Induction of rat hepatic epoxide hydratase by dietary antioxidants.

Supplementation of rat diet with butylated hydroxytoluene (BHT), butylated hydroxyanisole, or ethoxyquin resulted in increased liver epoxide hydratase activity. The increase was obvious at 0.1% and amounted to 200–400% at 0.5%. Increased activity was accompanied by increased proportion of the epoxide hydratase band in SDS polyacrylamide gels, indicating induction of the enzyme. Ethoxycoumarin deethylase activity and cytochrome b₅ concentrations were moderately elevated while cytochrome P-450 concentrations and aryl hydrocarbon hydroxylase activity remained at control levels. Preferential inhibition of monooxygenase activity by metyrapone and not 7,8-benzoflavone, as well as increased affinity of the reduced cytochrome P-450 for metyrapone as a ligand, indicated that the cytochrome P-450 population after BHT treatment was similar to that found after phenobarbital treatment. The antioxidants used in this study had no in vitro effect on epoxide hydratase activity and inhibited monooxygenase activity only in phenobarbital-stimulated microsomes but not in 3-methylcholanthrene-stimulated microsomes. Combined treatment with dietary antioxidants and intraperitoneally administered 3-methylcholanthrene resulted in marked induction of epoxide hydratase activity while the 3-methylcholanthrene-mediated increase in aryl hydrocarbon hydroxylase activity was partially depressed. Covalent binding of tritiated benzo[a]pyrene to calf thymus DNA was less effectively catalyzed by liver microsomes from animals fed antioxidants. The depression of covalent binding was marked after combined treatment with antioxidants and 3-methylcholanthrene. The shift in the microsomal enzyme pattern caused by antioxidants may be related to their inhibitory effects on chemical carcinogenesis.     

Regulation of rat liver epoxide hydratase activity by phenobarbital and 3-methylcholanthrene.

Phenobarbital-pretreatment of rats increased liver microsomal epoxide hydratase activity 2.6-fold over controls even after elimination of inherent latency problems. However. 3-methylcholanthrene pretreatment of rats does not alter the levels of hepatic epoxide hydratase activity. Epoxide hydratase was purified from control rats or rats pretreated with phenobarbital or 3-methylcholanthrene. The enzymes isolated from all three sources appear to be very similar in size, immunological activity and specific activity. These experiments strongly suggest that phenobarbital stimulates epoxide hydratase activity by selectively increasing microsomal content of a single form of the enzyme. The possible existence of multiple forms of epoxide hydratase is discussed.     

The apparent ubiquity of epoxide hydratase in rat organs

Using the recently developed sensitive assay with [³H] benzo [a] pyrene 4,5-oxide as substrate, epoxide hydratase was shown to be present in 26 rat (Sprague-Dawley) organs and tissues investigated. Only blood showed no detectable activity, which indicates that the low enzyme activity found in some organs is not due to the presence of blood components in the tissues. In earlier studies with a less sensitive assay, epoxide hydratase activity was detected only in rat liver and kidney but not in organs such as muscle, spleen, heart and brain. Epoxide hydratase was also measured in 6 organs of the mouse (NMRI). The distribution pattern was quantitatively quite different in the two species. The sp. act. in the rat were in the order liver > testis > kidney > lung > intestine ～- skin. In the mouse, very surprisingly, testis had the highest specific epoxide hydratase activity. Moreover, the order of sp. act. in the mouse organs was remarkably different from that in the rat, namely testis > liver > lung > skin > kidney > intestine. The fact that the sp. act. in kidney was much lower than in lung or skin is most striking. Pretreatment of rats with Aroclor 1254 (a mixture of polychlorinated biphenyls) increased the epoxide hydratase activity in the liver to 175 per cent of the control level. However, the enzyme activity in the 13 extrahepatic tissues investigated was not significantly changed. In organs possessing sufficiently high enzyme levels, epoxide hydratase activity was also measured with styrene oxide as substrate. The ratio of the sp. act. of the two substrates was very similar in rat liver, kidney, lung and lestis. This supports the assumption that in these organs a single enzyme is responsible for the hydration of both substrates—as was earlier shown by several methods for the rat liver.     

Effects of dietary R-goitrin on hepatic and intestinal glutathione S-transferase, microsomal epoxide hydratase and ethoxycoumarin O-deethylase activities in the rat

The influence of dietary R-goitrin on components of the xenobiotic-metabolizing system was examined in the liver and small intestine of male Sprague-Dawley rats. Given at a level of 200 ppm in the diet for 14 days, the R-goitrin caused a statistically significant (P less than 0.05) 21% increase in liver weight relative to body weight. A less pronounced, but statistically significant, 11% increase in relative liver weight resulted from the administration of R-goitrin at 40 ppm in the diet. Hepatic glutathione S-transferase (GST) activity was significantly increased 1.5- and 2-fold over the basal level at concentrations of 40 and 200 ppm R-goitrin, respectively. Hepatic microsomal epoxide hydratase (EH) activity was also significantly increased. Hepatic EH activity was 1.6- and 3.3-fold greater in the 40- and 200-ppm R-goitrin groups, respectively, than in the control group given the basal diet. R-Goitrin at 200 ppm in the diet produced significant 1.2- and 1.4-fold increases of GST and microsomal EH activities, respectively, in the mucosa of the small intestine. The administration of R-goitrin at 40 or 200 ppm in the diet had no significant effect on either hepatic or intestinal ethoxycoumarin O-deethylase activity.     

Drug metabolism in skin. Comparative activity of the mixed-function oxidases, epoxide hydratase, and glutathione S-transferase in liver and skin of the neonatal rat

Aryl hydrocarbon hydroxylase (AHH) activity in the skin of neonatal rats was compared to that in epidermis, dermis, and other body tissues. Following topical application of benzo[a]pyrene (BP) or the polychlorinated biphenyl mixture Aroclor 1254 there was induction of AHH in each tissue studied. There was a greater increase in the activity of skin enzyme as compared to other extrahepatic tissues. When whole-organ activity (pmol of 3-OH-BP per min per whole organ) was considered, skin represented 2%, 21%, and 27% or whole body activity in control, BP-treated, and Aroclor 1254-treated animals, respectively. Skin microsomes from control rats exhibited 2, 0.5, 24, and 6% of corresponding liver microsomal AHH, 7-ethoxycoumarin de-ethylase, NADPH-cytochrome c reductase, and epoxide hydratase activities, respectively. Glutathione S-transferase activity in skin cytosol was 15% of the corresponding hepatic activity. Following topical application of Aroclor 1254 there were increases in the activity of AHH and 7-ethoxycoumarin de-ethylase in the skin and the liver. Glutathione S-transferase (40-60%) and epoxide hydratase (83-94%) activities in neonatal rat liver were induced by skin application of Aroclor 1254.     

The effect of dietary protein and fat on the activity of aryl hydrocarbon hydroxylase in rat liver,kidney and lung

Feeding low protein diets reduces the activity of aryl hydrocarbon hydroxylase (3,4 benzo(α)pyrene hydroxylase) in rat liver and lung, but not in the kidney. In the kidney the level of aryl hydrocarbon hydroxylase activity is increased by feeding a diet containing fats. This increase in the kidney aryl hydrocarbon hydroxylase activity occurs when the fat content of the diet rises above 4 per cent and is maximal after feeding such diets for 7 days.     

Effect of dietary antioxidants on benzo[a]pyrene metabolism in rat liver microsomes

Feeding of rats with 1% ethoxyquin (EQ) and butylated hydroxytoluene (BHT) but not butylated hydroxyanisole (BHA) increases the formation rate of benzo[a]pyrene (BP)-4,5-dihydrodiol from BP in hepatic microsomes. The production of other BP-dihydrodiols and of BP phenols is decreased after treatment with EQ, BHT and BHA. EQ and BHT are more effective than BHA in inducing epoxide hydrolase (EH) activity towards styrene oxide as the substrate.     

The effect of aspartame on the activity of rat liver xenobiotic-metabolizing enzymes

Male, Wistar rats were administered aspartame (40 or 4000 mg/kg body weight) in their diet for 90 days. By 45 days, the activities of three microsomal enzymes, epoxide hydrolase, carboxylesterase, and p-nitrophenyl-UDP-glucuronosyltransferase, were significantly increased in rats consuming 4000 mg/kg of aspartame. By 90 days, however, the activity of the xenobiotic-metabolizing enzymes of the rats given aspartame did not differ significantly from the activity of control animals. From these results, we conclude that the consumption of aspartame does not substantially alter the function of the hepatic microsomal enzymes which protect the organism from foreign compounds found in its environment and food.     

Effect of dietary clofibrate on epoxide hydrolase activity in tissues of mice

The effects of dietary clofibrate on the epoxide-metabolizing enzymes of mouse liver, kidney, lung and testis were evaluated using trans-stilbene oxide as a selective substrate for the cytosolic epoxide hydrolase, cis-stilbene oxide and benzo[a]pyrene 4,5-oxide as substrates for the microsomal form, and cis-stilbene oxide as a substrate for glutathione S-transferase activity. The hydration of trans-stilbene oxide was greatest in liver followed by kidney greater than lung greater than testis. Its hydrolysis was increased significantly in the cytosolic fraction of liver and kidney of clofibrate-treated mice and in the microsomes from the liver. Isoelectric focusing indicates that the same enzyme is responsible for hydrolysis of trans-stilbene oxide in normal and induced liver and kidney. Clofibrate induced glutathione S-transferase activity on cis-stilbene oxide only in the liver. Hydrolysis of both cis-stilbene oxide and benzo[a]pyrene 4,5-oxide was highest in testis followed by liver greater than lung greater than kidney. Hydration of cis-stilbene oxide was induced significantly in both liver and kidney by clofibrate but that of benzo[a]pyrene 4,5-oxide was induced only in the liver. These and other data based on ratios of hydration of benzo[a]pyrene 4,5-oxide to cis-stilbene oxide in tissues of normal and induced animals indicate that there are one or more novel epoxide hydrolase activities which cannot be accounted for by either the classical cytosolic or microsomal hydrolases. These effects are notable in the microsomes of kidney and especially in the cytosol of testis.     

Specificity of human, rat and mouse skin epoxide hydratase towards K-region epoxides of polycyclic hydrocarbons

Epoxide hydratase activity has been measured in microsomal fractions of skin from mouse, rat and humans. The skin enzyme was able to hydrate all epoxides tested. The specific enzyme activities decreased in the order human > mouse > rat. The relative activity towards K-region epoxides of various polycyclic hydrocarbons in skin microsomal fractions from all three species decreased in the order phenanthrene 9,10-oxide > benz(a)anthracene 5,6-oxide ? benzo(a)pyrene 4,5-oxide ? 7-methylbenz(a)anthracene 5,6-oxide > 3-methylcholanthrene 11,12-oxide > dibenz(a,h)anthracene 5,6-oxide. The activity of epoxide hydratase in human skin microsomal fractions showed little pH dependence and was inhibited by small molecular weight inhibitors in a manner similar to that of the liver microsomal enzyme. Interindividual variation of epoxide hydratase activity in skin microsomal fractions from six human subjects was considerable, namely from 175 to 447 pmoles benzo(a)pyrene 4,5-dihydrodiol/min per mg protein. This variation was not due to skin disease or treatment and had no apparent correlation with age or sex. A possible correlation with the part of the body from which the skin sample was taken could not be excluded since the activity in skin samples from the abdomen seemed lower than that in samples from leg or breast.     

The effect of dietary fatty acids on the composition of adult rat lung lipids: Relationship to oxygen toxicity

Three groups of mature rats were placed on separate dietary regimens for a period of 33 days. One group was fed standard rat food, the second group was fed a diet high in saturated fatty acids provided by hydrogenated coconut oil, and the third group was fed a diet high in polyunsaturated fatty acids provided by cod liver oil. After 33 days of maintenance on these diets major changes in the fatty acid composition of lung triglycerides were observed. Fatty acids with two or more double bonds comprised 27.9% of the total lung triglyceride fatty acids in rats fed standard rat food, 24.7% in the rats fed cod liver oil, and 2.8% in the rats fed coconut oil. The fatty acid composition of lung phospholipids showed smaller changes which were confined to shifts in the relative content of arachidonic and docosahexaenoic acids in those rats fed cod liver oil. Exposure of all three groups of rats to 100% oxygen resulted in a greatly enhanced mortality in the group fed coconut oil compared with the other two groups. All of the rats fed coconut oil were dead within 68 hr, while, after 96 hr, only 54% of those fed either standard rat food or cod liver oil were dead. These data suggest that an increase in the saturated fatty acid content of lung triglycerides through dietery manipulation results in increased susceptibility to oxygen toxicity.     

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.     

Effect of dietary lipids on levels of UDP-glucuronosyltransferase in liver.

Others have shown recently that dietary fish oil protects against acetaminophen-induced liver injury in vivo. Fish oil was protective because it increased the clearance of acetaminophen via glucuronidation. This work left unresolved the basis for increased rates of glucuronidation in animals fed fish oil. We therefore have determined how the amount and type of lipid in the diet affect the activity of liver microsomal UDP-glucuronosyltransferase activity. Male Wistar rats were fed a fat-free diet or isocaloric diets containing 5% corn oil, olive oil or fish oil for 4 weeks. The activity of UDP-glucuronosyltransferase was highest in rats fed fish oil and lowest in rats fed the fat-free diet. Treatment with corn oil and olive oil resulted in intermediate levels of activity. Diet-induced differences in amounts of UDP-glucuronosyltransferase were shown by immunoblotting and kinetic measurements. Treatment with fish oil resulted in a 3-fold increase in the amount of UDP-glucuronosyltransferase versus the fat-free diet. Corn oil and olive oil diets caused 2-fold increases in the amount of UDP-glucuronosyltransferase versus the fat-free diet. Fatty acid analysis of microsomal lipids showed that the fat-free diet was associated with decreased levels of arachidonic acid versus the corn oil or olive oil diets. The fish oil diet resulted in increased levels of omega-3 fatty acids versus the other diets.     

Soluble epoxide hydrolase in rat inflammatory cells is indistinguishable from soluble epoxide hydrolase in rat liver.

Soluble epoxide hydrolase (sEH) is a ubiquitous mammalian enzyme for which liver and kidney are reported to have the highest activity. We have shown that the soluble epoxide hydrolase (sEH) activity present in rat neutrophils and macrophages is kinetically, immunologically, and physically indistinguishable from rat liver cytosolic sEH. Cytosol from rat liver or inflammatory cells and recombinant rat sEH were incubated with trans-diphenylpropene oxide (tDPPO), a selective substrate for sEH. The tDPPO hydration activity we observed in inflammatory cell cytosol was lower than that from liver. The Km for tDPPO hydration observed in rat inflammatory cell cytosol was the same as the Km for rat liver cytosol (10 microM). Recombinant rat sEH and cytosol from rat liver or inflammatory cells were incubated with the sEH inhibitors, chalcone oxide, 4-fluorochalcone oxide, and 4-phenylchalcone oxide. The IC50 values were 40, 8, and 0.4 microM, respectively, in all samples. Furthermore, sEH activity could be completely immunoprecipitated out of the samples, and the amount of antibody required to do so was apparently identical, regardless of the source of enzyme. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis revealed a single sEH band with a molecular weight of 62 kDa. Isoelectric focusing followed by Western blot analysis revealed multiple bands containing tDPPO-hydrating activity. Although the inflammatory cell bands had the same pattern as those from liver cytosol, the recombinant sEH showed a different banding pattern. These multiple bands were not an artifact of the IEF gel selected. Furthermore, in a 2-dimensional IEF gel, the bands re-migrated to the same position. The presence of sEH in inflammatory cells suggests that this enzyme may have an important endogenous function.