Effect of triton X-100 on the conjugation of tetrahydrocortisone, in vitro

The detergent, Triton X-100, increased the conjugation of estrone, estradiol and tetrahydrocortisone (THE) by uridine diphosphate glucuronyl transferase (UDPGT) in rat liver microsomes; the maximal increase of the conjugation of these substrates was measured when the concentration of Triton in the incubation mixtures was 0.05 per cent. However, the magnitude of the increase and the effect seen with varying Triton concentration were substrate dependent, which is consistent with the hypothesis that multiple forms of UDPGT may be present in hepatic microsomes. The effects of various compounds which had previously been shown to either increase or decrease the conjugation of THE in non-activated enzyme preparations were re-examined in Triton-activated preparations. Compounds such as β-diethylaminoethyldiphenylpropylacetate (SKF-525A) and 7-hydroxychlorpromazine which inhibited conjugation in non-activated preparations also inhibited conjugation in Triton-activated preparations. Alternatively, the demethylated metabolites of chlorpromazine, which increased activity in non-treated preparations, decreased activity slightly in preparations maximally stimulated by Triton. Bisubstrate kinetic analysis of the THE conjugation of UDPGT also revealed differences between the properties of the non-treated and Triton-activated enzyme preparations. Triton activation caused an increase in the V_max of the reaction in the forward direction while having an insignificant effect on the dissociation constant for THE.     

Ascorbic acid deficiency and hepatic UDP-glucuronyltransferase

The effect of dietary ascorbic acid on hepatic microsomal UDP-glucuronyltransferase (UDPGT) activity towards p-aminophenol, bilirubin, and acetaminophen was investigated. Ascorbate deficiency produced a 33% reduction in the specific activity of UDPGT towards p-aminophenol, whereas there was no difference between microsomes from ascorbate-deficient and supplemented guinea pigs in the activity towards bilirubin and acetaminophen. This suggests that the effect of the vitamin is on a specific isozyme. This reduction was correlated with the reduced quantity of hepatic microsomal cytochrome P-450, which has been previously reported for ascorbate-deficient guinea pigs. No difference was found in the apparent affinity for the substrate, p-aminophenol, or the cofactor, UDP-glucuronic acid. Differences in microsomal UDPGT activity towards p-aminophenol occurred between the two groups with membrane-perturbing processes such as sonication and Triton X-100. Sonication and magnesium chloride were found to increase activity 329% in ascorbate-supplemented animals and 138% in the ascorbate-deficient group. The addition of ascorbate acid in vitro, or its analog d-isoascorbic acid, could protect against the detrimental effects of excess substrate by maintaining a linear enzymatic rate over a 30-min time period; there was no significant effect on the initial rate of hepatic microsomal UDPGT activity in the ascorbate-supplemented animals whereas there was a significant increase in the ascorbate-deficient group. Glutathione was as effective as ascorbic acid in protecting against the detrimental effects of excess substrate whereas cysteine and dimethyltetrapteridine were only partially effective. Ascorbyl-2-sulfate and alpha-tocopherol had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes.

Prenylated chalcones from hops and beer were compared with non-prenylated flavonoids [chalconaringenin (CN), naringenin (NG), genistein (GS) and quercetin (QC)] for their ability to inhibit lipid peroxidation in rat liver microsomes. Chalcones with prenyl- or geranyl-groups (5 and 25 microM) were more effective inhibitors of microsomal lipid peroxidation than CN, NG or GS induced by Fe(2+)/ascorbate. Prenylated chalcones were effective inhibitors of microsomal lipid peroxidation induced by Fe(3+)-ADP/NADPH and by tert-butyl hydroperoxide (TBH) but to a lesser extent compared to the Fe(2+)/ascorbate system. An increase of prenyl substituents decreased antioxidant activity in the lipid peroxidation systems. Certain flavonoids behaved as prooxidants in the iron-dependent lipid peroxidation systems. For example, at 5 microM, NG enhanced iron/ascorbate-induced lipid peroxidation whereas CN, diprenylxanthohumol and tetrahydroxanthohumol enhanced Fe(3+)-ADP/NADPH-induced lipid peroxidation. None of the flavonoids (25 microM), except QC, inhibited NADPH cytochrome P450-reductase activity of rat liver microsomes, suggesting that the mechanism of inhibition of lipid peroxidation induced by Fe(3+)-ADP/NADPH is not due to inhibition of the reductase enzyme. Chalcones exhibiting antioxidant activity against TBH-induced lipid peroxidation such as xanthohumol and 5'-prenylxanthohumol, and NG, with no antioxidant property at 5 microM concentration protected cultured rat hepatocytes from TBH toxicity. Other antioxidants (desmethylxanthohumol and CN) in the TBH system were not cytoprotective. These results demonstrate the importance of prenyl groups in the antioxidant activity of hop chalcones in the various in vitro systems of lipid peroxidation. Furthermore, the antioxidant activity of the flavonoids has little or no bearing on their ability to protect rat hepatocytes from the toxic effects of TBH.     

Carbon disulphide induced activation of liver UDP glucuronosyltransferase in rats pretreated with phenobarbitone.

Carbon disulphide (CS2) exposure has been shown to activate the UDP glucuronosyltransferase of liver microsomes in rats pretreated with phenobarbitone. Now the nature of CS2 induced activation of the enzymes has been studied further. Phenobarbitone pretreated rats were exposed to 0.15% CS2 for 2 hrs on two successive days. The activity of UDP glucuronosyltransferase was measured from the liver microsomes after the enzymes was activated by incubation of the microsomes with various concentrations of the detergents Triton X-100, digitonin and cetylpyridinium chloride. The exposed animals showed an increased enzyme activity at all applied concentrations of the detergents; therefore in addition to membrane destruction by CS2 exposure, some other mechanism must also be involved in the CS2 induced activation of liver microsomal UDP glucuronosyltransferase. The changes in membrane lipid-protein interactions with l-anilino-8-naphthalene sulphonate (ANS) were also probed. The CS2 exposed animals had more high-affinity binding sites for ANS in their liver microsomal membranes, and in addition the quantum yield of ANS fluorescence was enhanced by CS2. The changes differed from those found after carbon tetrachloride exposure and suggest that, even if the two drugs have some common effects on microsomes, e.g. UDP glucuronosyltransferase activation, P-450 destruction and lipid peroxidation induction, the changes they cause in the microsomal micro-environment differ.     

Effect of N-methyltetrazolethiol on liver microsomal vitamin K reductase

NADH-dependent vitamin K reductase activity in rat liver microsomes was measured by detecting the amount of the reduced form of vitamin K from the oxidized form of the vitamin. The enzyme activity was not detected when intact microsomes were employed as the enzyme source, but the solubilization of the microsomal enzyme with 1.5% Triton X-100 caused a development of the activity. Although the enzyme activity decreased gradually with time after the solubilization, the enzyme was stabilized by the addition of 20% glycerol and 2 mM vitamin C. Some optimal assay conditions for the vitamin K reductase were determined using the solubilized enzyme, and the standard assay method is described. Vitamin K reductase activity was not affected by warfarin and N-ethylmaleimide (NEM), but pyridoxal-5-phosphate (PAL-P) inhibited the activity, especially when microsomes were preincubated with PAL-P. The enzyme activity was not inhibited by N-methyltetrazolethiol (NMTT) and NMTT-containing antibiotics, suggesting that the hypoprothrombinemia caused by beta-lactam antibiotics was not due to the inhibition of NADH-dependent vitamin K reductase.     

Ring- and N-Hydroxylation of 2-acetylaminofluorene by reconstituted mouse liver microsomal cytochrome P-450 enzyme system

The N- and ring-hydroxylation of 2-acetylaminofluorene (AAF) are examined with a reconstituted cytochrome P-450 enzyme system from liver microsomal fractions from both control and 3-methylcholanthrene (MC)-pretreated mice. Partial purification of cytochrome P-450 fraction is achieved by bacterial protease treatment of microsomes followed by Triton X-100 solubilization and ammonium sulfate precipitation. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions are required for optimum oxidative activity. Hydroxylation activity is determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from MC-pretreated mice is several fold more active than that from controls.     

Effects of detergents and organic solvents on rat liver microsomal UDP-glucuronosyltransferase activity towards phenolic substrates

1. The effects of several detergents (Brij 58, deoxycholate and Lubrol 12A9) and ether on the initial rate of UDP-glucuronosyltransferase activity towards fixed concentrations of five phenolic acceptor substrates of widely different octanol-buffer (pH 7.4) partition coefficient have been compared with those observed in non-activated and Triton X-100-and n-pentane-activated rat liver microsomes.

2. Enzymic activity was dependent on the lipid-solubility of acceptor substrate. Each activator, except Triton X-100, enhanced enzymic activity towards all substrates by a similar factor, which was independent of the octanol-buffer partition coefficient. For Triton X-100 microsomes, the activation was also partition-dependent.

3. The highest activation factor was seen with ether. Pre-incubation of ether-activated microsomes for 30?min at 37°C before assay resulted in inactivation of the enzyme towards more water-soluble substrates. Tryptic digestion (30?min at 37°C) of the ether-activated microsomes resulted in marked reduction of enzyme activity towards all substrates.

4. Ether, and the two detergents, Brij 58 and Lubrol 12A9, released small amounts of protein (5-12% total present); both detergents also released some (8-12%) phospholipid.

5. The K^app_m towards acceptor substrate also depended on the octanol-buffer partition coefficient, and was largely unchanged on activation by n-pentane. V_mak was not dependent on partition coefficient and was significantly increased on activation.     

Beta-glucuronidase latency in isolated murine hepatocytes

The physiological function of microsomal beta-glucuronidase is unclear. Substrates may be either glucuronides produced in the lumen of endoplasmic reticulum (ER) or those taken up by hepatocytes. In the latter case, efficient inward transport of glucuronides at the plasma membrane and the ER membrane would be required. Therefore, the potential role of beta-glucuronidase in ER was investigated. Isolated mouse hepatocytes and mouse and rat liver microsomal vesicles were used in the experiments. Selective permeabilization of the plasma membrane of isolated hepatocytes with saponin or digitonin resulted in an almost 4-fold elevation in the rate of beta-nitrophenol glucuronide hydrolysis, while the permeabilization of plasma membrane plus ER membrane by Triton X-100 caused a further 2-fold elevation. In microsomal vesicles, the p-nitrophenol glucuronide or phenolphthalein glucuronide beta-glucuronidase activity showed about 50% latency as revealed by alamethicin or Triton X-100 treatment. A light-scattering study indicated that the microsomes are relatively impermeable to both glucuronides and to glucuronate. On the basis of our results, the role of liver microsomal beta-glucuronidase in the deconjugation of glucuronides taken up by the liver seems unlikely. Hydrolysis of the glucuronides produced in the ER lumen may play a role in substrate supply for ascorbate synthesis or in "proofreading" of glucuronidation.     

UDP-glucuronosyltransferase activity toward digitoxigenin-monodigitoxoside. Differences in activation and induction properties in rat and mouse liver

Glucuronidation of digitoxigenin-monodigitoxoside (DT1), a metabolite of the cardiac glycoside digitoxin, is mediated by the microsomal isozymes, UDP-glucuronosyltransferase(s) (UDP-GT). The present studies examined the activation and induction properties of UDP-GT activity toward DT1 in hepatic microsomes of rats and mice. When compared to enzyme activity present in native (latent) microsomes of the rat (0.104 +/- 0.010 nmol/min/mg of protein), the activity toward digitoxigenin-monodigitoxoside in mouse native microsomes was 3.5-fold higher (0.379 + 0.44 mumol/min/mg of protein). After treatment with ionic (sodium cholate), zwitterionic [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)], or nonionic (Emulgen 911, Triton X-100) detergents, or with UDP-N-acetylglucosamine, enzyme activity in rat microsomes remained unchanged. In contrast, UDP-GT activity (DT1) in mouse liver microsomes treated with detergents or with the nucleotide was increased 2-3-fold above native enzyme activity. Pretreatment of rats with the microsomal enzyme inducers, 3-methylcholanthrene and phenobarbital, had no effect on this enzyme activity, whereas pretreatment with pregnenolone-16 alpha-carbonitrile (PCN) and dexamethasone (DEX) increased enzyme activity toward DT1 800 and 380%, respectively. These findings support the hypothesis that PCN and DEX induce a unique form of UDP-GT in the rat that selectively glucuronidates DT1. In marked contrast, the activity of this enzyme in mouse liver was not affected by pretreatment with any of the microsomal inducers, including PCN and DEX. In both rat and mouse, the P-450p-dependent N-ethylmorphine demethylase activity was increased 10-15-fold in PCN-pretreated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ascorbic acid deficiency and hepatic UDP-glucuronyl transferase. Qualitative and quantitative differences

The effect of dietary ascorbate on hepatic UDP glucuronyltransferase (UDPGT) appears to be selective in that only certain isozymes of UDPGT are jeopardized. In this study, ascorbic acid deficiency produced a 68% reduction in the specific activity of hepatic UDPGT towards p-nitrophenol. Earlier studies showed a reduction in UDPGT activity towards p-aminophenol in ascorbate-deficient guinea pigs, whereas bilirubin and acetaminophen glucuronidation were unaffected. Kinetic studies suggest that p-aminophenol and p-nitrophenol are metabolized by a single isozyme in that p-nitrophenol was found to be a competitive inhibitor of p-aminophenol glucuronidation. Both qualitative and quantitative studies on partially purified UDPGT from ascorbate-deficient and ascorbate-supplemented guinea pigs were carried out to investigate the biochemical role of the vitamin. Qualitative differences were observed in UDPGT from ascorbate-deficient animals and included an increased lability to: thermal inactivation; storage at 4 degrees; and purification with UDP-glucuronic acid agarose column chromatography. Furthermore, an analysis of the microsomal membrane showed a 14% increase in membrane fluidity in ascorbate deficiency. Ascorbic acid added in vitro could not reverse the increase in fluidity observed in ascorbate-deficient microsomal membranes; however, ascorbylpalmitate, a more lipophilic form of the vitamin, was effective. Palmitic acid had no effect on membrane fluidity in microsomes from either the ascorbate-supplemented or ascorbate-deficient animals. This increase in membrane fluidity could not be explained by differences in cholesterol, total phospholipid, or phosphatidylcholine content of hepatic microsomes. Furthermore, a quantitative reduction in UDPGT partially purified from ascorbate-deficient guinea pigs was indicated by a marked reduction in protein banding at 55,000 daltons when compared to UDPGT partially purified from ascorbate-supplemented animals.     

Carbon Disulphide Induced Activation of Liver UDP glucuronosyltransferase in Rats Pretreated with Phenobarbitone

Abstract Carbon disulphide (CS₂) exposure has been shown to activate the UDPglucuronosyltransferase of liver microsomes in rats pretreated with phenobarbitone. Now the nature of CS₂ induced activation of the enzymes has been studied further. Phenobarbitone pretreated rats were exposed to 0.15% CS₂ for 2 hrs on two successive days. The activity of UDPglucuronosyltransferase was measured from the liver microsomes after the enzymes was activated by incubation of the microsomes with various concentrations of the detergents Triton X-100, digitonin and cetylpyridinium chloride. The exposed animals showed an increased enzyme activity at all applied concentrations of the detergents; therefore in addition to membrane destruction by CS₂ exposure, some other mechanism must also be involved in the CS₂ induced activation of liver microsomal UDPglucuronosyltransferase. The changes in membrane lipid-protein interactions with 1-anilino-8-naphthalene sulphonate (ANS) were also probed. The CS₂ exposed animals had more high-affinity binding sites for ANS in their liver microsomal membranes, and in addition the quantum yield of ANS fluorescence was enhanced by CS₂. The changes differed from those found after carbon tetrachloride exposure and suggest that, even if the two drugs have some common effects on microsomes, e.g. UDPglucuronosyltransferase activation, P-450 destruction and lipid peroxidation induction, the changes they cause in the microsomal micro-environment differ.     

Developmental changes in ANP-stimulated guanylyl cyclase activity enhanced by ATP in rat lung membrane fractions.

1. ANP (atrial natriuretic peptides)- or ANP/ATP-stimulated guanylyl cyclase activities were compared in adult (2 month old) and neonatal (5-7 day old) rat lung membrane fractions. 2. The enzyme activities of both membranes depended on the incubation time and ATP concentration: although the activities of both membranes were similar after a short incubation time (4 min), those in adult membranes were lower than those of neonatal membranes after longer incubation times (10 and 30 min) or at lower concentrations of ATP. 3. ANP/ATP gamma S-stimulated guanylyl cyclase activities, which were much higher than ANP/ATP-stimulated activities, were similar in both membranes. 4. ATPase activity of adult membranes was higher than that of neonatal membranes, suggesting that hydrolysis of ATP leads to a decrease of ANP/ATP-guanylyl cyclase activity in adult membranes. Triton X-100 enhanced and diminished ANP/ATP-stimulated guanylyl cyclase activities of adult and neonatal membranes, respectively, and thereby abolished the adult/neonatal difference in the membrane response to ATP. 5. ANP-stimulated activities of both membranes were much more activated by pre-incubation with ATP gamma S than those induced by simultaneous addition of ATP gamma S. The former activities were decreased to levels of the latter by Triton X-100. The latter activities were not affected by Triton X-100. 6. The present results suggested that conformation of lung plasma membranes is related to activation of the ANP receptor/guanylyl cyclase system.     

On non-specific inhibition of rat liver microsomal UDP glucuronyltransferase by some drugs

The effect of imipramine. desipratnine, harmol, harmalol and some monoamine oxidase inhibitors of the hydrazine type on rat liver UDP glucuronyltransferase (EC 2.4.1.17) activity has been investigated. Substrates of the enzyme were p-nitrophenol and bilirubin; with p-nitrophenol only Triton X-100 activated microsomes were used as enzyme source, with bilirubin both not-activated and Triton X-100 activated microsomes were used. The degree of inhibition obtained with the various inhibitors was dependant on the substrate used and on the pretreatment of the microsomes. It is concluded that most of the effects were caused by the action of the compounds on microsomal membrane structure, which affects UDP glucuronyltransferase activity, and that the physiological relevance of inhibition of the enzyme in vitro is questionable.     

Inhibition of acetaminophen glucuronidation by oxazepam

Glucuronidation of [³H]acetaminophen (pHAA) in mouse liver microsomes is enhanced about 3-fold by 0.025% Triton X-100. Oxazepam inhibits microsomal glucuronidation of pHAA, yielding apparent competitive kinetics in native microsomes. Phenobarbital-pretreatment has no effect on the microsomal glucuronidation of pHAA. MH₁C₁ rat hepatoma cells also glucuronidate pHAA, approximately 40 nmoles per mg cell protein per hr being conjugated at a concentration ol 1 mM. Oxazepam also inhibits pHAA glucuronidation in the cell culture system. Intrapcritoneal injection of mice with oxazepam 15 min before subcutaneous injection of pHAA significantly increases the plasma hall-life of pHAA.     

UDP-glucuronosyltransferase(s) activities towards natural substrates in rat liver microsomes. Kinetic properties and influence of triton X-100 activation

We studied the in vitro capability of hepatic microsomal UDP-glucuronosyltransferase (UDPGT) in male rats to conjugate 22 natural xenobiotics which are known to be excreted as glucuronides in vivo. We clearly demonstrated that the Vmax can range in a decreasing scale for the following families of aglycones: 7-hydroxylated coumarins greater than 2-naphthol and phenols greater than monoterpenoid alcohols greater than 4-hydroxylated coumarins. The Km app. cannot be arranged in the same scale. This suggests that the catalytic mechanism of UDPGT is dependent on the hydroxyl group reactivity rather than on the binding interaction at the active site expressed by the Km app. The effects of various concentrations of detergent (Triton X-100) were determined on specificity (apparent Km) and activity (Vmax). For the 22 aglycones we showed that activation caused a variation in the Vmax which was a function of the concentration in detergent. The maximum of this activation did not always correspond to the same detergent/protein weight ratio. The impact of activation on Km app. was less clear since the variations observed were slightly different.     

HPLC法测定利多卡因及其代谢产物估算人肝微粒体CYP3A的活性

目的　以利多卡因 (LDC)与其代谢产物的比值估算人肝微粒体中CYP3A的活性。方法　以 1 0g·L- 1微粒体蛋白浓度 3 7℃孵育利多卡因 60min ,以HPLC测定利多卡因及其代谢产物单乙基甘氨二甲基苯酰胺 (MEGX)和甘氨二甲基苯酰胺 (GX)的含量。结果　LDC、MEGX和GX的标准曲线方程分别为 ^Y =0 2 93 4X -0 0 0 5661(r =0 9997)、^Y =0 7913X -0 0 0 8916(r =0 9993 )和 ^Y =0 6799X -0 0 0 7770 (r =0 9985)。体外孵育的最佳条件为 2 0mg·L- 1的LDC在浓度为 1 0 g·L- 1的微粒体中 ,孵育 60min ,代谢产物与利多卡因的平均比值为 3 2 8。结论　(MEGX +GX) /LDC可用来估算人肝微粒体CYP3A的活性。     

The adaptation of mussels Crenomytilus grayanus to cadmium accumulation result in alterations in organization of microsomal enzyme-membrane complex (non-specific phosphatase)

The kinetic parameters (V(m), K(m) and slope) of membrane-bound microsomal non-specific phosphatase (NPase, with G6P as the substrate) from the digestive gland of unexposed and cadmium adapted (45 days for 100 μg Cd(2+)/l) mussels were investigated. In vivo and in vitro approaches were used. Adaptation of mussels (Crenomytilus grayanus) to cadmium resulted in a 1.6-fold increase in NPase activity. V(m) was increased by 1.6-fold, but K(m) was the same in terms of enzyme kinetics. This indicates that the total concentration of the enzymes in the digestive gland increased. Cd(2+) (1 mM) did not significantly alter the activity of the membrane-bound enzyme in vitro both for unexposed and for cadmium adapted mussels, meaning that cadmium ions are not a direct inhibitor of the membrane-bound enzyme in this concentration. The microsomal NPase activity in both unexposed and cadmium adapted mussels was inhibited by in vitro solubilization of microsomes with non-ionic detergent (Triton X100, 0.01%). This inhibition was uncompetitive for microsomes of unexposed mussels (K(m) decreased 3.1-fold). The most drastic events were observed in cadmium adapted mussels, where inhibition was mixed (K(m) decreased 7.2-fold). The simultaneous actions of detergent and cadmium ions did not alter NPase activity significantly in comparison with action of the detergent alone. The differences in the types and the extents of inhibition of the enzymes activity by membrane disordering agent (Triton X100) indicated that the enzyme-membrane complex (NPase) has been altered as a result of adaptation of mussels to cadmium accumulation. We conclude that the mussels produced a new enzyme-membrane complex, with the same K(m) as the previous complex, but with other detergent sensitivity and greater amounts. Thus, the adaptation capacity of this enzyme is reduced as result of adaptation of mussels to cadmium accumulation.     

Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues

Abstract

1.?Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure–metabolic activity relationship was examined.

2.?Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains.

3.?In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben.

4.?Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity.

5.?Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes.

6.?Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens.

7.?Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively.     

Kinetic characteristics of UDP-glucuronosyltransferases towards a dithiol metabolite of malotilate in hepatic microsomes of rats and rabbits

1. The kinetic activity of UDP-glucuronosyltransferases (UDPGT) towards a dithiol metabolite of malotilate, 2,2-di(isopropoxycarbonyl)ethylene-1,1-dithiol, was investigated using rat and rabbit hepatic microsomes. The thio-glucuronide formed was analysed by h.p.l.c. The Km values obtained using rat and rabbit UDPGT were 36.3 +/- 3.3 and 443 +/- 43 microM, respectively. The Vmax values were 7.14 +/- 0.61 and 29.2 +/- 6.4 nmol/min per mg (mean +/- SD, n = 3). 2. Phenobarbital, an inducer of the GT2 isoform of UDPGT, increased rat microsomal UDPGT activity towards the dithiol. In inhibitory studies, menthol and borneol (specific substrates for GT2a isoform) competitively inhibited glucuronidation of the dithiol. Thus it was concluded that formation of the thio-glucuronide was catalysed mainly by the GT2a isozyme of UDPGT, which is involved in glucuronidation of monoterpenoid alcohols.     

Covalent binding of [14C]methoxychlor metabolite(s) to rat liver microsomal components

[14C]Methoxychlor was incubated with NADPH-fortified liver microsomes from male rats, and covalent binding to microsomal components was determined. The binding process was markedly enhanced when microsomes from phenobarbital-treated rats were employed. However, when microsomes from methylcholanthrene-treated rats were used the level of binding was not significantly affected. Incubation in the presence of glutathione, cysteine, or ascorbate markedly diminished binding. Metyrapone and SKF 525-A, inhibitors of hepatic cytochrome P-450-linked monooxygenase activity, inhibited the binding. Also, ethylmorphine and hexobarbital, alternate substrates of the monooxygenase system, inhibited binding. There was no binding to microsomal components in the absence of NADPH or oxygen. TCPO (1,1,1-trichloropropane-2,3-oxide), an inhibitor of epoxide hydrase activity, failed to enhance the binding process. However, N,N'-diphenyl-p-phenylenediamine (NDP) and n-propyl gallate (PG), both free radical scavengers, decreased binding at micromolar concentrations without altering the extent of formation of polar [14C]methoxychlor metabolites. It was concluded that methoxychlor undergoes a hepatic microsomal monooxygenase(s)-mediated activation and that the resultant reactive metabolites (possibly free radicals) bind covalently to microsomal components. By contrast, the binding resulting from the incubation of an impure mixture of polar [14C]methoxychlor metabolites with liver microsomes did not require NADPH and O2 and was not affected by NDP, Pg, ascorbate, or heat-treatment of microsomes. This finding suggested that the binding subsequent to the initial metabolic activation of methoxychlor does not require further enzymatic transformation. However, whether the binding with metabolites represents the same chemical species as the binding with [14C]methoxychlor remains to be established.