Hasan Mukhtar

trans-Stilbene oxide: an inducer of rat hepatic microsomal and nuclear epoxide hydrase and mixed-function oxidase activities.

The administration of trans-stilbene oxide to rats resulted in increased hepatic microsomal and nuclear epoxide hydrase (with styrene oxide (SO), benzo[a]pyrene 4,5-oxide (4,5-BP) as substrates) and aryl hydrocarbon hydroxylase (AHH) activities. Hepatic microsomal aminopyrine N-demethylase, benzphetamine N-demethylase, and ethylmorphine N-demethylase activities were also increased. These increases in microsomal enzyme activity were dose- and time-dependent (about 100% at 200 mg/kg body weight, administered for 2 consecutive days). However, only marginal increases in hepatic microsomal NADPH-cytochrome c reductase activity and cytochrome P-450 content were observed. No apparent proliferation of hepatic endoplasmic reticulum occurred in trans-stilbene oxide pretreated rats. The administration of trans-stilbene oxide has no effect on hepatic glutathione S-transferase activities (with SO or 4,5-BPO as substrates). None of the parameters were affected in pulmonary microsomes from treated rats. The in vitro addition of trans-stilbene oxide (10(-6)--10(-2) M) did not affect hepatic epoxide hydrase or glutathione S-transferase activities.

Arachidonic acid-dependent metabolism of (±) trans-7,8-dihydroxy-7,8- dihydro-benzo[a]pyrene (BP-7,8 diol) to 7,10/8,9 tetrols

The environmental pollutant benzo [alpyrene (BP) is not carcinogenic per se but requires metabolic activation by the cytochrome P-450-dependent monoxygenase system for the manifestation of carcinogenic activity [ 1,2]. The activation of BP results in the generation of reactive metabolites which can covalently bind to DNA. The covalent binding of some reactive BP intermediates to DNA is implicated with carcinogenesis [3,4]. Recent evidence suggests that the diol epoxides are the ultimate carcinogenic form of BP [4,5]. The pathway of diol epoxide formation involves epoxidation and hydration of BP catalyzed by the cytochrome P-450 monooxygenase system-dependent and epoxide hydrase . We have reported that BP, BP-7,8-diol and 7,12dimethyl benzanthracene are oxidized by guinea pig lung microsomes during prostaglandin (PC) biosynthesis to reactive metabolites which can covalently bind to tissue protein and nucleic acids [6]. The nature of the metabolites of BP-7,8-diol formed during PG biosynthesis is not known. The metabolites of NP-7,8-diol but not BP formed during PG biosynthesis by ram seminal vesicle microsomes are highly mutagenic to Salmonella typhimurium [7]. Therefore, we have investigated the nature of the metabolites generated from BP-7,8diol during PG biosynthesis in guinea pig lung (GPL) and ram seminal vesicle (RSV) microsomes. Our studies indicate 7,10/89-BP tetrol as the major metabolite formed by both tissues.

Epoxide hydrase and mixed-function oxidase activities of rat liver nuclear membranes.

Abstract Rat liver nuclei have 2 to 12% of the corresponding microsomal aryl hydrocarbon hydroxylase, aminopyrine and benzphetamine N -demethylase, NADPH-cytochrome c reductase, and epoxide hydrase activities. Nuclear membranes were prepared from isolated liver nuclei by a sucrose density centrifugation technique. A 2.5- to 10.2-fold increase in the specific enzyme activities was observed in nuclear membrane as compared to intact nuclei. Several properties of the rat liver nuclear membrane and microsomal epoxide hydrase have been compared. Nuclear epoxide hydrase was similar to the corresponding microsomal enzyme in being induced by phenobarbital whereas 3-methylcholanthrene did not produce any effects. Nuclear membrane and microsomal epoxide hydrase were inhibited to a similar degree by 1,1,1-trichloropropene oxide, cyclohexene oxide, an trans -stilbene oxide. The apparent K m value of nuclear membrane epoxide hydrase was 20 μ m for benzo( a )pyrene 4,5-oxide, which is 5.5-fold lower than the corresponding microsomal K m value (112 μ m ). Nuclear membranes were prepared from isolated nuclei of rat kidney, lung, spleen, and heart by the DNase digestion method. Epoxide hydrase activity in intact nuclei was in the following order: kidney > lung ⋙ spleen, or heart. Increases of 2.2- and 2.5-fold in specific epoxide hydrase activity were observed in kidney and lung when nuclear membranes were compared to intact nuclei. DMSO, dimethylsulfoxide

Serum glutathione S-transferases: Perinatal development, sex difference, and effect of carbon tetrachloride administration on enzyme activity in the rat☆

Abstract Glutathione S -transferase activity was determined in rat, rabbit, and guinea pig serum using styrene 7,8-oxide (SO) and benzo (a) pyrene 4,5-oxide (4,5-BPO) as substrates. Of the species tested, rat had the highest transferase activity (62.5 and 3.2 nmol/min/ml serum for SO and 4,5-BPO, respectively) and rabbit had the lowest activity. Glutathione S -transferase activity was 60% higher in serum from male rats than in female rats. In rats, serum enzyme specific activities (nmol/min/mg protein) were less than 1% of hepatic enzyme activities with SO, 4,5-BPO, 1,2-dichloro-4-nitrobenzene (DCNB), and 1-chloro-2,4-dinitrobenzene (DNCB). Glutathione S -transferase activity was also determined in rat serum during perinatal development. Serum from rats at 18 days of gestation or from 1- and 4-day-old animals had barely detectable transferase activity. Activity increased with age and reached a maximum in 140-day-old animals. The intraperitoneal administration of diethyl maleate (DEM) (0.8 ml/kg) or L-methionine-DL-sulfoximine (MS) (200 mg/kg) to male rats had no effect on serum or hepatic glutathione S -transferase activities 2 or 26 hr after dosing. Treatment with carbon tetrachloride (CCl4) (1 m1/kg) caused an 11-fold increase in serum transferase activity and a 40% decrease in liver specific activities 24 hr after administration.

Epoxide metabolizing enzyme activities in rat testes: postnatal development and relative activity in interstitial and spermatogenic cell compartments.

Microsomal epoxide hydrase (EH) and 176 000 g supernatant fraction glutathione-S-transferase (GSH-S-T) activities were determined with styrene oxide as substrate in rat testes during postnatal development. The development of these enzymes was also followed in liver for comparison. Testes of 6-day-old rats had high GSH-S-T activities (66 nmol/min/mg protein), which were about 50% of the adult levels. Transferase activity then developed slowly and reached a maximum by 165 days of age. Specific testicular GSH-S-T activities of 6-day-old rats were 3--4 times those of hepatic GSH-S-T activities in the same animals. In contrast, EH activities of both liver and testes were very low in prepubertal rats, but they increased dramatically at the onset of puberty and reached maximum activities by 45 days of age. Microsomal and microsomal supernatant fractions prepared from adult rat spermatogenic cells had about twice the EH and GSH-S-T specific activities (with styrene oxide or benzo[a]pyrene 4,5-oxide as substrates) of similar fractions prepared from interstitial cells. On the other hand, benzo[a]pyrene hydroxylase (AHH) activity and cytochrome P-450 content were at least 2-fold greater in microsomes from interstitial cells than in those from spermatogenic cells.

Effects of vitamin A deficiency on hepatic and extrahepatic mixed-function oxidase and epoxide-metabolizing enzymes in guinea pig and rabbit

Abstract Male guinea pigs and male rabbits were fed a vitamin A deficient diet for 9 weeks and for 12 weeks respectively. Hepatic levels of vitamin A were significantly reduced in the vitamin A deficient animals. The activities of some xenobiotic-metabolizing enzymes were measured in the liver, lung and small intestine. Aryl hydrocarbon hydroxylase, aniline hydroxylase, and 7-ethoxycoumarin deethylase activities were decreased in the vitamin A deficient guinea pig liver. However, in the guinea pig small intestine, aniline hydroxylase, 7-ethoxycoumarin deethylase, aminopyrine demethylase, and aryl hydrocarbon hydroxylase specific activities were increased. In rabbits, vitamin A deficiency decreased hepatic aniline hydroxylase and 7-ethoxycoumarin deethylase activities but increased intestinal aminopyrine demethylase activity. Enzyme activities in lung were not altered by vitamin A deficiency in guinea pig or rabbit. Microsomal epoxide hydrase and microsomal supernatant glutathione S -transferase activities in the three tissues of both species were not altered by vitamin A deficiency.

Epoxide-metabolizing enzyme activities and cytochrome P-450 content of rat ovaries during pregnancy.

Abstract Glutathione S -transferase activity was determined in 176,000 g supernatant fraction of ovaries, adrenals, and liver and in the serum of virgin, pregnant, lactating, and post-lactating female rats. A 90% increase in glutathione S -transferase specific activity was observed in the ovaries of pregnant rats whereas activity in the other tissues was not affected by pregnancy. The increase in ovarian glutathione S -transferase activity was not a function of the estrous cycle of the rat. The activity reached a maximum in midpregnancy and then remained constant until late pregnancy. The specific content of cytochromes P-450 and b5 in microsomes prepared from rat ovaries was induced 3.7- to 4.7-fold during mid- and late pregnancy. No concomitant changes occurred in mitochondrial cytochrome P-450 concentrations. Microsomal aryl hydrocarbon hydroxylase and epoxide hydrase activities of ovaries from pregnant rats were not affected.

Glutathione S-transferase activities in rat and mouse sperm and human semen

Glutathione S-transferase activity was found in sperm of the rat and DBA2J and C57 BL6J mice. In rat sperm activities with benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, and 1-chloro-2,4-dinitrobenzene were 0.88, 1.07, and 26.1 nmoles/min/mg protein, respectively. Δ5-3-Ketosteroid isomerase activity of rat sperm was 4.9 nmoles/min/mg protein. These specific glutathione S-transferase and Δ5-3-ketosteroid isomerase activities in sperm represent 0.4–4.1% of rat liver cytosol values. Human semen also contained significant glutathione S-transferase activity. It is postulated that these enzymes could function in the metabolism and detoxification of certain electrophilic xenobiotics, if present in sperm.

Thyroid hormone-induced changes in the hepatic monooxygenase system, heme oxygenase activity and epoxide hydrolase activity in adult male, female and immature rats

In 8-day-old rat pups, pretreatment with a single injection of L-triiodothyronine or L-thyroxine decreased hepatic cytochrome P-450 content, aminopyrine N-demethylase activity and epoxide hydrolase activity but increased hepatic microsomal cytochrome c reductase, 7-ethoxyresorufin O-deethylase and heme oxygenase activities without significantly altering UDP-glucuronosyltransferase activity (towards o-aminophenol) or the microsomal yield. In adult rats of either sex such single injections of L-triiodothyronine failed to significantly alter these enzyme activities. However, multiple injections evoked changes similar to those observed in the pups, in all these enzyme activities, except that 7-ethoxyresorufin O-deethylase activity was slightly decreased rather than increased. These findings demonstrate that: (1) The hepatic monooxygenase system in the rat pup is more responsive to thyroid hormones than that in adult. (2) Thyroid hormones can decrease rat liver cytochrome P-450 content and its dependent monooxygenase activity independently of sexual maturity. (3) Thyroid hormones also decrease hepatic epoxide hydrolase activity in both pups and adults. Thus, hyperthyroidism could render the rat pup more susceptible to hepatotoxicity from electrophilic epoxides which utilize microsomal epoxide hydrolase as the major detoxication pathway.