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.     

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.     

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.     

The effect of beclobric acid and clofibric acid on peroxisomal beta-oxidation and peroxisome proliferation in primary cultures of rat, monkey and human hepatocytes

The peroxisome-proliferating effects of clofibric acid and beclobric acid were studied in primary cultures of hepatocytes derived from rat, monkey (Macaca fascicularis) and human liver. Determination of peroxisomal fatty acid beta-oxidation and morphometrical analysis of the peroxisomal compartment were performed after incubation of 1-day-old hepatocyte cultures for 3 days with either compound. In rat liver cell cultures both compounds gave a 10-fold increase in peroxisomal beta-oxidation, a 3-fold increase in the relative number of peroxisomes and a 1.5-fold increase in the mean size of peroxisomes. Beclobric acid gave its maximal effect at a concentration of 10 microM, which is at least one order of magnitude lower than the maximum-effect concentration of clofibric acid. At concentrations greater than 300 microM beclobric acid was cytotoxic. No stimulation of peroxisomal fatty acid beta-oxidation was found in either monkey or human hepatocyte cultures. Morphometrical analysis also showed no increase in the peroxisomal compartment in cultures derived from these species, as indicated by the lack of increase in both relative number and size of peroxisomes. In all three species tested beclobric acid was equally cytotoxic for hepatocytes in vitro. These results are of relevance for the interpretation of the peroxisome-proliferating effects of clofibrate and similar compounds in rats. Since peroxisome proliferation may be correlated to increased hepatic tumour incidences in the rat, the absence of peroxisome proliferation in primates suggests the absence of tumourogenic activity by hypolipidemic compounds in these species.     

Toxicological studies on a benzofuran derivative. III. Comparison of peroxisome proliferation in rat and human hepatocytes in primary culture

Primary cultures of rat and human hepatocytes were used in our in vitro studies for investigating species differences in the response to a peroxisome proliferating benzofuran derivative, benzbromarone. Cyanide-insensitive palmitoyl coenzyme A oxidation (a marker of peroxisome fatty acid beta-oxidation) and electron microscopy were used to assess peroxisome proliferation. Hepatocytes were cultured essentially as described by Mitchell et al. (1984, Arch. Toxicol. 55, 239-246); clofibric acid and mono(2-ethylhexyl) phthalate (MEHP) were used as reference compounds, as they are well known to cause peroxisome proliferation in rat hepatocytes in primary culture. The benzofuran derivative, tested at drug concentrations ranging from 2.37 to 59.20 microM in rat hepatocyte primary cultures, induced, after 96 hr, a dose-related increase of the peroxisomal beta-oxidase activity correlated with an increased number of peroxisomes; this increase was much less marked than that obtained with clofibric acid or MEHP. By contrast, using the same range of concentrations, human hepatocytes in primary culture treated with benzbromarone revealed no enhancement of enzymatic activity and no concomitant statistically significant increase in the number of peroxisomes; the same observations were reported with clofibric acid and MEHP. These results demonstrate clearly that species differences in sensitivity to peroxisome proliferation with the benzofuran derivative do exist.     

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     

Effects of clofibric and beclobric acid in rat and monkey hepatocyte primary culture: influence on peroxisomal and mitochondrial β-oxidation and the activity of catalase,glutathione S-transferase and glutathione peroxidase

The effect of hypolipidaemic compounds on peroxisomal fatty acid β-oxidation and on peroxisome morphology in the liver differs widely between rodent and primate species. We studied the relative importance of peroxisomal and mitochondrial β-oxidation of palmitate in primary cultures of hepatocytes isolated from rat and monkey liver in the absence or presence of clofibric acid or beclobric acid. It was demonstrated that it is possible to differentiate between peroxisomal and mitochondrial β-oxidation activities in intact cells. Overall β-oxidation of palmitate was ca. 30% higher in rat hepatocytes than in monkey liver cells. In both monkey and rat cell cultures the mitochondrial component was over 90% of the total palmitate β-oxidation. In rat hepatocyte culture clofibric acid and beclobric acid caused a 5- to 8-fold stimulation of peroxisomal β-oxidation, while in monkey cells this activity was not significantly increased. However, in cells derived from both species mitochondrial palmitate β-oxidation was increased (rat 2.5-fold; monkey 1.5-fold). These results indicate that the species differences in the increase in peroxisomal fatty acid oxidation are not a result of an inability to metabolize fatty acids in rat liver cell mitochondria. A comparison of the activity of enzymes involved in the detoxification of hydrogen peroxide showed that catalase and glutathione-S-transferase activity is 2.9-fold higher in monkey hepatocytes than in rat liver cells, while glutathione peroxidase activity was 1.6-fold higher in rat cells. When a comparison between both species is made for the ratio of hydrogen peroxide production over catalase activity, it can be concluded that this peroxide will have much smaller possibilities to escape from the peroxisomal compartment in monkey hepatocytes. These findings suggest that species differences in these enzyme activities can contribute to differences in susceptibility for peroxisome proliferator-induced carcinogenicity between rodents and primates. Received: 3 January 1994/Accepted: 11 April 1994     

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     

Factors influencing peroxisome proliferation in cultured rat hepatocytes

A primary rat hepatocyte culture system has been developed for the study of peroxisome proliferation. Maximal induction of peroxisomal activity requires supplementation of the culture medium with hydrocortisone. The addition of clofibric acid (0.01–1 mM), mono-(2-ethylhexyl)phthalate (0.01–0.5 mM) and trichloroacetic acid (0.1–5 mM) to cultured rat hepatocytes resulted in a time- and dose-related increase in CN- insensitive palmitoyl CoA oxidation (maximal increases: 27-, 15.5-, and 5-fold respectively) and mitochondrial -glycerophosphate dehydrogenase activity (maximal increases: 7.3-, 5.8-, and 1.6-fold respectively). Electron microscopic examination revealed smooth endoplasmic reticulum proliferation and morphometric analysis indicated an increase in fractional peroxisomal volume of X 8 and X 4 for clofibric acid (1 mM) and trichloroacetic acid (2.5 mM), respectively. SDS-PAGE of cell homogenates revealed an intensified protein band of mol. wt. 76–78,000. The induction of peroxisomal -oxidation by clofibric acid was elevated from 9- to 12-fold by supplementation of the medium with l-carnitine (2mM).     

Quantitative assessment of enzyme induction by peroxisome proliferators and application to determination of effects on triglyceride biosynthesis in primary cultures of rat hepatocytes

Potencies for the induction of peroxisomal fatty acyl-CoA oxidase (FACO) and microsomal laurate hydroxylase (LH) were determined for clofibric acid (CPIB), ciprofibrate (Cipro) and gemfibrozil (Gem) in primary cultures of rat hepatocytes based on complete concentration-response analysis and determination of theoretical maximum inductive responses for Cipro. CPIB and Cipro each induced FACO and LH in a concentration-dependent manner. Scatchard analysis of the data allowed calculation of EC50 values (mM) of 0.82 and 0.028 (for FACO) and 0.22 and 0.0081 (for LH) for CPIB and Cipro respectively. The EC50 ratios (CPIB/Cipro) were identical (29-fold) for induction of FACO and LH, supporting the concept that these enzymes are induced by CPIB and Cipro through a common mechanism. By comparison, Gem was relatively ineffective as an inducer of FACO and LH. Furthermore, Gem did not antagonize Cipro-mediated enzyme inductions, suggesting that Gem is a peroxisome proliferator of low potency rather than a partial agonist. Based on the potency and time-course profiles observed for induction of FACO and LH, the effects of CPIB, Cipro and Gem on triglyceride (TG) biosynthesis were determined in the cultured rat hepatocytes. Conditions of maximal FACO and LH induction by the drugs did not result in inhibition of TG biosynthesis in the cells. These results support the in vivo evidence which indicates that FACO and LH induction are not causally linked to the hypotriglyceridemic actions of peroxisome proliferating drugs.     

Identification of the proximate peroxisome proliferator(s) derived from di (2-ethylhexyl) adipate and species differences in response.

Identification of the proximate peroxisome proliferator(s) derived from di (2-ethylhexyl) adipate (DEHA) has been achieved using primary hepatocyte cultures derived from different species and cyanide-insensitive fatty acyl CoA oxidase (PCO) as a marker enzyme for peroxisome proliferation. In rat and mouse hepatocytes, the parent compound (DEHA) had no effect on peroxisomal beta-oxidation, but primary metabolites of DEHA, mono (2-ethylhexyl) adipate (MEHA) and 2-ethylhexanol (EH), were approximately equipotent in PCO induction (5-fold at 0.5 mM final concentration). The secondary metabolite of DEHA, 2-ethylhexanoic acid (EHA), was in both species the most potent peroxisome proliferator (25- and 9-fold induction in mice and rats, respectively, at 1 mM final concentration). At 2 mM final concentration a tertiary metabolite of DEHA, 2-ethyl-5-hydroxyhexan-1-oic acid, was less effective in mouse and rat hepatocytes at inducing PCO (15- and 5-fold, respectively). 2-Ethyl-5-oxohexan-1-oic acid and 2-ethylhexan-1,6-dioic acid had little effect (2-3-fold in both rat and mouse hepatocytes). Thus, EHA was identified as the proximate peroxisome proliferator of DEHA and mouse hepatocytes were approximately twice as sensitive as rat hepatocytes to peroxisome proliferation due to MEHA, EH and EHA. We investigated further species differences in response to peroxisome proliferators by using guinea pig and marmoset primary hepatocyte culture. None of the chemicals studied stimulated peroxisomal beta-oxidation in these species up to a final concentration of 2 mM. Higher concentrations lead to cytotoxicity. This lack of sensitivity of guinea pig and marmoset hepatocytes is in agreement with previous studies with di (2-ethylhexyl) phthalate metabolites, suggesting the absence of a threat of hepatocarcinogenic damage to these species and confirming that primary hepatocytes cultures are useful models for investigating the phenomenon of peroxisome proliferation.     

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.     

Induction of peroxisomal acyl CoA oxidase activity and lipid peroxidation in primary rat hepatocyte cultures

Peroxisome proliferators have been suggested to induce liver carcinogenesis as a result of increased peroxisomal hydrogen peroxide production and cellular oxidative stress. Primary monolayer cultures of hepatocytes isolated from male F344 rats were incubated in medium containing one of three different peroxisome proliferators and examined for the induction of peroxisomal CoA oxidase activity and lipid peroxidation. The latter parameter was determined by measuring levels of conjugated dienes in lipid fractions extracted from harvested cells. The peroxisome proliferators used in these studies were nafenopin and clofibric acid (two hypolipidemic drugs) and mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of the industrial plasticizer, di(2-ethylhexyl)phthalate (DEHP). The relative specific activity of peroxisomal acyl CoA oxidase was increased by about 300% after incubation for 44 h with 200 microM nafenopin; lower levels of induction were observed with clofibric acid or MEHP. Relative to controls, the level of conjugated dienes was increased approximately 2-fold after incubation with 200 microM nafenopin; there was no apparent increase in conjugated dienes after incubation with up to 200 microM MEHP or 400 microM clofibric acid. The increase in conjugated dienes with 200 microM nafenopin was inhibited by co-incubation with the antioxidant, N,N'-diphenyl-p-phenylenediamine. Thus, peroxisomal enzyme induction by nafenopin can result in membrane lipid peroxidation and monolayer cultures of rat hepatocytes may provide a useful model system for studying relationships between peroxisome proliferation, enhanced hydrogen peroxide production and cellular changes due to hepatic oxidative stress.     

Cytosolic long-chain acyl-CoA hydrolase, a suitable parameter to measure hepatic response to peroxisome proliferators.

The possibility of using cytosolic long-chain acyl-CoA as a parameter to measure the response of liver to peroxisome proliferators was studied. A subcutaneous (s.c.) injection of perfluorooctanoic acid (PFOA) to male Wistar rats caused an increase in activity of cytosolic long-chain acyl-CoA hydrolase. This increase in activity seems to be due to enzyme induction, since it was prevented by simultaneous administration of cycloheximide or actinomycin D with PFOA. The activity of cytosolic long-chain acyl-CoA hydrolase was increased in a dose-dependent manner by the administration of three peroxisome proliferators with diverse chemical structures: alpha-(p-chlorophenoxy)isobutyric acid (clofibric acid), 2,2'-(decamethylenedithio)diethanol (tiadenol) and PFOA. The increased activity produced by clofibric acid lasted throughout a 22-week treatment. A good correlation was found between the activities of cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation induced by the administration of the peroxisome proliferators. These results indicate that cytosolic long-chain acyl-CoA hydrolase is a suitable parameter for measuring the response of rat liver to challenges by peroxisome proliferators.     

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.     

Effects of long-term administration of clofibric acid on peroxisomal beta-oxidation, fatty acid-binding protein and cytosolic long-chain acyl-CoA hydrolases in rat liver

Long-term effects of rho-chlorophenoxyisobutyric acid (clofibric acid) on inductions of peroxisomal beta-oxidation, fatty acid-binding protein and cytosolic acyl-CoA hydrolases in rat liver were studied. Male rats were fed clofibric acid at a dietary concentration of 0.25% for 22 weeks. The induction of peroxisomal beta-oxidation activity lasted throughout the long-term treatment of rats, the activity being a half that of rats treated with clofibric acid for 2 weeks. cytosolic long-chain acyl-CoA hydrolase I and II were both induced by the long-term and the short-term treatment of age-matched rats with clofibric acid, although the ability to induce hydrolase I decreased greatly by aging of rats. There was little difference in the inducing effect on fatty acid-binding protein between the long-term treatment and the short-term treatment. These results suggest that the inductions of peroxisomal beta-oxidation, fatty acid-binding protein and two cytosolic long-chain acyl-CoA hydrolases are essential responses of rats to clofibric acid (but not the brief events which occur in only the first stage of the continuous treatment with clofibric acid).     

In vivo and in vitro peroxisome proliferation properties of selected clofibrate analogues in the rat. Structure-activity relationships

We have examined, relative to clofibric acid (CPIB), the effects of a chemical series of phenoxyacetic acids and of two asymmetric CPIB analogues, the R(+)- and S(-)-enantiomers of 2-(4-chlorophenoxy)propionic acid (4-CPPA) and 2-(4-chlorophenoxy)butyric acid (4-CPBA), on hepatic peroxisome proliferation both in vivo and in vitro utilizing cholesterol-fed rats and primary cultured rat hepatocytes respectively. Peroxisome proliferation was assessed by measuring changes in peroxisomal fatty acyl-CoA oxidase (FACO) and microsomal laurate hydroxylase (LH) activities as well as by electron microscopic examination of 3,3'-diaminobenzidine-stained liver slices. CPIB and enantiomers of 4-CPPA and 4-CPBA (0.6 mmol/kg/day for 7 days) produced hepatomegaly, lowered serum cholesterol levels, and caused 4.7- to 12.9-fold and 2.9- to 6.1-fold increases in hepatic FACO and LH activities, respectively, in cholesterol-fed rats. Electron micrographs of liver cells showed an increased number of peroxisomes from cholesterol-fed rats given S(-)-4-CPBA and CPIB. Likewise, these compounds (0.03 to 1.0 mM) induced FACO and LH in primary rat hepatocyte cultures after 72 hr. R(+)- and S(-)-Enantiomers of 4-CPPA produced similar concentration-dependent and maximal increases in both FACO and LH activities, whereas enantiomeric selectivity [S(-) greater than R(+)] for the induction of these two enzymes was observed with the isomers of 4-CPBA. The increases in the activities of FACO and LH caused by S(-)-4-CPBA were similar to those elicited by 1.0 mM CPIB (58.6- and 9.8-fold respectively). These results show that the enantiomers of 4-CPPA and 4-CPBA induce the peroxisome proliferation-associated enzymes FACO and LH in vivo and in vitro, and that the S(-)-isomer of 4-CPBA causes a greater induction of FACO and LH in vitro than its corresponding R(+)-isomer, indicating that these two enzymes are induced in an enantioselective manner. Optimal induction of the peroxisome proliferation-associated enzymes FACO and LH in rat hepatocyte cultures was produced by phenoxyacetic acids possessing (1) a chlorine atom at the 4-position of the phenyl ring, (2) a dimethyl or mono-ethyl substitution at the alpha-carbon atom of the carboxylic acid side chain; and (3) an S(-)-orientation for chiral analogues possessing a mono-ethyl group at the alpha-carbon atom of the carboxylic acid side chain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppression of hepatocyte apoptosis and induction of DNA synthesis by the rat and mouse hepatocarcinogen diethylhexylphlathate (DEHP) and the mouse hepatocarcinogen 1,4-dichlorobenzene (DCB)

Nongenotoxic rodent hepatocarcinogens do not damage DNA but cause liver tumours in the rat and mouse, associated with the induction of hepatic DNA synthesis. Previously, we have demonstrated that nongenotoxic hepatocarcinogens such as phenobarbitone and the peroxisome proliferator (PP), nafenopin, also suppress rat hepatocyte apoptosis. The nongenotoxic chemicals 1,4-dichlorobenzene (DCB) and the PP, diethylhexyl phthalate (DEHP), both induce high levels of DNA synthesis in rat liver in vivo, but only DEHP is hepatocarcinogenic in this species. Here, we investigate whether the difference in rat carcinogenicity of these two hepatic mitogens may be due to differences in their ability to suppress hepatocyte apoptosis. In rat hepatocytes in vitro, MEHP (the active metabolite of DEHP) induced DNA synthesis 2.5-fold (P = 0.001) and suppressed 10- and 4-fold, respectively both spontaneous (P = 0.0008) and transforming growth factor β1 (TGFβ1)-induced (P = 0.0001) apoptosis. DCB gave a small (1.7-fold) increase in DNA synthesis (P = 0.03) and a small (1.7- to 2-fold) suppression of both spontaneous (P = 0.022) and TGFβ1-induced (P = 0.015) apoptosis. We next analysed the induction of DNA synthesis and the suppression of apoptosis in rat liver in vivo. Both DEHP and DCB were able to induce DNA synthesis although, as seen in vitro, the induction by DCB (4.2-fold; P = 0.023) was less marked than that with DEHP (13.4-fold; P = 0.007). Similarly, DEHP and DCB were both able to suppress rat hepatocyte apoptosis in vivo but the magnitude of the suppression was comparable; apoptosis was reduced to undetectable levels in four out of five animals with DCB and three out of five with DEHP. Since both chemicals suppressed apoptosis and induced DNA synthesis in rat liver but, overall, DCB was less potent, the disparate hepatocarcinogenic potential of these two chemicals could arise from differences in the magnitude of growth perturbation. To test this hypothesis, we repeated the studies in mouse, a species where both DCB and DEHP are hepatocarcinogenic. Both in vitro and in vivo, DCB and DEHP/MEHP were able to suppress apoptosis and induce hepatocyte DNA synthesis in the mouse with comparable potencies. The data support the hypothesis that the carcinogenicity of nongenotoxic hepatocarcinogens is associated strongly with the ability to perturb hepatocyte growth regulation. However, the ability to effect such changes is not unique to nongenotoxic carcinogens and is common to some noncarcinogenic chemicals, such as DCB, suggesting that the growth perturbation may need to exceed a threshold for carcinogenesis. Received: 9 June 1998 / Accepted: 23 September 1998