Increase In Epoxide Hydrolase Activity Research Articles

4-Methylsulfanyl-3-butenyl isothiocyanate derived from glucoraphasatin is a potent inducer of rat hepatic phase II enzymes and a potential chemopreventive agent

The objective of this study was to establish whether the phytochemical glucoraphasatin, a glucosinolate present in cruciferous vegetables, and its corresponding isothiocyanate, 4-methylsulfanyl-3-butenyl isothiocyanate, up-regulate enzymes involved in the detoxification of carcinogens and are thus potential chemopreventive agents. Glucoraphasatin and myrosinase were isolated and purified from Daikon sprouts and Sinapis alba L., respectively. Glucoraphasatin (0-10μM) was incubated for 24h with precision-cut rat liver slices in the presence and absence of myrosinase, the enzyme that converts the glucosinolate to the isothiocyanate. The intact glucosinolate failed to influence the O-dealkylations of methoxy- and ethoxyresorufin or the apoprotein expression of CYP1 enzymes. Supplementation with myrosinase led to an increase in the dealkylation of methoxyresorufin, but only at the highest concentration of the glucosinolate, and CYP1A2 expression. In the absence of myrosinase, glucoraphasatin caused a marked increase in epoxide hydrolase activity at concentrations as low as 1μM paralleled by a rise in the enzyme protein expression; at the highest concentration only, a rise was also observed in glucuronosyl transferase activity, but other phase II enzyme systems were unaffected. Addition of myrosinase to the glucoraphasatin incubation maintained the rise in epoxide hydrolase and glucuronosyl transferase activities, further elevated quinone reductase and glutathione S-transferase activities, and increased total glutathione concentrations. It is concluded that at low concentrations, glucoraphasatin, either intact and/or through the formation of 4-methylsulfanyl-3-butenyl isothiocyanate, is a potent inducer of hepatic enzymes involved in the detoxification of chemical carcinogens and merits further investigation for chemopreventive activity.

Differential response of human and rat epoxide hydrolase to polycyclic aromatic hydrocarbon exposure: Studies using precision-cut tissue slices

The potential of polycyclic aromatic hydrocarbons (PAHs) to modulate microsomal epoxide hydrolase activity, determined using benzo[a]pyrene 5-oxide as substrate, in human liver, was evaluated and compared to rat liver. Precision-cut liver slices prepared from fresh human liver were incubated with six structurally diverse PAHs, at a range of concentrations, for 24h. Of the six PAHs studied, benzo[a]pyrene, dibenzo[a,h]anthracene and fluoranthene gave rise to a statistically significant increase in epoxide hydrolase activity, which was accompanied by a concomitant increase in epoxide hydrolase protein levels determined by immunoblotting. The other PAHs studied, namely dibenzo[a,l]pyrene, benzo[b]fluoranthene and 1-methylphenanthrene, influenced neither activity nor enzyme protein levels. When rat slices were incubated under identical conditions, only benzo[a]pyrene and dibenzo[a,h]anthracene elevated epoxide hydrolase activity, which was, once again accompanied by a rise in protein levels. At the mRNA level, however, all six PAHs caused an increase, albeit to different extent. In rat, epoxide hydroxylase activity in lung slices was much lower than in liver slices. In lung slices, epoxide hydrolase activity was elevated following exposure to benzo[a]pyrene and dibenzo[a,l]pyrene and, to a lesser extent, 1-methylphenanthrene; similar observations were made at the protein level. At both activity and protein levels extent of induction was far more pronounced in the lung compared with the liver. It is concluded that epoxide hydrolase activity is an inducible enzyme by PAHs, in both human and rat liver, but induction potential by individual PAHs varies enormously, depending on the nature of the compound involved. Marked tissue differences in the nature of PAHs stimulating activity in rat lung and liver were noted. Although in the rat basal lung epoxide hydrolase activity is much lower than liver, it is more markedly inducible by PAHs.

METABOLISM OF STYRENE OXIDE TO STYRENE GLYCOL IN ENRICHED MOUSE CLARA-CELL PREPARATIONS

Styrene is a widely used chemical that has been shown to cause lung tumors in mice but not in rats. Styrene toxicity appears to be related to its bioactivation to styrene oxide, and this occurs almost exclusively in Clara cells. An important pathway in the detoxification of styrene oxide is via epoxide hydrolase to yield styrene glycol. When mouse Clara cells were incubated with racemic styrene oxide, R-styrene glycol was the predominant metabolite, giving an R/S ratio of 3.6. When the pure styrene oxide enantiomers were used as substrates, the corresponding styrene glycols were the predominant but not exclusive metabolites. Activity was slightly higher with the S-styrene oxide than with the R-styrene oxide. Addition of reduced glutathione to the incubation medium resulted in an increase in epoxide hydrolase activity, perhaps by decreasing oxidative stress. Mouse Clara cells thus show the capacity for detoxifying styrene oxide.

Treatment of lactating rats with PCBs induces CYP1A1 and enhances the formation of BP 7,8-dihydrodiol, the proximate carcinogen of benzo(a)pyrene

1. 1. Treatment of pregnant and lactating rats with a single i.p. dose of 250 mg/kg body weight produced 5-fold and 2-fold increases, respectively, in hepatic P-450 concentrations using microsomes isolated from pregnant, neonatal, lactating and foetal rats. 2. 2. Concomitantly, 26-fold, 20-fold and 14-fold increases in neonatal, maternal and foetal ethoxyre-sorufin- O-deethylation (EROD), respectively were found, but only 2.5-fold increases could be determined using microsomes isolated from lactating rats. 3. 3. The metabolism of [ 3H]benzo(a)pyrene was increased 9-fold and 2-fold in pregnant and foetal rats, respectively, but only 2-fold increases were measured for lactating rats. 4. 4. Western blot analysis of microsomal proteins obtained from lactating rats showed significant CYP1A1 and CYP1A2 induction and for the same hepatic tissue 62-fold and 44-fold increases in cDNA hybridized CYP1A1 and CYP1A2 mRNA, respectively, were found. 5. 5. Treatment of lactating rats with PCBs resulted in enhanced formation of all BP-metabolites, but the ratio of diol to total BP-metabolites was more than 3-fold greater. 6. 6. The formation of the proximate carcinogen BP-7,8-dihydrodiol was 5-fold increased and a similar 3-fold increase in epoxide hydrolase activity was estimated for lactating rats. 7. 7. The results of the present study indicates that lactation protects, in part, against the inductive effect of PCBs, possibly by enhanced clearance of these chemicals via lactation.

Effects of m-dichlorobenzene and its sulfur-containing metabolites on hepatic drug-metabolizing enzymes in mice.

Sulfur-containing metabolites, 2, 4-dichlorophenyl methyl sulfoxide, 2, 4-dichlorophenyl methyl sulfone (2, 4-DCPSO2CH3), 3, 5-dichlorophenyl methyl sulfoxide and 3, 5-dichlorophenyl methyl sulfone (3, 5-DCPSO2CH3) were all found in the urine of both male and female mice given orally a 200 mg/kg dose of m-dichlorobenzene (m-DCB). m-DCB (200 mg/kg) given to mice of both sexes by either oral or intraperitoneal (i.p.) route increased the activities of aminopyrine N-demethylase (AD) and aniline hydroxylase, and the content of cytochrome P-450 in the liver microsomes. 3, 5-DCPSO2CH3 (100μmol/kg, i.p.) induced markedly the enzyme system in mice of both sexes. On the other hand, 2, 4-DCPSO2CH3 (100 μmol/kg, i.p.) decreased significantly the AD activity in the males and the cytochrome b5 content in the females. An i.p. administration of m-DCB, 2, 4-DCPSO2CH3 or 3, 5-DCPSO2CH3 also caused a marked increase in epoxide hydrolase activity in the livers of the male and female mice, except that 2, 4-DCPSO2CH3 had no effect on the enzyme activity in the females. Thus, the differences in the effects of 2, 4- and 3, 5-DCPSO2CH3 on the enzyme system were demonstrated and the sex differences in the effects of the sulfones were also found in mice.

Effect of length of exposure to malathion on xenobiotic biotransformation in male rat liver

The effect of exposure to malathion on several parameters of hepatic xenobiotic biotransformation was studied in male Sprague-Dawley rats. Groups of rats dosed i.p. daily for 1 or 2 weeks with 40 or 200 mg/kg malathion showed an increase in epoxide hydrolase activity (1 week, 200 mg/kg) and glutathione S-transferase activity (1 week, 200 mg/kg; 2 weeks 40 and 200 mg/kg). Aldrin epoxidation was decreased after 1 week of exposure to 200 mg/kg and by both dosage regimens after 2 weeks. After 9 weeks exposure to 40 mg/kg malathion administered i.p. 3 times per week, however, no changes in hepatic xenobiotic biotransformation were noted. The results demonstrate that only continuous exposure to high doses of malathion results in an induction of epoxide hydrolase and glutathione S-transferase activities. Inductive effects on hepatic cytochrome P-450 monooxygenase activity were not observed irrespective of whether exposure was short- or medium-term.

Determination of epoxide hydrolase activity in whole cells (human lymphocytes) and activation by benzoflavones

Epoxide hydrolase (epoxide hydratase, epoxide hydrase, E.C. 3.3.2.3.) activity so far has only been measured in subcellylar preparations. We show here that, with the highly lipophilic substrate ( 3H)-benzo( a )pyrene 4,5-oxide, the activity can be determined in intact cells. Whole human lymphocytes hydrolyze it at a similar rate to that in lymphocyte homogenate. We have previously reported that cultivation of lymphocytes in a medium containing 5,6-benzoflavone leads to an increase in epoxide hydrolase activity. We now demonstrate that this stimulation is due to enzyme activation and that enzyme induction does not contribute to this increase to any measurable extent. Moreover, both 5,6-benzoflavone and 7,8-benzoflavone activate epoxide hydrolase. This activation occurs not only in cell homogenate, but also - with a similar concentration-response relationship - in whole lymphocytes. Hence measurement of epoxide hydrolase activity in subcellular preparations reflects the activity in these intact cells. Furthermore, insofar as a concentration of 1 μM of the benzoflavones is sufficient to cause a measurable (10 to 20 %) activation, it appears likely that foreign compounds can activate epoxide hydrolase in man.