Effects of environmentally encountered epoxides on mouse liver epoxide-metabolizing enzymes

Male mice were treated (i.p.) for 3 days with 15 different environmentally encountered epoxides, and the effects of these compounds on liver microsomal and cytosolic epoxide hydrolase (mEH and cEH), glutathione S-transferase (mGST and cGST) and carboxylesterase (mCE) activities were determined. The epoxides included the pesticides: heptachlor epoxide, dieldrin, tridiphane, and juvenoid R-20458; the natural products: disparlure, limonin, nomilin, and epoxymethyloleate; the endogenous steroids: lanosterol epoxide, cholesterol-alpha-epoxide, and progesterone epoxide; and the industrial or synthetic epoxides: epichlorohydrin, araldite, trans-stilbene oxide, and 4'-phenylchalcone oxide. The pesticide epoxides were the most effective inducers of liver weight, microsomal protein, and the enzyme activities measured, with mEH and cEH activities towards cis-stilbene oxide (mEHcso and cEHcso), cGST activities towards four of five substrates, and mCE towards clofibrate (mCEclof) and p-nitrophenylacetate (mCEpna) increased following treatment with most of the pesticides. The synthetic epoxides increased some of the same activities, while the natural products, except for increases in cGST activities, and endogenous steroid epoxides were generally not inductive. cEH activity towards trans-stilbene oxide (cEHtso) was increased only following treatment with the peroxisome proliferator, tridiphane, but decreased following treatment with several of the epoxides, while microsomal cholesterol epoxide hydrolase (mEHchol) was increased only moderately by disparlure. Microsomes could effectively conjugate glutathione to chlorodinitrobenzene (mGSTcdnb) and cis-stilbene oxide (mGSTcso). These two activities were differentially induced by a few of the epoxides, suggesting that they may be selective substrates for different isozymes of mGST. Correlation coefficients were determined for the relative response of liver weight, subfraction protein, and enzyme activities. A relatively high correlation was found between the response of liver weight and cytosolic hydrolysis of trans-stilbene oxide (r = 0.73) and cis-stilbene oxide (r = 0.62), and cytosolic glutathione conjugation of dichloronitrobenzene (r = 0.66) and trans-stilbene oxide (r = 0.75). In addition, relatively high correlations were found between the different cGST activities, in particular for dichloronitrobenzene with trans-stilbene oxide (r = 0.89). These studies show that there exists a wide variation in the response of xenobiotic-metabolizing enzymes to environmentally encountered epoxides and that a fairly strong correlation exists between the increases in liver size and increases in certain cytosolic enzyme activities; they also suggest further studies concerning the possibility of an additional isozyme of mGST.     

Different induction of microsomal carboxylesterases, palmitoyl-CoA hydrolase and acyl-L-carnitine hydrolase in rat liver after treatment with clofibrate

The levels of hepatic carboxylesterases, including palmitoyl-CoA hydrolase and decanoyl-D,L-carnitine hydrolase, were studied in total homogenates and subcellular fractions prepared from the livers of male rats fed diets containing 0.3% clofibrate. The microsomal carboxylesterase as well as the fatty acyl-thioesterase are differently induced by clofibrate feeding. The specific activities of acetanilide carboxylesterase and decanoyl-D,L-carnitine hydrolase increased more than 3-fold in the microsomal fraction, compared to pellet-fed control animals. The microsomal activities of palmitoyl-CoA hydrolase and propanidid hydrolase were decreased by about 20 to 40% in clofibrate-treated rats. The specific clofibrate hydrolase activity remained unchanged after clofibrate administration, indicating that this microsomal carboxylesterase is not induced by its own substrate. The data suggest a different distribution of the differing carboxylesterase along the endoplasmic reticulum.     

The effect of hypolipidemic agents on the hepatic microsomal drug-metabolizing enzyme system of the rat. Induction of cytochrome(s) P-450 with specificity toward terminal hydroxylation of lauric acid

The effect of chronic dietary administration of the hypolipidemic agents, clofibrate, methylclofenapate, fenofibrate, and tibric acid, on the hepatic drug-metabolizing enzyme system of the albino rat has been studied. Each compound caused dose-dependent increase in liver size and cytochrome P-450 (methylclofenapate = fenofibrate = tibric acid greater than clofibrate). NADPH-cytochrome c reductase activity was increased only after clofibrate and methylclofenapate treatment. There was no overall increase in the metabolism of a number of commonly used model substrates in parallel with the cytochrome P-450 induction. Aminopyrine and ethoxyresorufin dealkylation, biphenyl 4-hydroxylation, testosterone 16 alpha-hydroxylation, and o-aminophenol and chloramphenicol glucuronidation showed no change or inhibition, whereas ethoxycoumarin and phenacetin dealkylation and testosterone 6 beta-hydroxylation were increased (only up to 2-fold). Using clofibrate as a representative of this class of pharmacological agent, the enzymatic changes were essentially reversed within 6 days after removal of drug from the diet. Clofibrate administration also increased liver size and, to a lesser extent, hepatic cytochrome P-450 content in the albino (CD-1) mouse but had no effect in the marmoset monkey. In the rat, clofibrate administration specifically increased the hepatic microsomal omega-hydroxylation of lauric acid approximately 28-fold, which contrasted with the specific increase in (omega - 1)-hydroxylation caused by phenobarbital administration. The specific increase in microsomal cytochrome P-450-mediated omega-oxidation of a medium length, straight chain, saturated fatty acid is similar to the documented increase in peroxisomal and mitochondrial fatty acid beta-oxidation caused by administration of hypolipidemic agents.     

Apparent induction of microsomal carboxylesterase activities in tissues of clofibrate-fed mice and rats

Treatment with 0.5% (w/w) dietary clofibrate, a peroxisome proliferator, for 14 days induced microsomal carboxylesterase activities for five substrates including malathion, clofibrate, diethylsuccinate, diethylphthalate, and p-nitrophenylacetate in liver and kidney of male Swiss-Webster mice and Sprague-Dawley rats. The induction was substrate, tissue, and species dependent. The carboxylesterase activity was induced in mouse from 1.2- to 2.2-fold (liver) and from 1.1- to 1.7-fold (kidney) depending upon substrate used. Analogous values from rat ranged from 1.0- to 1.4-fold (liver) and from 1.1- to 1.8-fold (kidney). Enzyme activities were either decreased or not affected in testes of treated mice and rats. Substituted trifluoroketones ("transition-state" inhibitors of carboxylesterase) were found to be very potent inhibitors of clofibrate-metabolizing carboxylesterase(s) and to be potentially useful in distinguishing among isozymes. The inhibition data suggested that changes in carboxylesterase activity following clofibrate treatment were both qualitative and quantitative.     

Differential increase of hepatic peroxisomal,mitochondrial and microsomal carnitine acyltransferases in clofibrate-fed rats

Hepatic peroxisomes, mitochondria and microsomes from control and clofibrate-treated animals were separated by isopycnic sucrose gradient centrifugation and the carnitine acyltransferase system studied in each of these organelles. Clofibrate treatment produced a 13-fold increase in the total activity of carnitine acetyltransferase and a 5-fold increase in carnitine octanoyl- and palmitoyl-transferase activities. The specific activities of the transferases in all three subcellular locations increased, but to different extents. Peroxisomal and microsomal carnitine acetyltransferases doubled in specific activity; the mitochondrial enzyme increased 10-fold. Peroxisomal, mitochondrial and microsomal carnitine octanoyltransferases all increased 3-fold in specific activity. Carnitine palmitoyltransferase, which is found only in mitochondria, increased 3-fold in specific activity. These differential increases changed the per cent distribution of total carnitine acetyltransferase from 50 per cent in the mitochondria of control livers to 90 per cent in treated livers. Peroxisomes from clofibrate-treated livers had a consistently greater isopycnic density in sucrose gradients. Total catalase activity increased 2-fold upon treatment and a greater percentage of it was found in the paniculate fractions. The specific activity of peroxisomal catalase and urate oxidase remained the same as in controls. Carnitine acetyl- and octanoyltransferases are the first reported enzymes whose peroxisomal specific activity increases with clofibrate treatment. Preliminary results of treatment with another membrane-inducing drug, phenobarbital, indicated no change in peroxisomal density, catalase distribution and activity, and no effect on the specific activities of the peroxisomal, mitochondrial and microsomal carnitine acyltransferases.     

Effect of clofibrate administration on the esterification and deesterification of steroid hormones by liver and extrahepatic tissues in rats

Treatment of rats with clofibrate markedly stimulated the liver microsomal esterification of estradiol, testosterone, pregnenolone, dehydroepiandrosterone, and corticosterone by acyl-CoA:steroid acyltransferase. This enzyme catalyzes the esterification of estradiol with long-chain fatty acids in both liver and extrahepatic tissues. In untreated control rats, brain had the highest acyltransferase activity per milligram of microsomal protein for estradiol esterification (3- to 4-fold higher than in the liver). Although, treatment of rats with clofibrate stimulated the esterification of estradiol by 9- to 14-fold in the liver, estradiol esterification in kidney, lung, brain, uterus, fat, and mammary glands was not increased, indicating that liver may be uniquely sensitive to induction of acyl-CoA:estradiol acyltransferase by clofibrate. In additional studies, esterase activity for hydrolysis of the oleoyl ester of estradiol was determined in control and clofibrate-treated rats. Clofibrate administration increased esterase activity by an average of 107% in fat and 70% in liver. The results indicate that treatment of rats with clofibrate stimulates the hepatic formation of highly lipophilic fatty acid esters that can be hydrolyzed in the liver and in extrahepatic tissues to the parent steroid hormone by a clofibrate-inducible esterase.     

Epoxide metabolism in the liver of mice treated with clofibrate (ethyl-alpha-(p-chlorophenoxyisobutyrate], a peroxisome proliferator

An increase in cytosolic epoxide hydrolase (cEH) activity occurs in the livers of mice treated with peroxisome proliferating-hypolipidemic-nongenotoxic carcinogens. As increases in activity of epoxide metabolizing enzymes may reflect the carcinogenic mechanism, a detailed comparison of the response of cEH, microsomal epoxide hydrolase (mEH), and cytosolic glutathione S-transferase (cGST) activities using the geometrical isomers trans- and cis-stilbene oxide as substrates has been performed in livers from mice treated with clofibrate (ethyl-alpha-(p-chlorophenoxyisobutyrate]. The maximal increase of cEH activity occurred at lower dietary doses of clofibrate (0.5%) and within a shorter time (5 days) than mEH and cGST (2%, 14 days) activity. After 14 days at 0.5% clofibrate, cEH, mEH, and cGST activities were 250, 175, and 165% and 290, 220, and 75% of control values in male and female mice, respectively. Withdrawal of clofibrate from the diet resulted in a reversion of activities to control values within 7 days. Clofibrate treatment shifted the apparent subcellular compartmentation of all three enzymatic activities with an increase in the ratio of soluble to particulate activity. In particular, the relative specific activity of all three enzymes decreased in the light mitochondrial (peroxisomal) cell fraction, and an increase of a mEH-like activity (benzo[a]pyrene-4,5-oxide and cis-stilbene oxide hydrolysis) in the cytosol occurred. Both the increase of cEH activity and the appearance of mEH-like activity in the cytosol are novel responses of epoxide metabolizing enzymes, which may be related to the novel cellular responses that follow clofibrate treatment, peroxisome proliferation, hypolipidemia, and nongenotoxic carcinogenesis.     

Subchronic Toxicity of Di(2-ethylhexyl)phthalate in Common Marmosets: Lack of Hepatic Peroxisome Proliferation, Testicular Atrophy, or Pancreatic Acinar Cell Hyperplasia

To evaluate the toxicological effect, di(2-ethylhexyl)phthalate(DEHP) was administered orally at 100, 500, and 2500 mg/kg tofour male and four female marmosets in each group for 13 weeks.Its potentials of hepatic peroxisome proliferation, testicularatrophy, and pancreatic acinar cell hyperplasia were evaluatedmore closely. Clofibrate, which potently causes peroxisome proliferationin rodents, was administered in like manner at 250 mg/kg asa reference drug. DEHP induced significant suppression of weightgain in males at 2500 mg/kg. However, the increase in livermass and hypertrophy of hepatocytes were not detected in organweight measurements or histopathological examination. The numberof peroxisomes, volume density, peroxisome morphology, and peroxisomalenzyme activities were not different from those in the controlgroup, though the males treated with 500 and 2500 mg/kg DEHPshowed 1.3- and 1.4-fold increases in mean peroxisome volume,respectively. In contrast, clofibrate induced 2.2 (in male)-and 1.9-fold (in female) increases in hepatic cyanide-insensitiveacyl CoA oxidation system activity, 1.2 (in male)- and 1.7-fold(in female) increases in hepatic carnitine-dependent acetyltransferaseactivity, and 1.8 (in male)- and 3.0-fold (in female) increasesof carnitine-dependent palmitoyltransferase activity. CytochromeP-450 contents tended to increase in all males and females administered500 and 2500 mg/kg of DEHP and clofibrate associated with theincrease in hepatic microsomal protein content, suggesting arelationship with the treatment The atrophic change in the testisor proliferative change in the pancreatic acinar cells seenin rodents were not seen histopathologically; also, no changeswere observed in testes weight, testicular zinc level, bloodlevels of testosterone and estradiol, pancreas weight, and bloodlevels of cholecystokinin. Finally, no changes considered tobe due to the administration of DEHP were noted in blood chemicalexamination or pathological examination of other organs.     

Hepatic epoxide hydrolase activities and their induction by clofibrate and diethylhexylphthalate in various strains of mice

The presence of epoxide hydrolase activity in cytoplasm, microsomes and mitochondrial fraction in livers from twelve strains of mice (AKR/J, A/J, BALB/cByJ, CBA/J, C3H/HeJ, G57BL/6J, C57BL/10J, DBA/2J, NZB/B1NJ, PL/J, SEC/1ReJ and SW), and the influence of orally administered clofibrate and di(2-ethylhexyl)phthalate (DEHP) (0.5 and 2%, respectively, in diet) on epoxide hydrolase activities, were studied. Significant differences in basal cytosolic epoxide hydrolase activity, which ranged from 5.6 to 11.2 nmol diol.min-1.(mg protein)-1 using trans-stilbene oxide (TSO) as substrate, were noted among the mice. The highest and lowest enzyme levels were observed in the A/J and DBA/2J strains respectively. Similarly, microsomal epoxide hydrolase activity, monitored with cis-stilbene oxide (CSO), varied with the mouse strain, with the highest and lowest microsomal epoxide hydrolase activity being observed in A/J and SW strains respectively. Variations were also noted in the epoxide hydrolase activity in the mitochondrial fraction (monitored with TSO) with the highest and lowest levels observed in C57BL/6J and SW strains respectively. Clofibrate or DEHP treatment induced both cytosolic and microsomal epoxide hydrolases in nearly all of the strains examined. In contrast, the hydrolysis of TSO by the mitochondrial fraction in these strains was either not affected or decreased by clofibrate or DEHP treatment. The induction of cytosolic epoxide hydrolase was found to range between 1.2- and 2.8-fold, with generally a higher level of induction in mouse strains with low basal levels of cytosolic epoxide hydrolase activity. This level of cytosolic epoxide hydrolase activity, monitored with TSO as substrate, closely reflected the level of cytosolic epoxide hydrolase protein detected by immunoblot. There were also no significant differences observed in the molecular weight, immunological characteristics, pH-dependence and heat stability of hepatic cytosolic epoxide hydrolase activities of control and clofibrate-treated mice from various strains. These results suggest that clofibrate and DEHP induce both cytosolic and microsomal epoxide hydrolases but not the epoxide hydrolase in the mitochondrial fraction.     

Distribution and inducibility of cytosolic epoxide hydrolase in male Sprague-Dawley rats

Cytosolic epoxide hydrolase (cEH) activity has been determined in liver and various extrahepatic tissues of male Sprague-Dawley rats using trans-stilbene oxide (TSO) and trans-ethylstyrene oxide (TESO) as substrates. Large interindividual differences in the specific activity of cytosolic epoxide hydrolase in the liver from more than 80 individual rats were observed varying by a factor of 38. In a randomly selected group of five animals liver cEH varied by a factor of 3.9 and kidney cEH by a factor of 2.7, whereas liver microsomal epoxide hydrolase and lactate dehydrogenase showed only very low variations (1.4- and 1.1-fold, respectively). The individual relative activity of kidney cEH was related to that of the liver. Cytosolic epoxide hydrolase activity was present in all of six extrahepatic rat tissues investigated. Interestingly specific activities were very high in the heart and kidney (higher than in liver), followed by liver greater than brain greater than lung greater than testis greater than spleen. TSO and TESO hydrolases in subcellular fractions of rat liver were present at highest specific activities in the cytosolic and the heavy mitochondrial fraction. As indicated by the marker enzymes, catalase, urate oxidase and cytochrome oxidase, this organelle-bound epoxide hydrolase activity may be of peroxisomal and/or mitochondrial origin. In the microsomal fraction, TSO and TESO hydrolase activity is very low, whereas STO hydrolase activity is highest in this fraction and very low in cytosol. In kidney, subcellular distribution is similar to that observed in liver. None of the commonly used inducers of xenobiotic metabolizing enzymes caused significant changes in the specific activities of rat hepatic cEH (trans-stilbene oxide, alpha-pregnenolone carbonitrile, 3-methylcholanthrene, beta-naphthoflavone, isosafrole, butylated hydroxytoluene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, dibenzo[a,h]anthracene, phenobarbitone). However, clofibrate, a hypolipidemic agent, very strongly induced rat liver cEH (about 5-fold), whereas microsomal epoxide hydrolase activity was not affected. Specific activity of kidney cEH was increased about 2-fold.     

Co-induction of microsomal cytochrome P-452 and the peroxisomal fatty acid beta-oxidation pathway in the rat by clofibrate and di-(2-ethylhexyl)phthalate. Dose-response studies

Male Wistar rats have been pretreated with either clofibrate or diethylhexylphthalate and the dose-dependency of induction of the microsomal, cytochrome P-452-driven fatty acid hydroxylase and peroxisomal fatty acid beta-oxidation system investigated. Both clofibrate and DEHP specifically induced (approximately 10-fold) the 12-hydroxylation of lauric acid in a dose-dependent manner and only marginally increased the associated 11-hydroxylase activity. This dose-dependent increase in fatty acid hydroxylase activity was accompanied by a similar ten-fold increase in the specific content of the cytochrome P-452 isoenzyme responsible for this activity, as assessed by an immunochemical-based ELISA method. Similarly, both clofibrate and DEHP induced the peroxisomal fatty acid beta-oxidation pathway in a dose-dependent manner. Furthermore, our results provide evidence that, after oral administration, clofibrate has a higher in vivo potency in inducing the above enzymes of fatty acid metabolism than is exhibited by DEHP. A correlation matrix analysis of the above data indicated a close association between the induction of microsomal cytochrome P-452 (and its associated fatty acid hydroxylase activity) and peroxisomal beta-oxidation enzymes, implicating a mechanistic inter-relationship between changes in fatty acid metabolising enzymes in these two hepatic subcellular organelles.     

Reduced activity of hepatic microsomal fatty acid chain elongation synthesis in clofibrate-fed rats.

Na-clofibrate dissolved in drinking water was administered at a dose of 12 mg per day per animal to adult male Wistar rats. After different periods of drug administration, measurement was made of the activity of hepatic acetyl-CoA carboxylase, fatty acid synthetase and both microsomal and mitochondrial fatty acid chain elongation systems. Data obtained indicate a significant reduction in all synthetic activities, with the exception of the mitochondrial, even after dialysis of the investigated subcellular fractions. This reduction was found to increase with increased drug-administration periods, reaching the maximum after 7–8 days. A similar effect was previously shown by clofibrate in vitro [7]. The present results indicate an increase of 66.4% in hepatic cyclic AMP level as well after seven days of drug feeding to rats. Similarly, both serum aspartate aminotransferase and alanine amino-transferase activities were found to increase by about 30% after 13 days. The hypothesis is advanced that in vivo clofibrate probably reduces the synthetic activities under investigation by firmly binding to active enzyme-protein sites. In addition, the possibility that also the altered cyclic AMP level induced by this drug is responsible for both the above reductions and other metabolic variations reported elsewhere should not be excluded.     

Effects of perfluoro fatty acids on xenobiotic-metabolizing enzymes, enzymes which detoxify reactive forms of oxygen and lipid peroxidation in mouse liver.

Male mice were exposed via their diet to perfluoro fatty acids of various chain-lengths (2-10 carbon atoms) at different doses (0.02 and 0.1% weight) and for different periods of time (2-10 days). Thereafter, we monitored effects on liver and body weights and a number of hepatic parameters, including mitochondrial protein content, microsomal contents of cytochromes P450 and b5, NADPH-cytochrome P450 reductase activity [measured as NADPH-cytochrome c reductase (EC 1.6.2.3)], microsomal and cytosolic epoxide hydrolase (EC 3.3.2.3) activities, cytosolic DT-diaphorase (EC 1.6.99.2), glutathione transferase (EC 2.5.1.18), glutathione peroxidase (EC 1.11.1.9) and superoxide dismutase (EC 1.15.1.1) activities, and levels of thiobarbituric acid-reactive material (as an indicator of lipid peroxidation) in the mitochondrial subfraction. The most dramatic changes observed were a 5-9-fold increase in mitochondrial protein, a 3-6-fold increase in the microsomal content of cytochrome P450, a 3-10-fold increase in cytosolic DT-diaphorase activity, an approximately 2-fold increase in cytosolic epoxide hydrolase activity and as much as a 60% decrease in the level of thiobarbituric acid-reactive compounds in the mitochondrial fraction. Smaller increases in microsomal epoxide hydrolase activity and decreases in cytosolic glutathione peroxidase activity were also observed. Of the perfluoro fatty acids tested, perfluorooctanoic acid caused the largest changes in the parameters examined here. Dietary exposure of mice to a 0.02% dose of this substance for 10 days results in a maximal or near-maximal effect in most cases.     

Hydrolysis of pyrethroids by human and rat tissues: examination of intestinal, liver and serum carboxylesterases

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are approximately 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts (approximately 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected. Together, these results demonstrate that extrahepatic esterolytic metabolism of specific pyrethroids may be significant. Moreover, hepatic cytosolic and microsomal hydrolytic metabolism should each be considered during the development of pharmacokinetic models that predict the disposition of pyrethroids and other esterified compounds.     

Effects of diispropyl 1, 3-dithiol-2-ylidene malonate (NKK-105) on the drug-metabolizing enzymes and fine structure of rat liver (author's transl)]

The mechanism of liver enlargement and anti-fatty liver effect of NKK-105 in the rat were investigated by the mesurement of drug-metabolizing enzyme activities and morphological changes in liver tissue detected using electron microscopy. A single administration of NKK-105(250, 500, 1000 mg/kg, p.o.) induced an apparent increase in liver weight. The elevation of aminopyrine demethylase activity and slight increase in microsomal cytochrome b5 and cytochrome P-450 content were seen with the administration of NKK-105. NKK-105 inhibited lipid peroxide formation in mitochondrial and microsomal fractions. Total lipid content of liver decreased at 12 hr after the administration of NKK-105. Lipid peroxide formation in mitochondrial and microsomal fractions was markedly inhibited by the addition of NKK-105 (1 X 10(-3)M), in vitro. Disarrangement of rough endoplasmic reticulum and increase in smooth endoplasmic reticulum were observed by the administration of NKK-105. The decrease in drug-metabolizing enzymes caused by CCl4 or ethionine was protected in the combination with NKK-105. NKK-105 markedly inhibited the elevation of lipid peroxide formation caused by CCl4 or ethionine. Similar effects on lipid peroxide formation were also obtained in vitro. These results suggest that the enlargement induced by NKK-105 indicates a functional not a toxic response. The inhibition of lipid peroxide formation in mitochondrial and microsomal fractions may thus play an important role in the mechanism of anti-fatty liver effect of NKK-105 on the CCl4 or ethionine-induced fatty liver.     

Comparison of gamma-glutamyl transferase induction by phenobarbital in the rat, guinea pig and rabbit

Serum and hepatic γ-glutamyl transferase (GGT) activities were correlated with the microsomal markers cytochrome P-450 and aminopyrine N-demethylase after i.p. injection of phenobarbital (PB) to rats, guinea pigs and rabbits. The response to PB in the regimen employed was greatest in the rabbit and least in the guinea pig. Great disparities were observed in the microsomal protein contents following PB administration to the three species, masking the responses of the other indices when these were related to protein contents rather than to tissue weights. The increased hepatic GGT activities in PB-treated guinea pigs and rabbits were reflected in increased serum activities of this enzyme; the hepatic and serum GGT activities showed an excellent correlation with cytochrome P-450 and aminopyrine N-demethylase activities, supporting the view that the changes in GGT activity were related to enzyme induction. Although hepatic GGT activity in PB-treated rats also showed good correlation with enzyme induction indices, activity of this enzyme in rat serum was undetectable in control and PB-treated animals. Analysis of ribosome-free microsomal proteins by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis confirmed the marked increase in three bands in the PB-treated rat, but quite different changes were noted in the guinea pig and the rabbit. Our results extend knowledge about the heterogeneous response to PB shown by different animal species. The data provide further evidence that GGT is a PB-inducible enzyme, and suggest that the rabbit is the best model for elucidating the relationship between enzyme induction and GGT activity occurring in several human clinical situations.     

Proliferation of peroxisomes and induction of cytosolic and microsomal epoxide hydrolases in different strains of mice and rats after dietary treatment with clofibrate

1. The effects of dietary clofibrate (0.5%, w/w, for 10 days) on seven inbred strains of mice--C57BL/6, C57BL/B10A(5R), ATL/OLA, C3H/HE/OLA, BALB/C, CBA/CA and A/J/OLA--and three strains of rats--Sprague-Dawley, Wistar and LOU/OLA--have been investigated. Liver weight, peroxisome proliferation, catalase activity, cytosolic, microsomal and mitochondrial epoxide hydrolase activities, cytochrome oxidase activity, microsomal cytochrome P-450 content and cytosolic glutathione transferase activity in liver were determined, together with cytosolic and microsomal epoxide hydrolase and cytosolic glutathione transferase activities in the kidneys. 2. In all cases peroxisome proliferation and induction of cytosolic epoxide hydrolase were observed in livers of rodents exposed to clofibrate. Thus, no non-responsive strains were found and further evidence for a coupling between these two phenomena was provided. In many cases significant increases in the liver microsomal cytochrome P-450 content and decreases in the hepatic cytosolic glutathione transferase activity were also seen. 3. High levels of cytosolic epoxide hydrolase were found in the rat kidney. In several strains of mice and rats renal cytosolic epoxide hydrolase activity was increased by clofibrate. 4. There were often considerable strain differences. However, in general mice had higher cytosolic epoxide hydrolase and glutathione transferase activities, whereas rats had higher microsomal epoxide hydrolase activities.     

Isolation of nuclear fractions from human fetal tissues. Ultrastructure and presence of epoxide hydrolase and aryl hydrocarbon hydroxylase

The aryl hydrocarbon hydroxylase (AHH) and epoxide hydrolase (EH) activities with styrene oxide and benzo[a]pyrene-4,5-oxide as substrates were investigated and compared in the nuclear and microsomal fractions isolated from the human fetal liver, adrenals, kidneys and lungs. The purity of the fractions was estimated by electron microscopy and found to be around 85% for the nuclear and 90% for the microsomal fractions. All tissues catalyzed the hydration of the two epoxides at significant rates. The EH followed Michaelis-Menten kinetics in all fractions. The highest activities were seen in the liver and the adrenals. The nuclear/microsomal ratios of the EH activity was tissue dependent, being highest in the kidneys and lungs. AHH was measurable in the microsomes of all investigated tissues. As to the nuclear fraction it was detectable only in the adrenals and the liver. The nuclear/microsomal ratio of AHH was four times higher in the adrenals than in the liver. It is concluded that not only the microsomal but also the nuclear fraction of several human fetal tissues have the potential of catalyzing formation and elimination of epoxides.     

Proliferation of peroxisomes and induction of cytosolic and microsomal epoxide hydrolases in different strains of mice and rats after dietary treatment with clofibrate

1. The effects of dietary clofibrate (0.5%, w/w, for 10 days) on seven inbred strains of mice—C57BL/6, C57BL/B10A(5R), ATL/OLA, C3H/HE/OLA, BALB/C, CBA/CA and A/J/OLA—and three strains of rats—Sprague-Dawley, Wistar and LOU/OLA—have been investigated. Liver weight, peroxisome proliferation, catalase activity, cytosolic, microsomal and mitochondrial epoxide hydrolase activities, cytochrome oxidase activity, microsomal cytochrome P-450 content and cytosolic glutathione transferase activity in liver were determined, together with cytosolic and microsomal epoxide hydrolase and cytosolic glutathione transferase activities in the kidneys.

2. In all cases peroxisome proliferation and induction of cytosolic epoxide hydrolase were observed in livers of rodents exposed to clofibrate. Thus, no non-responsive strains were found and further evidence for a coupling between these two phenomena was provided. In many cases significant increases in the liver microsomal cytochrome P-450 content and decreases in the hepatic cytosolic glutathione transferase activity were also seen.

3. High levels of cytosolic epoxide hydrolase were found in the rat kidney. In several strains of mice and rats renal cytosolic epoxide hydrolase activity was increased by clofibrate.

4. There were often considerable strain differences. However, in general mice had higher cytosolic epoxide hydrolase and glutathione transferase activities, whereas rats had higher microsomal epoxide hydrolase activities.