / Accepted: 21 September 1998     

Comparison of the effects of various peroxisome proliferators on peroxisomal enzyme activities, DNA synthesis, and apoptosis in rat and human hepatocyte cultures.

Peroxisome proliferators (PPs) are a class of rodent nongenotoxic hepatocarcinogens that cause hepatocyte peroxisome proliferation, increased DNA synthesis, and decreased spontaneous apoptosis. We examined the effects of various PPs such as the hypolipidemic agents clofibric acid (CLO), bezafibrate (BEZA), ciprofibrate (CIPRO), and nafenopin (NAFE) and the plasticizer di-(2-ethylhexyl)phthalate (DEHP) on the various parameters in vitro in rat and human hepatocyte cultures. In rat hepatocyte cultures, after 72 h of treatment with the various PPs at 100-500 microM, a compound-dependent increase in acyl CoA oxidase (ACO) and carnitine acetyl transferase (CAT) activities, markers of peroxisome proliferation, was observed with the following potencies: CIPRO = NAFE > BEZA > CLO > DEHP. A minor (120-150%), but significant, no concentration-dependent increase in DNA synthesis and a marked, no compound-dependent and, with the exception of NAFE, no concentration-dependent 60-80% decrease in spontaneous apoptosis was observed with all tested compounds (50-250 microM) after 48 h of treatment. Inhibition of spontaneous apoptosis in PP-treated versus control rat hepatocyte cultures was also observed morphologically. Furthermore, PPs inhibited transforming growth factor beta (TGFbeta)-induced apoptosis but not tumor necrosis factor alpha (TNFalpha)/alpha Amanitine (alphaAma)-induced apoptosis in rat hepatocyte cultures. In human hepatocyte cultures, the various PPs at 50-500 microM did not affect peroxisomal enzyme activities, DNA synthesis, or spontaneous and induced (TGFbeta or TNFalpha/alphaAma) apoptosis. The compound-dependent peroxisome proliferation but no compound-dependent disruption of the mitogenic/apoptotic balance elicited by PPs in primary rat hepatocyte cultures supports the hypothesis that oxidative stress is directly linked to the hepatocarcinogenic potential of a given PP in rodents and that disruption of the mitogenic/apoptotic balance contributes to the development of PP-induced hepatocarcinogenesis. In addition, the absence of effects of all PPs on both peroxisome proliferation-associated parameters and mitogenic/apoptotic balance supports the hypothesis that human liver cells are refractory to PP-induced hepatocarcinogenesis.     

Quantitative proteomic analysis of mouse liver response to the peroxisome proliferator diethylhexylphthalate (DEHP)

Peroxisome proliferators (PPs) are a diverse group of chemicals that cause hepatic proliferation, suppression of apoptosis, peroxisome proliferation and liver tumours in rodents. The biochemical response to PPs involves changes in the expression of peroxisomal beta-oxidation enzymes and fatty acid transport proteins such as acyl-CoA oxidase and liver fatty acid binding protein. The response to PPs is mediated by the peroxisome proliferator-activated receptor alpha (PPARalpha) and the livers of PPARalpha-null transgenic mice do not develop tumours in response to PPs. In order to identify the molecular pathways underlying the adverse effects of PPs in rodent liver, we carried out two-dimensional differential gel electrophoresis to provide quantitative proteomic analyses of diethylhexylphthalate (DEHP)-treated wild-type or PPARalpha-null mouse livers. Since tumourigenesis is both PP- and PPARalpha-dependent, analyses were focused on these changes. Fifty-nine proteins were identified where altered expression was both PPARalpha- and PP-dependent. In addition, six proteins regulated by the deletion of PPARalpha were identified, possibly indicating an adaptive change in response to the loss of this receptor. The proteins that we identified as being regulated by PPARalpha are known to be involved in lipid metabolism pathways, but also in amino acid and carbohydrate metabolism, mitochondrial bioenergetics and in stress responses including several genes not previously reported to be regulated by PPARalpha. These data provide novel insights into the pathways utilised by PPs and may assist in the identification of early markers rodent nongenotoxic hepatocarcinogenesis.     

Peroxisome proliferators induce apoptosis and decrease DNA synthesis in hepatoma cell lines

We examined the effects of various peroxisome proliferators (PPs) such as the hypolipidaemic agents clofibric acid (CLO), bezafibrate (BEZA), ciprofibrate (CIPRO) and nafenopin (NAFE) and the plasticizer di-(2-ethylhexyl)phthalate (DEHP) on peroxisomal enzyme activities, apoptosis and DNA synthesis in rat FaO and human HepG2 hepatoma cell lines. Both growing and confluent cultures were treated with PPs (250 microM) for 48 or 72 h. In accordance with our previous observations in PP-treated primary hepatocyte cultures of rat and human origin, the various PPs increased peroxisomal enzyme activities in rat FaO cells but not in human HepG2 cells. PPs strongly induced apoptosis in FaO cells. They did not affect TGFbeta-induced apoptosis, with the exception of DEHP and NAFE, respectively blocking and increasing induced apoptosis in confluent cultures. Moreover, PPs produced a minor, but significant, decrease in DNA synthesis in FaO cells. PPs also decreased DNA synthesis in growing HepG2 cells, and CLO, CIPRO and NAFE induced apoptosis in confluent HepG2 cultures. This is in opposition with the effects of PPs on primary hepatocyte cultures, i.e. inhibition of both spontaneous and TGFbeta-induced apoptosis and increases in DNA synthesis in rat hepatocytes, and unchanged mitosis-apoptosis balance in human hepatocytes.     

Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphthalate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro

Diethylhexylphthalate (DEHP) and diisononylphthalate (DINP) are plasticizers with many important commercial, industrial and medical applications. However, both DEHP and DINP are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent liver tumours associated with peroxisome proliferation, induction of hepatic DNA synthesis and the suppression of apoptosis. Despite these effects in the rodent, humans appear to be nonresponsive to the adverse effects of PPs. Previously, we have shown that the fibrate hypolipidaemic peroxisome proliferator, nafenopin, induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes. In this work, we have examined species differences in the response of rat and human hepatocytes to DEHP and DINP in vitro. In rat hepatocytes in vitro, both DINP and MEHP (a principle metabolite of DEHP and the proximal peroxisome proliferator) caused a concentration-dependent induction of DNA synthesis and suppression of both spontaneous and transforming growth factor β1 (TGFβ1)-induced apoptosis. Similarly, both MEHP and DINP caused a concentration-dependent induction of peroxisomal β-oxidation although the response to DINP was less robust. In contrast to the pleiotropic response noted in rat hepatocytes, neither DINP nor MEHP caused an induction of β-oxidation, stimulation of DNA synthesis and suppression of apoptosis in human hepatocytes cultured from three separate donors. These data provide evidence for species differences in the hepatic response to the phthalates DEHP and DINP, confirming that human hepatocytes appear to be refractory to the hepatocarcinogenic effects of PPs first noted in rodents. Received: 16 August 1999 / Accepted: 21 September 1999     

Involvement of the peroxisome proliferator-activated receptor alpha in the immunomodulation caused by peroxisome proliferators in mice

Peroxisome proliferators (PPs) are a large class of structurally diverse chemicals, which includes drugs designed to improve the metabolic abnormalities linking hypertriglyceridemia to diabetes, hyperglycemia, insulin-resistance and atherosclerosis. We have recently demonstrated that exposure of rodents to potent PPs indirectly causes a number of immunomodulating effects, resulting in severe adaptive immunosuppression. Since the peroxisome proliferator-activated receptor alpha (PPARalpha) plays a central role in mediating the pleiotropic responses exerted by PPs, we have compared here the immunomodulating effects of the PPs perfluorooctanoic acid (PFOA) and Wy-14,643 in wild-type and PPARalpha-null mice. The reductions in spleen weight and in the number of splenocytes caused by PP treatment in wild-type mice was not observed in PPARalpha-null mice. Furthermore, the reductions in thymus weight and in the number of thymocytes were potently attenuated in the latter animals. Similarly, the dramatic decreases in the size of the CD4(+)CD8(+) population of cells in the thymus and in the number of thymocytes in the S and G2/M phases of the cell cycle observed in wild-type mice administered PPs were much less extensive in PPARalpha-null mice. Finally, in contrast to the case of wild-type animals, the response of splenocytes isolated from the spleen of PP-treated PPARalpha-null mice to appropriate T- or B-cell activators in vitro was not reduced. Altogether, these data indicate that PPARalpha plays a major role in the immunomodulation caused by PPs. The possible relevance of these changes to the alterations in plasma lipids also caused by PPs is discussed.     

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.     

The role of tumor necrosis factor alpha and the peroxisome proliferator-activated receptor alpha in modulating the effects of fumonisin in mouse liver

Fumonisins are mycotoxins that are produced by Fusarium verticillioides found in corn and corn-based foods, and are suspected human esophageal carcinogens. Exposure of rodents to fumonisin B1 causes hepatotoxicity and results in alterations in the balance between cell proliferation and apoptosis in the liver. As the cytokine tumor necrosis factor alpha (TNFalpha) and the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) also modulate hepatocyte proliferation and apoptosis, we tested the hypothesis that fumonisin-induced hepatotoxicity in the liver is modulated by these factors. We examined the effects of dietary exposure to a fumonisin-containing culture material (CM) of the fungus F. verticillioides for 8 days or 5 weeks in the livers of mice lacking either TNFalpha or PPARalpha. Compared to wild-type mice TNFalpha-null mice exhibited increased hepatocyte proliferation and apoptosis. In contrast, PPARalpha-null and wild-type mice were found to exhibit similar patterns of hepatocyte apoptosis and proliferation when fed the CM diet. Overall, these findings provide evidence that TNFalpha, but not PPARalpha, plays a role in modulating fumonisin-induced hepatotoxicity in mice.     

Hepatocyte Spheroids: Prolonged Hepatocyte Viability for in Vitro Modeling of Nongenotoxic Carcinogenesis

To explore peroxisome proliferator-perturbed hepatocyte growthregulation, robust in vitro models of liver are required. Thishas always posed a problem since isolated hepatocytes show arapid loss of viability and differentiation status and ceaseto be useful after 3–4 days in culture. We now describea model system in which rat hepatocytes are maintained as three-dimensionalspheroids. The maintenance of hepatocyte viability and morphologyin these cultures is considerably prolonged over that seen inmonolayer culture and is comparable to that obtained by theuse of collagen gels or dimethyl sulfoxide. The spheroid systemis, however, free of any additives that may lead to artifactand free of excessive exogenous protein that may compromisesubsequent analyses. Ultrastructural examination reveals extensiveinterhepatocyte junctional complexes and in terdigitation ofadjacent membranes together with the presence of bile cannalicularstructures. Furthermore, hepatocytes maintained as spheroidsretain expression of liver markers such as albumin and alsoretain their ability to respond to peroxisome proliferators:even after 12 days in culture, treatment with the peroxisomeproliferator nafenopin causes a 4.5-fold increase in cytoplasmicvolume fraction of peroxisomes. There is a concomitant inductionof peroxisomal bifunctional enzyme and cytochrome P4504A, theenzyme markers associated with peroxisome proliferation. Thespheroids also maintain expression of the peroxisome proliferator-activatedreceptor and preliminary data indicate that they are able toundergo replicative DNA synthesis in response to nafenopin.Hepatocyte spheroids will provide us with a model system forstudying the early changes in rodent liver nongenotoxic carcinogenesis.     

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat,mouse and human hepatocytes

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.     

Effect of Rodent Hepatocarcinogenic Peroxisome Proliferators on Fatty Acyl-CoA Oxidase, DNA Synthesis, and Apoptosis in Cultured Human and Rat Hepatocytes

The effects of the rodent hepatocarcinogens clofibric acid and ciprofibrate on the activity of the peroxisomal fatty acyl-CoA oxidase, DNA synthesis, and apoptosis were compared in cultured rat and human hepatocytes. Rat hepatocytes expressed a 10-fold greater level of the peroxisomal fatty acyl-CoA oxidase compared to human hepatocytes. At the highest concentration (1.0 mM), both drugs induced a two- to threefold increase in this enzyme activity in both rat and human hepatocytes. Ciprofibrate (0.1 and 0.2 mM) caused a twofold increase in DNA synthesis in rat hepatocytes, whereas clofibric acid had no effect on DNA synthesis in these cells. In contrast, increasing concentrations of both clofibric acid and ciprofibrate produced inhibition of DNA synthesis in human hepatocytes. By using the terminal transferase dUTP–biotin nick end labeling technique, it was observed that 0.1 and 0.2 mM clofibric acid and ciprofibrate suppressed transforming growth factor-β (TGFβ)-induced apoptosis by 50% in rat hepatocytes, but they had no effect on TGFβ-induced apoptosis in human hepatocytes. Although clofibric acid and ciprofibrate diminished TGFβ-induced apoptosis, they had no effect on the basal apoptotic levels in the rat hepatocyte cultures. However, both drugs significantly increased the percent of apoptotic cells in the human hepatocyte cultures. It is concluded that primary rat and human hepatocyte cultures respond differently to peroxisome proliferators. The differences in effects on DNA synthesis and apoptosis support the hypothesis that human liver cells are refractory to peroxisome proliferator-induced hepatocarcinogenesis.