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.     

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.     

CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes

1. We investigated the nature and roles of various xenobiotic acyl-CoA hydrolases in liver subcellular fractions from rat treated with sulphur-substituted (thia) fatty acids. To contribute to our understanding of factors influencing enzymes involved in the degradation of activated fatty acids, the effects on these activities of the oppositely acting thia fatty acid analogues, the peroxisome proliferating 3-thia fatty acids (tetradecylthioacetic acid and 3- dithiacarboxylic acid), which are blocked for β-oxidation, and a non-peroxisomeproliferating 4-thia fatty acid (tetradecylthiopropionic acid), which undergoes one cycle of β-oxidation, were studied. 2. The hepatic subcellular distributions of palmitoyl-CoA, tetradecylthioacetyl-CoA and tetradecylthiopropionyl-CoA hydrolase activities were similar to each other in the control and 3-thia fatty acid-treated rat. In control animals, most of these hydrolases were located in the microsomal fraction, but after treatment with the 3-thia fatty acids, the specific activities of the mitochondrial, peroxisomal, and cytosolic palmitoyl-CoA, tetradecylthioacetyl-CoA, and tetradecylthiopropionyl-CoA hydrolase activities were significantly increased. This increase in activity was seen mostly for the enzymes using tetradecylthiopropionyl-CoA and tetradecylthioacetyl-CoA as substrates. The increased mitochondrial activities for these two substrates were seen already after 1 day of treatment, whereas the peroxisomal activities increased after 3 days. No stimulation was seen after treatment with the 4-thia fatty acid analogue, tetradecylthiopropionic acid, but a decrease in peroxisomal hydrolase activities for all three substrates was observed. 3. The cellular distributions of clofibroyl-CoA, POCA-CoA, and sebacoyl-CoA hydrolase activities were different from those of the 'long-chain acyl-CoA' hydrolases mentioned above both in the normal and 3-thia fatty acid treated rat. This group of hydrolases was found in the mitochondrial, peroxisomal, and cytosolic fractions. 3-Thia fatty acid treatment increased the activities of clofibroyl-CoA and sebacoyl-CoA hydrolases in all three fractions. Clofibroyl-CoA and sebacoyl-CoA hydrolase activities were increased after 1 day of treatment. Only the cytosolic POCA-CoA hydrolase was stimulated after 3- thia fatty acid treatment after only 1 day of treatment, whereas treatment with the 4-thia fatty acid led to an increase of enzyme activity in the mitochondrial and peroxisomal fractions. 4. Based on the subcellular distributions and specific activities, we suggest that several enzymes exist which may act as regulators of intracellular acyl-CoA levels.     

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.     

Effects of long-term administration of clofibric acid on stearoyl-CoA desaturase, 1-acylglycerophosphorylcholine acyltransferase and fatty acyl composition of microsomal phosphatidylcholine in rat liver

Long-term effects of p-chlorophenoxyisobutyric acid (clofibric acid) on inductions of stearoyl-CoA desaturase and 1-acylglycerophosphorylcholine (1-acyl-GPC) acyltransferase, and on changes in fatty acyl composition of microsomal lipid in rat liver were studied. Male rats were fed clofibric acid at a dietary concentration of 0.25% for 2 or 22 weeks. Inductions of stearoyl-CoA desaturase and 1-acyl-GPC acyltransferase lasted throughout the long-term treatment and were the same as those of either young or aged rats which were treated with clofibric acid for 2 weeks. The long-term treatment of rats with clofibric acid scarcely affected components of stearoyl-CoA desaturation system other than terminal desaturase. In accordance with the induction of stearoyl-CoA desaturase, the increase in the proportion of octadecenoic acid in hepatic lipid lasted throughout the 22-week treatment. In the case of both of the long-term treatment and the short-term treatment of rats, the increase in the proportion of octadecenoic acid in microsomal phosphatidylcholine was due to the marked increase in the proportion of octadecenoic acid in position 2, but not position 1, of phosphatidylcholine. These changes in fatty acyl composition of phosphatidylcholine were not due to the alteration of the content of phosphatidylcholine in liver.     

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.     

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.     

'Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data

1. In this study we explored the relationship between specific acyl-CoA esters and induction of acyl-CoA binding protein (ACBP) and enzymes related to the proliferation of peroxisomes. Male Wistar rats were administered a single dose (150mg/day/kg) of sulphur-substituted fatty acid analogues, and the effects of tetradecylthioacetic acid and 3-thiadicarboxylic acid, which both act as peroxisome proliferators, were compared with the effects of tetradecylthiopropionic acid and palmitic acid which do not induce peroxisome proliferation.

2. The hepatic level of total long-chain acyl-CoA was significantly increased within 12?h of feeding these fatty acids, except in rat fed tetradecylthioacetic acid. Hplc chromatograms of liver extracts prepared from rat fed tetradecylthioacetic acid showed that tetradecyl-thioacetyl-CoA ester accumulated in the liver 4h after feeding and had disappeared after 24h. In liver extracts of the tetradecylthiopropionic acid-treated rat tetradecylthiopropionyl-CoA was not observed, but the appearance of a new long-chain acyl-CoA ester, probably a metabolite of tetradecylthiopropionic acid, was detected. This new peak reached a maximum 4?h after feeding. In rat fed tetradecylthioacetic acid and 3-thiadicarboxylic acid the hepatic level of fatty acyl-CoA oxidase mRNA increased 8h after feeding, while the acyl-CoA oxidase activity had increased after 12h.

3. The early accumulation of specific tetradecylthioacetyl-CoA suggests that this ester may be a possible mediator of the induction of fatty acyl-CoA oxidase. The level of hepatic acyl-CoA binding protein, long-chain acyl-CoA hydrolase activity and long-chain acyl-CoA synthetase activity did not change after a single dose of all four fatty acids. Prolonged administration of 3-thia fatty acids resulted, however, in a dose- and time-dependent increase in hepatic ACBP content and ACBP mRNA level. The amount of ACBP increased in parallel to the long-chain acyl-CoA hydrolase activity. The correlated induction of fatty acyl-CoA binding protein and long-chain acyl-CoA hydrolase seems to be dependent on a sustained accumulation of total long-chain acyl-CoA esters.     

Alkylthio acetic acids (3-thia fatty acids)--a new group of non-beta-oxidizable peroxisome-inducing fatty acid analogues--II. Dose-response studies on hepatic peroxisomal- and mitochondrial changes and long-chain fatty acid metabolizing enzymes in rats

The activity of key enzymes involved in oxidation and esterification of long-chain fatty acids was investigated after male Wistar rats were treated with different doses of sulfur substituted fatty acid analogues, 1,10-bis(carboxymethylthiodecane) (BCMTD, non-beta-oxidizable and non-omega-oxidizable), 1-mono(carboxymethylthiotetradecane) (CMTTD, trivial name, alkylthio acetic acid, non-beta-oxidizable) and 1-mono(carboxyethylthiotetradecane) (CETTD trivial name, alkylthio propionic acid, beta-oxidizable). The sulfur substituted dicarboxylic acid and the alkylthio acetic acid induced in a dose-dependent manner the mitochondrial, microsomal and especially the peroxisomal palmitoyl-CoA synthetase activity, the mitochondrial and cytosolic palmitoyl-CoA hydrolase activity, the mitochondrial and especially the microsomal glycerophosphate acyltransferase activity and the peroxisomal beta-oxidation, especially revealed in the microsomal fraction. Morphometric analysis of randomly selected hepatocytes revealed that BCMTD and CMTTD treatment increased the number, size and volume fraction of peroxisomes and mitochondria. Thus, the observed changes in the specific activity of fatty acid metabolizing enzymes with multiple subcellular localization can partly be explained as an effect of changes in the s-values of the organelles as proliferation of mitochondria and peroxisomes occurred. The most striking effect of the alkylthio propionic acid was the formation of numerous fat droplets in the liver cells and enhancement of the hepatic triglyceride level. This was in contrast to BCMTD treatment which decreased the hepatic triglyceride content. In conclusion, the results provide evidence that administration of non-beta-oxidizable fatty acid analogues had much higher in vivo potency in inducing hepatomegaly and key enzymes involved in fatty acid metabolism, including proliferation of peroxisomes and mitochondria than is exhibited in the beta-oxidizable, alkylthio propionic acid. Moreover, the dicarboxylic acid was apparently three to six times more potent than the alkylthio acetic acid in inducing peroxisomal beta-oxidation and peroxisome proliferation when considered on a mumol/day basis. As palmitic acid and hexadecanedioic acid only marginally affected these hepatic responses, it is conceivable that the potency of the selected compounds as proliferators of peroxisomes and inducers of the associated enzymes depends on their accessibility for beta-oxidation.     

Different effects of fibrates on the microsomal fatty acid chain elongation and the acyl composition of phospholipids in guinea-pigs.

1. The effects in vitro and in vivo of three fibric acid derivatives, clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on some enzyme activities related to fatty acid biosynthesis, namely palmitoyl-CoA synthetase and hydrolases (microsomal and cytosolic), NADH and NADPH cytochrome c reductases and acyl-CoA elongases were investigated in guinea-pigs. 2. The three fibrates inhibited acyl-CoA elongation in vitro, irrespective of the substrate of elongation used (saturated, monounsaturated, polyunsaturated) and with an order of potency GFB > BFB > CFB. In the case of GFB, inhibition occurred at concentrations that can be reached in vivo. 3. Microsomal palmitoyl-CoA hydrolase and synthetase were also inhibited in vitro (GFB > or = BFB > CFB), whereas NADH cytochrome c reductase activity was increased by GFB. Nevertheless, the magnitude of changes were lower than those observed in elongation activities. 4. Treatment with fibrates did not produce peroxisomal proliferation in guinea-pigs, as measured by peroxisomal beta-oxidation activity and liver weight/body weight ratio. Nevertheless, fibrates provoked a reduction in plasma cholesterol and triglycerides, at least in GFB- and BFB-treated animals. 5. Fatty acid elongation was significantly modified by GFB treatment in vivo. The remaining enzyme activities studied were only slightly changed by fibrate treatment. 6. Treatment with BFB and to a lesser extent with CFB, increased the relative proportion of MUFA (palmitoleic and oleic acids) in microsomal phospholipids, whereas PUFA (mainly linoleic acid) decreased. GFB behaved differently, increasing palmitic and linoleic acids and decreasing stearic and oleic acids. The latter changes are attributable to an inhibition of elongation activity by GFB. 7. The changes observed after fibrate treatment in both rats and guinea-pigs, as they are not directly related to peroxisome proliferation, could be more reliably extrapolated to man than those observed only in rats.     

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)     

高糖饮食导致大鼠肝脏脂代谢紊乱实验研究

目的 探讨高糖饮食对大鼠肝脏过氧化物酶体脂肪酸B-氧化的影响.方法 用高糖饮食饲养大鼠24周,观察体重,血清总胆固醇(TC)、三酰甘油(TG)、游离脂肪酸(FFA),肝脏过氧化氢酶、脂酰CoA氧化酶和过氧化物酶体脂肪酸β-氧化活性的变化.结果 试验组TG、T℃、FFA与对照组相比有不同程度升高(P<0.01).试验组大...     

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.     

Hepatic induction potency of hypolipidaemic drugs in the rat following long-term administration: influence of different dosing regimens

1. The effects of different dosing regimens of three hypolipidaemic, peroxisome-proliferator drugs on hepatic enzymes in the Fischer rat following 26 weeks treatment have been studied. 2. In study 1, with once-daily dosing (dose levels based on comparative antisecretory activity), the liver/body weight ratio and peroxisomal beta-oxidation were significantly increased in the order: ciprofibrate greater than bezafibrate greater than clofibric acid. Glutathione peroxidase activity was decreased to 65% and 77% control after treatment with ciprofibrate and bezafibrate, respectively, but not after treatment with clofibric acid. 3. In study 2, dosing regimens were adjusted to compensate for the different drug pharmacokinetic profiles in rat, with clofibric acid and bezafibrate administered twice daily and ciprofibrate once every 48 h. Liver enlargement and increases in peroxisomal beta-oxidation were similar with all three drugs when compensation for differences in drug clearance was made. Glutathione peroxidase activity was decreased to similar extents by all three compounds. 4. The induction profiles of these hypolipidaemic drugs, largely different with once-daily dosing, were shown to be similar after adjusting the frequency of dosing with respect to drug half-life.     

Characterization of the induction of cytosolic and microsomal epoxide hydrolases by 2-ethylhexanoic acid in mouse liver

When mice were exposed to 1% 2-ethylhexanoic acid in the diet, cytosolic and microsomal epoxide hydrolase (EC 3.3.2.3) activities were increased maximally (2-2.5- and 0.5-1-fold, respectively) after 3 days. Immunochemical quantitation of these enzymes indicated that the process involved was a true induction in both cases. Maximal levels of peroxisome proliferation (as indicated by carnitine acetyltransferase activity) were obtained after 7 days of exposure. All three of these activities returned to control levels within 4 days after termination of the treatment. The liver somatic index was slightly increased after 4 days of administration of 1% 2-ethylhexanoic acid, but the protein contents of the "mitochondrial," microsomal, and cytosolic fractions were unaffected. The activity of peroxisomal palmitoyl-CoA beta-oxidation was increased 2-fold, whereas peroxisomal catalase activity was unaffected. Exposure to 2-ethylhexanoic acid also increased cytochrome oxidase activity, suggesting an effect on mitochondria. Other parameters of detoxication--i.e. total microsomal cytochrome P-450 content, cytosolic glutathione transferase activity toward 1-chloro-2,4-dinitrobenzene, and the "cytosolic" epoxide hydrolase activity localized in the "mitochondrial" fraction--were not affected by 4 days of treatment with 1% 2-ethylhexanoic acid.     

Comparison of the inducing effect of dehydroepiandrosterone on hepatic peroxisome proliferation-associated enzymes in several rodent species. A short-term administration study.

The in-vivo effect of dehydroepiandrosterone (DHEA) on hepatic enzyme activities of rats, mice, hamsters and guinea pigs was investigated. After DHEA treatment (300 mg/kg body weight, per os, 14 days), the activities of peroxisomal beta-oxidation, catalase, carnitine acetyltransferase, carnitine palmitoyltransferase, lauric acid omega-hydroxylation, 1-acylglycerophosphocholine acyltransferase, malic enzyme and cytosolic palmitoyl-CoA hydrolase were increased in rats and in mice although to a smaller extent in the latter. These enzyme activities, however, were unchanged in hamsters with the exception of omega-hydroxylation (2.5-fold increase) and 1-acylglycerophosphocholine acyltransferase (2.0-fold increase). No significant changes were observed in any of these enzyme activities in guinea pigs. Immunoblot analysis confirmed the induction of peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme in rats and mice. These results indicate that there are species differences in the inducing effect of DHEA on hepatic peroxisome proliferation-associated enzymes, which correlates well with the enzyme induction observed with other peroxisome proliferators.     

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.     

Methylated eicosapentaenoic acid and tetradecylthioacetic acid: effects on fatty acid metabolism.

We introduced methyl or ethyl groups to the 2- or 3-position of the eicosapentaenoic acid (EPA) molecule to investigate whether the branching of EPA could influence its hypolipidemic effect in rats. The most effective branching involved two methyl groups in the 2-position and one methyl group in the 3-position. These EPA derivatives increased hepatic mitochondrial and peroxisomal beta-oxidation and decreased plasma lipids concomitant with suppressed acetyl-coenzyme A (CoA) carboxylase (EC 6.4.1.2) and fatty acid synthase (EC 2.3.1.85) activities. This was followed by elevated activities of camitine O-palmitoyltransferase (EC 2.3.1.21) and possibly 2,4-dienoyl-CoA reductase (EC 1.3.1.34), as well as induced mRNA levels of these enzymes and fatty acyl-CoA oxidase. The fatty acid composition in liver changed, with an increased 18:1 n-9 content, whereas the expression of delta9-desaturase remained unchanged. We investigated the flux of fatty acids in cultured hepatocytes, and found that oxidation of [1-14C]-labeled palmitic acid increased but the secretion of palmitic acid-labeled triglycerides decreased after addition of 2-methyl-EPA. The fatty acyl-CoA oxidase (EC 1.3.3.6) activity in these cells remained unchanged. A significant negative correlation was obtained between palmitic acid oxidation and palmitic acid-labeled synthesized triglycerides. To investigate whether the hypolipidemic effect occurred independently of induced peroxisomal beta-oxidation, we fed rats 2-methyl-tetradecylthioacetic acid. This compound increased the peroxisomal but not the mitochondrial beta-oxidation, and the plasma lipid levels were unchanged. In conclusion, EPA methylated in the 2- or 3-position renders it more potent as a hypolipidemic agent. Furthermore, this study supports the hypothesis that the mitochondrion is the primary site for the hypolipidemic effect.     

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.     

Fatty acid oxidation in hepatic peroxisomes and mitochondria after treatment of rats with di(2-ethylhexyl)phthalate

Rats were fed a diet containing di(2-ethylhexyl)-phthalate, which increases the number of peroxisomes and mitochondria in the liver. This proliferation does not change the ratio of phospholipid to protein in mitochondria or microsomes, but causes certain changes in the fatty acid composition of the phospholipids. The highest rates of peroxisomal and mitochondrial beta-oxidation are obtained with 12:0 and 16:0 fatty acids as substrates, respectively. A 3-4 fold increase in the rate of beta-oxidation by both organelles is caused by DEHP treatment, but there are no qualitative changes in the relative rates of oxidation of individual fatty acids. Short- and medium-chain carnitine acyltransferases in peroxisomes, microsomes and mitochondria, as well as the mitochondrial long-chain carnitine acyltransferase are induced to various extents. These results indicate that the increased beta-oxidation of fatty acids caused by phthalate treatment involves the same peroxisomal and mitochondrial pathways which operate under normal conditions.