Concomitant induction of cytosolic but not microsomal epoxide hydrolase with peroxisomal beta-oxidation by various hypolipidemic compounds

The effects of two cholesterol-lowering (probucol and 1-benzyl-imidazole), three triglyceride- and cholesterol-lowering (clofibrate, tiadenol and fenofibrate) and one triglyceride-lowering (acetylsalicylic acid) compounds on the specific activities of two lipid-metabolizing enzymes (cyanide-insensitive peroxisomal beta-oxidation and palmitoyl-CoA hydrolase) and two xenobiotic metabolizing enzymes (cytosolic (cEH) and microsomal epoxide hydrolase (mEHb] from the livers of male Fischer F-344 rats were investigated. With the exception of probucol and acetylsalicylic acid, all compounds tested caused a dose-dependent hepatomegaly. Taken on a weight basis fenofibrate was the most effective inducer, causing a 20-fold induction of peroxisomal beta-oxidation, a 13-fold induction of cEH activity and a 16-fold induction of palmitoyl-CoA hydrolase activity. The other compounds with triglyceride-lowering activity also induced cEH as well as peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. The potency of each individual drug was similar for induction of cEH activity as compared with that of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity, but very dissimilar for mEHb, which upon treatment with any of the triglyceride-lowering compounds was either not or only minimally (less than 1.5-fold) induced. 1-Benzylimidazole possessing exclusively cholesterol-lowering activity increased mEHb much more than either cEH or peroxisomal beta-oxidation. The absence of an enhancement of cEH activity in in vitro studies confirmed that the increase in enzyme activity by the test compounds is not caused by activation. cEH activity was also induced in the kidney but only about 2-fold by fenofibrate, tiadenol and acetylsalicylic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental studies on pharmacology, metabolism and toxicology with tiadenol-disulfoxide. Dissociation of lipid lowering effects and the induction of peroxisomal and microsomal drug-metabolizing enzymes

The hypolipidemic activity of tiadenol-disulfoxide, the major metabolite of 1,10-bis(hydroxyethylthio)decane (tiadenol, Eulip) in man and in the rat was assessed in various experimental models versus the corresponding activity of tiadenol. Tiadenol-disulfoxide in the normolipidemic rats lowers total serum cholesterol and serum and liver triglycerides in an extent comparable to that of the reference compound. Likewise, it is equally effective as tiadenol in preventing Triton-induced hyperlipidemia and Nath diet induced hypercholesterolemia; in addition tiadenol-disulfoxide is slightly more effective than tiadenol in increasing HDL-cholesterol in hypercholesterolemic rats. At hypolipidemic doses the compound causes no hepatomegaly, no induction of peroxisomal catalase and palmitoyl-CoA oxidase activities, no smooth endoplasmic reticulum proliferation and no induction of microsomal cytochrome P-450 and of cytochrome P-450 dependent enzyme activities: aminopyrine (aminophenazone) N-demethylase, aniline hydroxylase, zoxazolamine hydroxylase and hexobarbital oxidase. At the suprapharmacological dose of 300 mg/kg tiadenol-disulfoxide, if compared to the reference compound, shows a generally lower order of toxicity on these hepatic parameters. Orally administered tiadenol-disulfoxide is well absorbed by the gastrointestinal tract and is eliminated in urine at 45% of the dose in unchanged form, and the remaining being: glucuron-conjugated tiadenol-disulfoxide (10%), S-oxidized metabolites (15%) and sulfoxidized carboxylic metabolites (15%). The compound is well tolerated both in mice and rats. The results of this comparative study demonstrate that: 1. tiadenol-disulfoxide is a substance with promising hypolipidemic properties; 2. tiadenol-disulfoxide is largely responsible for the hypolipidemic activity of tiadenol; 3. hepatomegaly consequent to tiadenol administration is the consequence of the response of the liver cell to the increased functional demand of the mixed function oxidase (MFO) system involved in the metabolism of the drug; 4. peroxisomal enzyme activities induction observed with both drugs at non-pharmacological doses does not play any role in their hypolipidemic action and is not associated with hepatomegaly.     

Influence of chronic administration of valproate on ultrastructure and enzyme content of peroxisomes in rat liver and kidney. Oxidation of valproate by liver peroxisomes

Chronic administration to rats of the anticonvulsant drug, valproate, induced proliferation of liver peroxisomes and selectively increased the activity of the enzymes involved in beta-oxidation in these organelles. In kidney cortex, only a moderate increase in enzyme activity could be recorded. Valproate (1% w/w in the diet for 25 to 100 days) caused the appearance on electron micrographs of unusual tubular inclusions in the matrix of liver peroxisomes. SDS-PAGE analysis of purified peroxisomal fractions from treated rats demonstrated an increase in the content of five polypeptides; four of which most likely correspond to enzymes of the peroxisomal beta-oxidation. It is suggested that the peroxisomal inclusions correspond to the accumulation of these polypeptides in the matrix of the organelle. An in vivo evaluation of the peroxisomal hydrogen peroxide production suggested that valproate itself or one of its metabolites is substrate for peroxisomal beta-oxidation. This was confirmed by in vitro studies. Activation of valproate or its metabolites by liver acyl-CoA synthetase could be demonstrated, although it was 50 times slower than that of octanoate. This reaction further led to a small, but significant production of H2O2 by the action of peroxisomal acyl-CoA oxidase.     

Effect of hypolipidemic compounds on lauric acid hydroxylation and phase II enzymes

Treatment of male Fischer 344 rats with various hypolipidemic drugs of different peroxisome proliferating potency (1-benzylimidazole, acetylsalicylic acid, clofibrate, tiadenol) led to an induction of liver lauric acid hydroxylase, whereas probucol, which is not a peroxisome proliferator, did not induce this enzyme. Activity of bilirubin UDP-glucuronosyltransferase was increased by all the compounds tested. The highest increase was observed after treatment with acetylsalicylic acid (2.3-fold). High correlation (r = 0.953) was observed between the activities of lauric acid hydroxylase and the corresponding activities of cytosolic epoxide hydrolase reported previously. The amount of microsomal epoxide hydrolase was not changed by any of the compounds. Whereas clofibrate and tiadenol decreased glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate, 1-benzylimidazole and probucol increased this activity. With 4-hydroxynonenal as a substrate qualitatively the same results were obtained with the exception that probucol did not affect the enzyme activity. When glutathione S-transferase activity was measured with cis-stilbene oxide as substrate only the more than five-fold increase after treatment with 1-benzylimidazole was significantly different from control values. Activity of dihydrodiol dehydrogenase was increased after treatment of rats with 1-benzylimidazole (1.5-fold), whereas application of tiadenol led to a decrease of enzyme activity. Feeding of male guinea pigs with clofibrate did not change the activity of peroxisomal beta-oxidation, cytosolic epoxide hydrolase or lauric acid hydroxylase. However, treatment with tiadenol caused an increase of these activities.     

Induction of rat hepatic long-chain acyl-CoA hydrolases by various peroxisome proliferators

Induction of cytosolic long-chain acyl-CoA hydrolases was investigated in rat liver after administration of various peroxisome proliferators and related compounds. Treatment of rats with di-(2-ethylhexyl)-phthalate, di-(2-ethylhexyl)-adipate or tiadenol induced hydrolases I and II, while acetylsalicylic acid induced only hydrolase II. Among the various phenoxyacetic acid derivatives and related compounds, 2,4,5-trichlorophenoxyacetic acid, 2-(4-chlorophenoxy)-2-methylacetic acid, 2-(2-chlorophenoxy)-2-methylpropionic acid and clofibric acid induced both hydrolases I and II, whereas 2, 4-dichlorophenoxyacetic acid induced only hydrolase II. All nine of the above-mentioned inducers also markedly increased the activity of peroxisomal beta-oxidation. Other compounds tested (2-chlorophenoxyacetic acid, 4-chlorophenoxyacetic acid, 4-chlorophenol, phenoxyacetic acid and phenoxy-2-methylacetic acid) were ineffective as inducers. These results suggest that inducers of acyl-CoA hydrolase II also enhance peroxisomal beta-oxidation activity, but do not necessarily induce acyl-CoA hydrolase I. The structure-inducing activity relationships of these compounds are discussed.     

Peroxisome proliferating sulphur- and oxy-substituted fatty acid analogues are activated to acyl coenzyme A thioesters.

In liver homogenates from untreated rats the sulphur-substituted fatty acid analogues tetradecylthioacetic acid (CMTTD) was activated to its acyl-coenzyme A thioester. The activation was found to take place in the mitochondrial, microsomal and peroxisomal fractions. The activity of CMTTD-CoA synthetase was 50% compared to palmitoyl-CoA synthetase in all cellular fractions. When rats were treated with the peroxisome proliferating sulphur-substituted fatty acid analogues CMTTD and 3-dithiahexadecanedioic acid (BCMTD), the CMTTD-CoA synthetase activity was induced in mitochondrial, peroxisomal and microsomal fractions. Palmitoyl-CoA synthetase was induced proportionally. In rats treated with tetradecylthiopropionic acid (CETTD) of low peroxisome proliferating potency, the activities of CMTTD-CoA synthetase and palmitoyl-CoA synthetase were inhibited in mitochondrial and microsomal fractions. In contrast, all three sulphur-substituted acids induced the activity of palmitoyl-CoA synthetase and CMTTD-CoA synthetase in peroxisomes. Both the CMTTD-CoA and palmitoyl-CoA synthetase activities were induced by CMTTD and BCMTD, in close correlation to the induction of peroxisomal beta-oxidation. During the three treatment regimes, CMTTD-CoA synthetase activity ran parallel to the palmitoyl-CoA synthetase activity at a rate of 50% in all cellular fractions. Thus, CMTTD is assumed to be activated by the long-chain acyl-CoA synthetase enzyme. Rats were treated for 5 days with sulphur- and oxy-substituted fatty acid analogues, clofibric acid and fenofibric acid. All compounds which induced peroxisomal beta-oxidation activity in vivo could be activated to their respective CoA thioesters in liver homogenate. CETTD which induced peroxisomal beta-oxidation only two-fold, was activated at a rate of 50% compared to palmitate. Fenofibric acid induced peroxisomal beta-oxidation 9.6-fold, while it was activated at a rate of only 10% compared to palmitate. Thus, no correlation was found between rate of activation in vitro and induction of peroxisomal activity in vivo. On the other hand, tetradecylsulfoxyacetic acid (TSOA) and tetradecylsulfonacetic acid (TSA) (sulphuroxygenated metabolites of CMTTD) with no inductive effects, were not activated to their respective CoA derivatives. Altogether the data suggest that the enzymatic activation of the peroxisome proliferating compounds is essential for their proliferating activity, but the rate of activation does not determine the potency of the proliferators. The role of the xenobiotic-CoA pool in relation to the whole coenzyme A profile during peroxisome proliferation is discussed.     

Lack of induction of hepatic DNA damage on long-term administration of peroxisome proliferators in male F-344 rats

In order to evaluate the relationship between hydrogen peroxide (H2O2) generation and subsequent DNA damage caused by peroxisome proliferation, we examined DNA damage and changes in peroxisomal beta-oxidation activity in rat liver. Male F-344 rats were given orally clofibrate, bezafibrate or di(2-ethylhexyl)phthalate (DEHP) for up to 78 weeks. In rats fed DEHP for 52 or 78 weeks hepatocarcinomas or neoplastic nodules were found. In rats treated for 2 weeks with peroxisome proliferators, peroxisomal beta-oxidation activity was increased 10-17 times over control levels. After long-term treatment (20-78 weeks), the level of peroxisomal beta-oxidation activity remained 3-13-times higher in each group. When single strand DNA breaks were measured by a DNA-alkaline elution technique, no increase in DNA damage was observed in livers from rats fed peroxisome proliferators for 2, 40 or 78 weeks. In rats bearing hepatocarcinomas induced by DEHP, the hepatic DNA showed significant breaks; the rate of DNA-alkaline elution was found to increase approximately 5-fold. No significant increase in hepatic lipid peroxide level was observed in each group. These results show that although prolonged treatment with peroxisome proliferators induces markedly peroxisomal beta-oxidation activity, the active oxygen species from peroxisomal beta-oxidation are not enough to give rise to significant DNA damage. Moreover, the change in the activity of peroxisomal beta-oxidation may not relate to hepatocarcinogenesis induced by peroxisome proliferators.     

Enzymatic changes in rat liver associated with low and high doses of a peroxisome proliferator

The activities of a number of lipid-metabolizing and subcellular marker enzymes were measured in total homogenates and subcellular fractions prepared from the livers of male rats fed diets containing 0.05, 0.1, 0.3, and 0.5% of the hypolipidemic drug tiadenol, resulting in mean drug intake of 45, 90, 330, and 530 mg/day/kg body wt, respectively. In the total homogenates, a massive induction of palmitoyl-CoA hydrolase and peroxisomal palmitoyl-CoA oxidation accompanied by increased free CoASH and long-chain acyl-CoA content was observed at the highest dose levels whereas little change occurred up to 90 mg/day/kg/body wt. The palmitoyl-CoA synthetase activity increased slightly up to 90 mg/day/kg body wt, but higher doses resulted in decreased enzyme activity. Catalase activity increased with the dose to be elevated by a factor of approximately 1.6 at 330 mg/day/kg, whereas the activities of urate oxidase decreased. The specific activities of palmitoyl-CoA hydrolase and peroxisomal palmitoyl-CoA oxidation increased in all fractions, but most markedly in the cytosol. The changes in the activities and the distribution of subcellular marker enzymes and the increase of the peroxisome-associated polypeptide (PPA-80) are in keeping with a peroxisome proliferating effect resulting in formation of premature organelles with altered properties. Since high doses of many hypolipidemic drugs produce hepatic tumors and peroxisomal proliferation in rodents and since no increase in peroxisomes is found in human liver after therapeutic use of lower doses, the dose-response relationship is of interest for the evaluation of the toxicology of this class of agents.     

Metabolism of the hypolipidemic agent tiadenol in man and in the rat

The metabolism of the hypolipidemic agent 1,10-bis(hydroxyethylthio)decane (tiadenol, Eulip) has been studied in vivo in man and in the rat and in vitro in the rat. Following oral administration, in both species tiadenol was completely absorbed, extensively metabolized by the liver and more than 95% of the dose was eliminated in this form via kidneys within 48 h. Insignificant was the excretion of the unchanged drug in urine (approximately 1%) as well as that of its metabolites in the feces. 8 metabolites were isolated from human or rat urine and their structures were elucidated by means of electron impact, field desorption and positive and negative fast atom bombardment mass spectrometry. Both in man and in the rat the main metabolic pathway was the oxidation of the thioether sulfur, followed by oxidation or conjugation of the primary alcohol group(s). The urinary excretion of S-oxidized metabolites and sulfoxidized carboxylic metabolites accounted for 75% of the dose and that of S-oxidized conjugated metabolites for 20%. Rat in vitro studies showed that hepatic microsomal cytochrome P-450-dependent monooxygenase catalyzes the S-oxidative pathway, which governs the in vivo elimination of the drug in both species. Thus cytochrome P-450 is the key enzyme in the hepatic detoxification of tiadenol.     

Induction by perfluorooctanoic acid of microsomal 1-acylglycerophosphocholine acyltransferase in rat kidney. Sex-related difference.

Response of rat kidney to the challenges by perfluorooctanoic acid (PFOA) was studied using microsomal 1-acyglycerophosphocholine (1-acyl-GPC) acyltransferase as a parameter. Marked induction of the enzyme was brought about in kidney of male rats, whereas the induction in kidney of female rats was far less pronounced. The sex-related difference in the response of kidney to PFOA was much more marked than those seen with p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethy-lenedithio)diethanol (tiadenol). Hormonal manipulations revealed that the sex-related difference in the response of kidney to PFOA was strongly dependent on the state of gonadal hormones of rats. Even after a prolonged administration of PFOA for up to 26 weeks, this sex-related difference was still evident. Induction of peroxisomal beta-oxidation was brought about concurrently with microsomal 1-acyl-GPC acyltransferase and a high correlation was confirmed between the inductions of these two parameters.     

Metabolism of sameridine to monocarboxylated products by hepatocytes isolated from the male rat.

The metabolism of sameridine (LPB) (an amide-type local anesthetic-analgesic agent with a hexyl side chain) to carboxylic acid derivatives by isolated male rat hepatocytes was studied using gradient reversed-phase HPLC and mass spectrometry. Incubation of sameridine with hepatocytes resulted in the formation of numerous different metabolites. Two carboxylic acids, i.e., the C(6) and C(4) carboxylated derivatives of sameridine (LPB-6'-oic acid and LPB-4'-oic acid), were found to be produced from the intermediate omega-hydroxy metabolite (6'-hydroxy-LPB). Shortening of the alkyl chain in LPB-6'-oic acid by two carbon atoms resulted in LPB-4'-oic acid. However, incubation of rat hepatocytes with 5'-hydroxy-LPB [the (omega-1)-hydroxy derivative of sameridine] did not give rise to any carboxylated derivative. Addition of SKF525A inhibited the metabolism of sameridine by rat hepatocytes, indicating that the initial step is catalyzed by cytochrome P450. Furthermore, the metabolism of sameridine to LPB-4'-oic acid was enhanced in hepatocytes isolated from rats treated with clofibrate, an up-regulator of peroxisomal fatty acid beta-oxidation and of microsomal cytochrome P450 4A. L-Carnitine (which increases the rate of mitochondrial fatty acid beta-oxidation) had no effect on the level of LPB-4'-oic acid produced by isolated rat hepatocytes. The metabolism of 6'-hydroxy-LPB to LPB-6'-oic acid was inhibited almost completely by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. Considered together, our findings suggest that cytochrome P450 4A, cytosolic dehydrogenases, and the enzymes involved in peroxisomal fatty acid beta-oxidation catalyze the metabolism of sameridine to LPB-4'-oic acid.     

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.     

Contribution of peroxisomal beta-oxidation system to the chain-shortening of N-(alpha-methylbenzyl)azelaamic acid in rat liver

Hepaptic peroxisomal and mitochondrial beta-oxidation of N-(alpha-methylbenzyl)azelaamic acid (C9), which is a possible metabolic intermediate of Melinamide, a potent hypocholesterolemic drug, were investigated. Isolated hepatocytes generated H2O2 when incubated with C9, indicating that C9 served as the substrate for peroxisomal beta-oxidation. Also with isolated peroxisomes a significant activity of peroxisomal beta-oxidation for C9-CoA measured by following cyanide-insensitive NAD reduction was observed, when the chain-shortened products such as C7 and C5 were detected from the incubation mixture of C9-CoA, and so NADH, acetyl-CoA and C2 units split off from C9-CoA were produced in stoichiometric amounts. In contrast, the mitochondrial beta-oxidation for C9 measured by following ketone body production and antimycin A-sensitive O2 consumption was not detectable, indicating that C9 is not metabolized by mitochondrial beta-oxidation. Comparative study of beta-oxidation capacities in peroxisomes and mitochondria indicate that the beta-oxidation of C9 occurs exclusively in peroxisomes. Also, the formation activity of C2 units liberated from C9 in intact hepatocytes reflects the peroxisomal beta-oxidation activity of liver homogenates with a highly close correlation. Therefore, it is concluded that C9 can be an excellent substrate for estimating peroxisomal beta-oxidation activity in intact cells.     

Oxidation and glucuronidation of valproic acid in male rats--influence of phenobarbital, 3-methylcholanthrene, beta-naphthoflavone and clofibrate

The influence of phenobarbital, clofibrate, 3-methylcholanthrene and beta-naphthoflavone on omega- and beta-oxidation as well as on glucuronidation of valproic acid (n-dipropylacetic acid) was evaluated in male Sprague-Dawley rats by determination of urinary excretion of its metabolites by GC-MS after administration of 100 mg/kg. In controls 12% of the dose was excreted within 24 hours, primarily as glucuronides; metabolites formed by oxidation amounted to about 4%. Phenobarbital treatment led to stimulation of 4-hydroxyvalproic acid [(omega-1)-oxidation], 5-hydroxyvalproic acid and n-propylglutaric acid (omega-oxidation) excretion. Clofibrate enhanced the excretion of 4-hydroxyvalproic acid and 3-keto-valproic acid, a product of peroxisomal beta-oxidation. beta-Naphthoflavone slightly increased the excretion of 5-hydroxyvalproic acid. The most specific effect was found for 3-methylcholanthrene, which was effective in stimulating the formation of 3-hydroxyvalproic acid which might be formed by (omega-2)-oxidation. The addition of fatty acids (olive oil in which 3-methylcholanthrene and beta-naphthoflavone were suspended) led to increased excretion of 3-keto-valproic, 4-hydroxyvalproic and n-propylglutaric acid. The excretion of 3-hydroxyvalproic acid was completely suppressed by olive oil. Such specific effects were not observed for glucuronidation of valproic acid and its metabolites, although stimulation was attained after phenobarbital, clofibrate and 3-methylcholanthrene treatment, because of instability of glucuronide conjugates. Stimulation of valproic acid metabolism was also shown by increased plasma clearance after treatment with phenobarbital. In contrast, clofibrate given once 1 hr before valproic acid inhibited excretion of valproic acid, possibly by competition during renal tubular secretion. Determination of valproic acid metabolites in urine provides a useful tool for evaluation of inducer specificity of short chain fatty acid metabolism and differentiation between microsomal and peroxisomal enzyme activity.     

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).     

Induction of triglyceride accumulation in the liver of rats by perfluorinated fatty acids with different carbon chain lengths: comparison with induction of peroxisomal beta-oxidation

The potency to accumulate triglyceride (TG) was compared between perfluorinated fatty acids (PFCAs) with different carbon chain lengths in the liver of male and female rats and induction of peroxisomal beta-oxidation. In male rats, either perfluoroheptanoic acid (C7) or perfluorooctanoic acid (C8) had no effect, although perfluorononanonic acid (C9) and perfluorodecanoic acid (C10) markedly accumulated TG. In female rats, C7, C8, and C9 did not cause TG accumulation, whereas C10 caused TG accumulation at the same level as in male rats. TG accumulation induced by C9 was regulated by the level of testosterone in male rats. In contrast with TG accumulation, peroxisomal beta-oxidation was induced by C8, C9, and C10 in male rats and by C9 and C10 in female rats. Only a slight difference was observed in the induction by C9 between male and female rats. The induction of TG accumulation by these PFCAs occurred in a dose-dependent manner and significantly correlated with hepatic concentrations of PFCA regardless of their carbon chain length, as was observed with induction of peroxisomal beta-oxidation. There is, however, a striking difference in the hepatic concentration of PFCA required to cause induction of TG accumulation and that of peroxisomal beta-oxidation. The concentration of PFCA required to induce TG accumulation is much higher than that to induce peroxisomal beta-oxidation. These results strongly suggest that TG accumulation induced by PFCAs, as well as the induction of peroxisomal beta-oxidation, is dependent only on the number of PFCA molecules in hepatocytes, but is not due to the difference in their chemical structures, and that there is a marked difference in the PFCA threshold to cause distinct biological effects.     

Effect of diabetes and starvation on the activity of rat liver epoxide hydrolases, glutathione S-transferases and peroxisomal beta-oxidation

The activities of peroxisomal beta-oxidation, cytosolic and microsomal epoxide hydrolase as well as soluble glutathione S-transferases have been determined in the livers of alloxan- and streptozotocin-diabetic male Fischer-344 rats. Five, seven and ten days after initiation of diabetes serum glucose levels were elevated 3.6-, 5.7- to 6.2- and 6-fold, while the activities of peroxisomal beta-oxidation and cytosolic epoxide hydrolase were elevated 1.5- and 2.5-fold, 1.4- and 2.7-fold and 1.3- and 2.0-fold, respectively. The activities of microsomal epoxide hydrolase and glutathione S-transferases were reduced to about 71% and 80% of controls. Application of 10 I.U./kg depot insulin twice a day for 10 consecutive days to alloxan-diabetic individuals approximately restored the initial glucose levels and enzyme activities except for peroxisomal beta-oxidation. Starvation of Fischer-344 rats for 48 hours and 5 days similarly resulted in a 1.3-fold to 2.1-fold and 1.2- to 1.6-fold increase in peroxisomal beta-oxidation and cytosolic epoxide hydrolase activity, respectively. Microsomal epoxide hydrolase was significantly decreased to 57% and 61% of control activity whereas glutathione S-transferase was only marginally reduced to 91% and 92%. Except for glutathione S-transferases initial enzyme activities were restored upon refeeding within 10 days. These results are similar to those obtained upon feeding of hypolipidemic compounds with peroxisome proliferating activity, and may indicate that high levels of free fatty acids or their metabolites which are known to accumulate in liver in both metabolic states may act as endogenous peroxisome proliferators.     

Effects of 5-bromo-2'deoxyuridine (BrdU) implants on hepatic cytochrome P-450 content and beta-oxidation activity in rats and mice.

Standard regulatory toxicity tests are frequently supplemented with additional compound specific analysis. Analysis of hepatic cytochrome P-450 content, hepatic beta-oxidation activity (biochemical analysis), and cell proliferation rates are examples of these analyses that are included when past experience or similarity to other compounds, suggest that a presently tested compound may have an effect. Until now, separate subsets of animals have been designated for cell proliferation analysis and biochemical analysis, because it was unknown if implantation of 5-bromo-2'deoxyuridine (BrdU) filled osmotic pumps (BrdU implants) would effect the rate of hepatic-beta or hepatic cytochrome P-450 content. The purpose of the current study was to determine if BrdU implants had an effect on hepatic cytochrome P-450 content, beta-oxidation activity, or the measurement of these enzymes in rats and mice. The BrdU was administered through subcutaneous osmotic pump implants. The rate of hepatic peroxisomal beta-oxidation was not altered in male or female rats or mice with the BrdU implants when compared to those of the control groups. The total hepatic cytochrome P-450 content was also not altered in male or female rats or mice with the BrdU implants when compared to those of the control groups. BrdU implants do not appear to have an effect on the rate of hepatic peroxisomal beta-oxidation or the total hepatic cytochrome P-450 content in male or female rats and mice. It can be concluded that in future studies, rats or mice which are designated for cell proliferation analysis using BrdU implants are also suitable for use in evaluating chemically induced effects on hepatic peroxisomal beta-oxidation activity and/or total hepatic cytochrome P-450 content.     

Long-term effects of peroxisome proliferators on the balance between hydrogen peroxide-generating and scavenging capacities in the liver of Fischer-344 rats

In order to clarify whether peroxisomal hydrogen peroxide (H2O2) plays an important role in peroxisome proliferator-induced hepatocarcinogenesis, we examined the change in metabolism of peroxisomal H2O2 in vivo and in vitro using male Fischer-344 rats fed clofibrate, bezafibrate and di(2-ethylhexyl)phthalate (DEHP) for up to 78 weeks. Hepatic peroxisomal fatty acyl-CoA oxidase activity increased 12-20-fold after 2 or 4 weeks treatment; later this level gradually decreased toward controls, and at 78 weeks activity was 3-10-times of control. Although hepatic H2O2 levels were increased slightly by clofibrate, bezafibrate and DEHP, the changes did not correlate with the changes in peroxisomal fatty acyl-CoA oxidase activity. In isolated hepatocytes, the rate of leakage of peroxisomal H2O2 from peroxisomes into the cytosol and the hepatocellular H2O2 content was measured. The rate of leakage of peroxisomal H2O2 into cytosol increased 2.5-4-fold when peroxisomal beta-oxidation activity was induced by peroxisome proliferators, and the increases in this rate corresponded with changes in the peroxisomal beta-oxidation activity. In contrast, the hepatocellular H2O2 contents were not affected by induced peroxisomal beta-oxidation. These data show that H2O2 leaking from peroxisome into cytosol would be quickly decomposed, and thus peroxisomal H2O2 does not appear to play an important role in hepatocarcinogenesis by such an oxidative stress mechanism after the long-term treatment with peroxisome proliferators.     

Changes in lipid metabolizing enzymes of hepatic subcellular fractions from rats treated with tiadenol and clofibrate

the levels of hepatic lipid metabolizing enzymes including palmitoyl-CoA hydrolase, palmitoyl-l-carnitine hydrolase as well as some other enzymes were studied in the 100,000 g × 1 hr sediment, the corresponding supernatant and lipid-rich floating layer from rats fed tiadenol or clofibrate-containing diets (0.3 per cent $\text{w}\text{w}$). Tiadenol administration resulted in a large increase of the total activity of palmitoyl-CoA hydrolase, and of peroxisomal-CoA oxidation, while only a moderate enhancement was obtained after clofibrate feeding. the total activity of palmitoyl-l-carnitine hydrolase was increased more by tiadenol than by clofibrate. the specific activity of the two former enzymes was decreased in the particulate MLP-fraction (100,000 g × 1 hr sediment containing mitochondria, peroxisomes and microsomes) after treatment with tiadenol. The specific activity of palmitoyl-CoA hydrolase was increased more than 10-fold in the cytosolic fraction after administration of tiadenol. Tiadenol increased the specific activity of palmitoyl-l-carnitine hydrolase considerably in the cytosolic fraction, but the activity of this enzyme was little affected by clofibrate treatment. the specific activity of palmitoyl-CoA hydrolase and palmitoyl-l-carnitine hydrolase increased in the lipid-rich floating layer. Since there was also a shift in the distribution of peroxisomal palmitoyl-CoA oxidation and catalase, but not of urate oxidase after treatment with the drugs, it is suggested that the drugs induce peroxisomes with altered membrane characteristics.