Metabolism of tridiphane (2-(3,5-dichlorophenyl)-2(2,2,2-trichloroethyl)oxirane) by hepatic epoxide hydrolases and glutathione S-transferases in mouse

The transformation of the herbicide tridiphane (Tandem, Dowco 356, 2-(3,5-dichlorophenyl)-2(2,2,2-trichloroethyl)oxirane by the epoxide-metabolizing enzymes, epoxide hydrolases (EH) and glutathione S-transferases (GST), was investigated in mouse liver microsomes and cytosol. The microsomal EH catalyzed the formation of tridiphane diol. The production of this metabolite was prevented by cyclohexene oxide at 1 mM, a known inhibitor of microsomal EH. The structure of the diol was verified by comparison of retention time or Rf of the compound with those of an authentic standard using gas-liquid chromatography or thin-layer chromatography techniques. The diol formed a diester with 1-butane boronic acid or an aldehyde with lead tetraacetate. Mass spectral analysis supported the structural assignment. After optimization of the assay conditions, kinetic constants for the hydration of tridiphane by the microsomal EH were determined (Km = 65 microM and Vmax = 0.9 nmol/min/mg protein). Dietary exposure of mice to the hypolipidemic drug clofibrate at a dose of 0.5% (w/w) for 2 weeks increased by 173% the metabolism of tridiphane to tridiphane diol by the microsomal fraction. No diol could be detected following incubation of tridiphane with the cytosolic EH, even after induction by clofibrate. Tridiphane was also a substrate for GST, but administration of clofibrate did not change the specific activity for the formation of the glutathione conjugate. The herbicide was a rather weak inhibitor of the microsomal EH and the cytosolic GST activities measured with cis-stilbene oxide and trans-stilbene oxide as substrates with I50's of 3.0 x 10(-5) and 1.8 x 10(-4)M, respectively. Tridiphane diol was a poor inhibitor of the enzymes studied, and the glutathione conjugate of tridiphane caused marked inhibition of only the GST activity (I50, 2.0 x 10(-5)M). By contrast the activity of cytosolic EH (trans-stilbene oxide) was relatively insensitive to the addition of tridiphane or of tridiphane metabolites.     

Microsomal and cytosolic epoxide hydrolase and glutathione S-transferase activities in the gill, liver, and kidney of the rainbow trout, Salmo gairdneri. Baseline levels and optimization of assay conditions

Microsomal and cytosolic epoxide hydrolase (mEH and cEH respectively) and glutathione S-transferase (GST) activities were measured in the liver, kidney, and gills of rainbow trout. Assays were optimized for time, pH, and temperature, using trans-stilbene oxide (TSO) and cis-stilbene oxide (CSO) as substrates for cEH and mEH, respectively. Optimal pH values for mEH, cEH, and GST were similar to mammalian values (i.e. 8.5, 7.5, and 9). Temperature optima differed between tissues and cell fractions. Specific activity of cEH-TSO was 3-14 times greater than mEH-CSO for all three tissues, and 8-60 times greater on a tissue weight basis. Liver and, to a lesser extent, kidney mEH were active against benzo[a]pyrene 4,5-oxide, whereas gill mEH was not active against this substrate. Liver cytosolic GST was active against CSO and 1-chloro-2,4-dinitrobenzene (CDNB) but not TSO, whereas gill and kidney cytosolic GST were active only against CDNB. Liver and kidney microsomal GST were active against CDNB, but no activity was found in gill microsomes. The results are discussed in relation to possible endogenous substrates and uninduced xenobiotic metabolizing capacities of different trout tissues.     

Effect of aging on testicular epoxide hydrolase and glutathione-S-transferase activities in mice

The effect of aging on epoxide hydrolase (EH) and glutathion-S-transferase (GST) activities was investigated in testes of C57BL/6 mice 1-30 months of age. Microsomal EH (mEH) activity, as monitored with cis-stilbene oxide (CSO), showed statistically insignificant changes throughout the lifespan of mice. Although cytosolic EH (cEH) was detected in testes by immunoblotting, the enzyme activity towards trans-stilbene oxide (TSO) could not be measured under the experimental conditions used. Gonadal GST monitored with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate displayed an increasing trend until the mice reached senescence, showing a 3.7-fold increase in the enzyme activity in old animals (30 months) when compared with that in young animals (2 months). However, with CSO as substrate, GST showed no change in activity in mice of different ages.     

Activities of hepatic epoxide hydrolase and glutathione S-transferase in rats under the influence of tetramethyl thiuramdisulfide, tetramethyl thiurammonosulfide or dimethyl dithiocarbamate

The epoxide hydrolase (EH) activity in the liver of adult female Wistar rats significantly increased 18 h after the administration by gavage of tetramethyl thiuramdisulfide (TMTD, 1 mmol/kg) or tetramethyl thiurammonosulfide (TMTM, 2 mmol/kg). No increase was observed 5 h after administration of Na-dimethyl dithiocarbamate (Na-DMDTC, 4 mmol/kg). The glutathione S-transferase (GST) activity in the cytosol and microsomes of the liver was slightly enhanced after oral (gavage) administration of TMTD, TMTM or Na-DMDTC (doses up to 4 mmol/kg). In vitro, TMTD, TMTM, and Na-DMDTC significantly enhanced the hepatic activity of EH prepared from adult female Wistar rats. Cytosolic and microsomal GST activities from the liver were significantly raised in vitro by Na-DMDTC. The results have a bearing on the evaluation of the risk to health of these chemicals in the workplace.     

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.     

Kinetics of propylene oxide metabolism in microsomes and cytosol of different organs from mouse, rat, and humans

Kinetics of the metabolic inactivation of 1,2-epoxypropane (propylene oxide; PO) catalyzed by glutathione S-transferase (GST) and by epoxide hydrolase (EH) were investigated at 37 degrees C in cytosol and microsomes of liver and lung of B6C3F1 mice, F344 rats, and humans and of respiratory and olfactory nasal mucosa of F344 rats. In all of these tissues, GST and EH activities were detected. GST activity for PO was found in cytosolic fractions exclusively. EH activity for PO could be determined only in microsomes, with the exception of human livers where some cytosolic activity also occurred, representing 1-3% of the corresponding GST activity. For GST, the ratio of the maximum metabolic rate (V(max)) to the apparent Michaelis constant (K(m)) could be quantified for all tissues. In liver and lung, these ratios ranged from 12 (human liver) to 106 microl/min/mg protein (mouse lung). Corresponding values for EH ranged from 4.4 (mouse liver) to 46 (human lung). The lowest V(max) value for EH was found in mouse lung (7.1 nmol/min/mg protein); the highest was found in human liver (80 nmol/min/mg protein). K(m) values for EH-mediated PO hydrolysis in liver and lung ranged from 0.83 (human lung) to 3.7 mmol/L (mouse liver). With respect to liver and lung, the highest V(max)/K(m) ratios were obtained for GST in mouse and for EH in human tissues. GST activities were higher in lung than in liver of mouse and human and were alike in both rat tissues. Species-specific EH activities in lung were similar to those in liver. In rat nasal mucosa, GST and EH activities were much higher than in rat liver.     

Altered enzyme activities of xenobiotic biotransformation in kidneys after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to rats

Activities of the xenobiotic metabolizing enzymes were measured in the liver, kidney, duodenum and lung microsomes and cytosol fractions of Wistar rats after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent bacterial mutagen in chlorinated drinking water. MX was administered by gavage at the dose level of 30 mg/kg for 18 weeks (low dose), or at the dose level which was raised gradually from 45 mg/kg for 7 weeks via 60 mg/kg for 2 weeks to a clearly toxic dose of 75 mg/kg for 5 weeks (high dose). Microsomal and cytosolic preparations were made and the activities of 7-ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD), NADPH-cytochrome-c-reductase, UDP-glucuronosyltransferase (UDPGT) and glutathione-S-transferase (GST) were measured. Kidneys were affected most. A dose-dependent decrease was observed in EROD (90% in males, 80% in females at the high dose) and in PROD (58% in females, at the high dose) in kidneys. An increase was, however, detected in kidney NADPH-cytochrome-c-reductase (66% in females at high dose), UDPGT (89% in males and 97% in females at high dose) and GST activities (56% in males and 50% in females at high dose). MX caused only a few changes in the enzyme activities of the liver. The EROD activity was decreased 25% to 37%, both in the livers of males and females, but the total content of P450s was not altered. Hepatic GST activity was elevated in females in a dose-dependent manner (31% and 44%). GST activity was elevated in duodenum in females (59%) at the high dose. There were no marked changes in the enzyme activities in the lungs. MX was a weak inhibitor of EROD activity both in the liver and kidney microsomes in vitro, decreasing the EROD activity by 53% and 43%, respectively at the concentration of 0.9 mM. The results indicate that MX decreases the activity of phase I metabolism enzymes, but induces phase II conjugation enzyme activities, particularly in kidneys in vivo. It is possible that these changes contribute to metabolism of MX in kidneys and renders them susceptible to MX in the course of repeated exposure.     

Differential induction of cytochrome P-450 by the enantiomers of trans-stilbene oxide

Optically pure (+)- and (-)-trans-stilbene oxide (TSO) enantiomers were administered to immature male Sprague-Dawley rats. (+)-TSO was the more potent inducer of liver microsomal cytochrome P-450-dependent monooxygenases. The greater potency of (+)-TSO may be explained on the basis of stereoselective metabolism since a far greater concentration of TSO was found in liver microsomes of (+)-TSO-treated rats. Furthermore, of the enzymes known to metabolize TSO, cytosolic epoxide hydrolase turned over the (-)-TSO enantiomer at a faster rate, consistent with the greater persistence of the (+)-enantiomer. Although this report is of chiral effects in potency of enzyme induction, stereoselective metabolism (i.e. disposition) rather than inherent structural characteristics (recognition) may be responsible for these effects.     

Age-related changes in the activities of epoxide hydrolases in different tissues of mice

Age-related changes in epoxide hydrolase (EH) activity in the liver, kidney, lung, and intestine were studied in male C57BL/6 mice of 1 through 30 months of age. Hepatic cytosolic EH activity increased until 15 months after which there was a decline of 59% during senescence (30 months). Hepatic EH activity in the mitochondrial fraction increased until 4 months and decreased thereafter with a 43% decline by 30 months. The hepatic microsomal EH activity increased until 6 months followed by decline of 32% by 30 months. All of these increases and declines were statistically significant. Renal cytosolic EH showed maximum activity at 6 months after which the activity decreased significantly with age. However, renal EH activity in the mitochondrial fraction, in general, did not change substantially with age. Although changes in renal microsomal EH activity were small, the decrease in activity at 9-18 months was significantly lower than at 1 month and 23-30 months. EH activity in the cytosolic, mitochondrial and microsomal fractions of kidney was 2.5-, 2-, and 10-fold less, respectively, than that found in similar subcellular fractions of liver. Cytosolic EH activity in lung and intestine and lung microsomal EH showed variations with age. The intestinal microsomal EH was not detectable under the experimental conditions used.     

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.     

Piperine effects on the expression of P4502E1, P4502B and P4501A in rat

Treatment of rat with piperine (PIP) (1·4 mmol/kg, 3 days ip injections) resulted in an approximate two-fold increase in total liver microsomal P450 content relative to that in uninduced animals.

2. 4-Nitrophenol and aniline hyroxylase activities in the hepatic microsomes prepared from rat treated with PIP decreased by 30 and 28% respectively as compared with control. Immunoblot analyses also revealed decreased P4502E1 levels in hepatic microsomes from PIP-treated animals.

3. In contrast with P4502E1 suppression, hepatic 2B1 and 2B2 levels were significantly increased in PIP-induced animals, as evidenced by both metabolic activity and immunoblot analysis of the liver microsomal fractions. The rate of hexobarbital hydroxylase activity in microsomes from PIP-treated animals was markedly elevated and was inhibited by approximately 62% in the presence of monoclonal anti-P4502B IgG. Immunoblot analyses demonstrated that P4502B1 ana 2B2 levels in hepatic microsomes from PIP-treated animals were comparable with those from phenobarbital-treated animals.

4. 7-Ethoxycoumarin deethylase activity was elevated approximately two-fold in PIP-induced animals and was 17% of that derived from 3-methylcholanthrene-induced animals. 7-ethoxycoumarin deethylase activity in PIP-induced hepatic microsomes was inhibited 63% in the presence of monoclonal anti-P4501 A antibody. Immunoblot analysis confirmed the increase in P4501A levels by PIP, which was 15% of that in hepatic microsomes from 3-methylcholanthrene-induced animals.

5. PIP treatment failed to affect microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GST) expression, as indicated by immunoblot analyses using polyclonal antibodies toward mEH and GST subunits Ya, Yb1, Yb2 and Yc.

6. These results demonstrate that PIP treatment suppressed P4502E1 expression and enhanced 2B and 1A expression, whereas this agent failed to affect hepatic mEH and GST expression.     

Identification of a glutathione S-transferase associated with microsomes of tumor cells resistant to nitrogen mustards

Walker 256 rat mammary carcinoma cells resistant to chlorambucil (WR) exhibited an approximate 4-fold increase in glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene as compared to the sensitive parent cell line (WS). WR cells maintained without biannual exposure to chlorambucil (WRr) reverted to the sensitive phenotype and possessed GST levels equivalent to WS. Mitochondria, microsomes and cytosol were isolated from WS, WR and WRr cell lines and analyzed for their GST composition. GST activity in each subcellular compartment of resistant cells was increased over the sensitive cells. Antibodies raised against total rat liver cytosolic GST crossreacted in resistant cells with two microsomal proteins (25.7 kD and 29 kD). The 29 kD protein was not detected in microsomal fractions from either WS or WRr and this protein was found to be dissimilar from cytosolic GST subunits in its isoelectric point (pI 6.7) and migration on two-dimensional polyacrylamide gels. In addition, the 29 kD microsome-associated GST from WR cells was immunologically distinct from a 14 kD GST subunit previously identified in rat liver microsomes. These data implicate the induction of a specific microsomal GST subunit in WR cells following drug selection and suggest its potential involvement in the establishment of cellular resistance to chlorambucil.     

Positional metabolism of benzo(a)pyrene in rat placenta and maternal liver. Comparison of induction effects

The biotransformation of [14C]benzo(a)pyrene (BP) was studied in vitro in the presence of microsomes prepared from isolated labyrinth and basal zone tissues of the rat placenta, as well as from maternal liver. Pregnant rats, day 14 of gestation, received beta-naphthoflavone (beta NF; 15 mg/kg, ip) or 3-methyl-cholanthrene (3MC; 30 mg/kg, ip). On day 15, placentae were dissected and microsomes were incubated with 17 microM [14C]BP and 2 mM NADPH. Metabolites formed in the incubation flasks were extracted and separated by HPLC utilizing a reverse phase column. Only trace BP metabolism occurred in basal zone microsomes from control, beta NF-, or 3MC-pretreated animals, as well as in labyrinth microsomes from control animals. In contrast, the preadministration of beta NF and 3MC increased labyrinth microsomal BP metabolism by 10- to 15-fold. Labyrinth and maternal liver microsomes from beta NF- and 3MC-treated animals actively converted BP to eight separate metabolites which co-chromatographed primarily with quinones and phenols. The overall formation of BP diol and phenolic metabolites by labyrinth microsomes was appreciably less than was observed for liver preparations. The very low activity of BP-4,5-oxide hydrolase in labyrinth microsomes compared to liver may in part explain the low level of formation of BP diols in placental microsomes. Labyrinth microsomes catalyzed the covalent binding of [3H]BP to calf thymus DNA, and this activity increased 5-fold following beta NF pretreatment. A comparison of induced tissues indicates that the amount of DNA binding in labyrinth microsomes is more extensive than would be expected by the level of total BP metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

Organ distribution of epoxide hydrolases in cytosolic and microsomal fractions of normal and nafenopin-treated male DBA/2 mice

Using trans-stilbene oxide and styrene oxide as substrates, epoxide hydrolase activities were measured in cytosolic and microsomal fractions from liver, kidney, heart, lung and testis of male DBA/2 mice. The activities towards these two substrates are remarkably organ specific: trans-stilbene oxide was most effectively hydrolyzed in subcellular fractions from liver, kidney and heart, whereas styrene oxide was predominantly hydrolyzed in those from liver, lung and testis. Immunoblotting experiments were performed with two polyclonal antibodies isolated from goat antisera. Using an anti-mouse liver cytosolic epoxide hydrolase antibody, the corresponding antigen protein was predominantly detected in both cytosolic and microsomal fractions from liver, kidney and heart. An anti-rat liver microsomal epoxide hydrolase antibody proved to be cross-reactive with the mouse enzyme and stained SDS-gels run with microsomal fractions from liver, lung and testis. The anti-mouse liver cytosolic epoxide hydrolase antibody precipitated cytosolic epoxide hydrolase activities from liver, kidney and heart cytosolic fractions. Dietary exposure to the hypolipidemic agent nafenopin (2000 ppm/10 days) caused an induction of trans-stilbene oxide hydrolase and styrene oxide hydrolase activities in cytosolic and microsomal liver fractions whereas, in the other organs, the same activities were unaffected by this treatment. This finding was in accordance with the increased amounts of antigen protein as detected with the antibodies in liver fractions from treated animals. The anti-mouse liver cytosolic epoxide hydrolase antibody was found to precipitate the whole trans-stilbene oxide hydrolase activity also from liver cytosol of nafenopin-treated mice, which indicates the presence of a single cytosolic epoxide hydrolase following induction.     

Induction of xenobiotic-metabolizing enzymes and peroxisome proliferation in rat liver caused by dietary exposure to di(2-ethylhexyl)phosphate

Exposure of rats to 1% or 3% (w/w) di(2-ethylhexyl)phosphate in the diet for five days results in two- to three-fold inductions of liver cytosolic epoxide hydrolase activity and microsomal cytochrome P-450 content. Cytochromes P-450b + e were induced 20- to 35-fold, but no increase was observed in cytochrome P-450c. Considerably smaller effects were obtained on NADPH-cytochrome c reductase, microsomal epoxide hydrolase and microsomal cytochrome b5 content, and there was no effect on cytosolic glutathione transferase activity, under the same conditions. A dramatic increase in cyanide-insensitive palmitoyl-CoA oxidation and total mitochondrial protein, together with smaller increases in total catalase and cytochrome oxidase activities, were observed after treatment with di(2-ethylhexyl)phosphate, indicating that this compound causes proliferation of both peroxisomes and mitochondria. It is suggested that the induction of cytosolic epoxide hydrolase and the proliferation of peroxisomes may be related processes.     

Biochemical characterization of the hepatic effects in mice and rats of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a hepatic neoplasm promoter

The biochemical effects on the liver of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), a phenobarbital-like enzyme inducer, were studied in the mouse and rat. In mice a single dose of TCPOBOP (3 mg/kg, ip) caused marked and long-lasting (at least 7 days) induction of liver cytochrome P-450, 7-ethoxycoumarin-O-deethylase, and epoxide hydrolase. TCPOBOP had much less effect in rats than in mice, even at a higher dose (30 mg/kg) TCPOBOP also induced DNA synthesis in mice and rats but ornithine decarboxylase activity only in mice. In addition, in mice but not in rats, TCPOBOP increased microsomal membrane fluidity, as detected by fluorescence polarization measurements.