Cytochrome P450 isozymes responsible for the metabolism of toluene and styrene in human liver microsomes

1. Cytochrome P450 isozymes from Asian (31 Chinese subjects) and Caucasian (14 Finnish subjects) livers were examined for their roles in the metabolism of toluene (rates of benzyl alcohol, o- and p-cresol formation) and styrene (rates of styrene glycol formation). 2. For toluene, the overall rate of metabolism was higher in samples from Finnish than from Chinese subjects. At 0·20 mM toluene, the rate of o-cresol formation was significantly higher in Finnish microsomes than in Chinese ones. The formation rates of benzyl alcohol and p-cresol in Finnish samples were also higher than those of Chinese samples, but only at a high substrate concentration (5·0 mM). For styrene metabolism, the Chinese liver microsomes showed higher metabolic rates than the Finnish ones at 0·085 mM styrene, but not at the higher substrate concentration. 3. Mean expression levels of immunochemically detected CYP1A2 1 and CYP2B6 were almost 3-fold higher in Finnish microsomes, whereas CYP2E1 was 1 7-fold higher in Chinese samples. 4. Correlation analysis showed that CYP2E1 (benzyl alcohol formation) and CYP1A2/1 (o-cresol formation) contributed to the metabolism of toluene at the low substrate concentration, whereas CYP2C8 was the form more actively involved at the higher toluene concentrations. At the higher concentration (1·8 mM) of styrene, CYP2B6 was most active isozyme to catalyse the formation of styrene oxide from styrene. 5. These results suggest that CYP2E1 and CYP1A2/1 are the main isoforms responsible for the metabolism of toluene at low substrate concentrations in human liver microsomes, CYP2E1 at low styrene concentration, and CYP2C8 and CYP2B6 at high concentrations of toluene and styrene respectively.     

Immunochemical detection of cytochrome P450 isozymes induced in rat liver byn-hexane, 2-hexanone and acetonyl acetone

Cytochrome P450 isozymes induced in rat liver by treatment withn-hexane, 2-hexanone and acetonyl acetone (given intraperitoneally 5 mmol/kg for 4 days) were investigated using enzyme assays (benzene, toluene, 7-ethoxyresorufin and 7-pentoxyresorufin metabolism) and monoclonal antibodies (anti-P450IA1/2, anti-P450IIB1/2, anti-P450IIC11/6, anti-P450IIE1(91) and anti-P450IIE1(98)).n-Hexane treatment enhanced the activities of low-K _m benzene aromatic hydroxylase and toluene side-chain oxidase, but not 7-ethoxyresorufin O-deethylase or 7-pentoxyresorufin O-depentylase. 2-Hexanone or acetonyl acetone treatment enhanced the activities of low-and high-K _m benzene aromatic hydroxylases, toluene side-chain oxidase and 7-pentoxyresorufin O-depentylase, but not of 7-ethoxyresorufin O-deethylase. Immunoblot analysis showed that anti-P450IA1/2 did not bind liver microsomal protein from either control and treated rats in the region of cytochrome P450s, whereas with anti-P450IIE1(98) a clear-cut band was seen in liver microsomes from control and treated rats, with intensities in the following order: 2-hexanone=acetonyl acetone n-hexane > control > phenobarbital. With anti-P450IIB1/2, a band was detected in microsomes from phenobarbital-treated rats, and to a lesser extent, in microsomes from 2-hexanone-and acetonyl acetone-treated rats. Like the immunoblot analysis, anti-P450IIE1(91) inhibited toluene side-chain hydroxylase activity in all microsomes, except in preparations from phenobarbital-treated rats and anti-P450IIB1 in microsomes from phenobarbital-, 2-hexanone- and acetonyl acetone-treated rats. Anti-P450IIC11/6 also inhibited toluene side-chain hydroxylase activity: the inhibited activity in the five different microsome preparations was as follows:n-hexane=control > acetonyl acetone=2-hexanone=phenobarbital. These results indicate thatn-hexane induces only quantitative alterations in the constitutive cytochrome P450 isozyme (P450IIE1), whereas its metabolites 2-hexanone and acetonyl acetone induce not only quantitative changes in constitutive cytochrome P450 (P450IIE1 and P450IIC11/6) but also a different type of isozyme (P450IIB1/2).     

Identification of Human UDP-Glucuronosyltransferase Responsible for the Glucuronidation of Niflumic Acid in Human Liver

Purpose To assess the uridine diphosphate (UDP)-glucuronosyltransferase (UGT) isozymes involved in the glucuronidation of niflumic acid in human liver. Methods The glucuronidation activity of niflumic acid was determined in liver microsomes and recombinant UGT isozymes by incubation of niflumic acid with UDP-glucuronic acid (UDPGA). Results Incubation of niflumic acid with liver microsomes and UDPGA produced one peak, which was identified as a glucuronide from mass spectrometric analysis. A study involving a panel of recombinant human UGT isozymes showed that glucuronidation activity was highest in UGT1A1 among the isozymes investigated. The glucuronidation in human liver microsomes (HLMs) followed Michaelis-Menten kinetics with a K_m value of 16 μM, which is similar to that found with recombinant UGT1A1. The glucuronidation activity of niflumic acid in microsomes from eight human livers significantly correlated with UGT1A1-catalyzed estradiol 3β-glucuronidation activity (r=0.78, p<0.05). β-Estradiol inhibited niflumic acid glucuronidation with an IC₅₀ of 25 μM in HLMs, comparable to that for UGT1A1. Conclusions These findings indicate that UGT1A1 is the main isozyme involved in the glucuronidation of niflumic acid in the human liver.     

Monoclonal antibody-directed characterization of cytochrome P450 isozymes responsible for toluene metabolism in rat liver

Monoclonal antibodies (MAbs) were used to study the contribution of cytochromes P450IA1/IA2, P450IIB1/IIB2, P450IIC11/IIC6 and P450IIE1 to toluene side-chain (benzyl alcohol, BA, formation) and ring (o- and p-cresol formation) oxidation in liver microsomes from fed, one-day fasted, and phenobarbital (PB)-, 3-methylcholanthrene (MC)- and ethanol-treated rats. All rats were fed synthetic liquid diets. MAb 1-7-1 against P450IA1/IA2 inhibited markedly o-cresol formation and slightly p-cresol formation but not BA formation only in microsomes from MC-treated rats. MAbs 2-66-3, 4-7-1 and 4-29-5 against P450IIB1/IIB2 strongly inhibited BA, o-cresol and p-cresol formation only in PB-induced microsomes. MAb 1-68-11 against P450IIC11/IIC6 inhibited BA formation at high toluene concentration in the following order: fed greater than fasted greater than ethanol = MC greater than PB, and ethanol greater than or equal to fed = fasted greater than MC greater than PB on the basis of the percentage and net amount inhibition, respectively. MAb 1-91-3 against P450IIE1 inhibited BA formation at low toluene concentration, but not at high concentration, in the following order: ethanol greater than fasted = fed greater than MC, and ethanol greater than fasted greater than fed greater than MC on the basis of percentage and net inhibition, respectively. MAbs 1-68-11 and 1-91-3 also inhibited p-cresol formation at high and low toluene concentrations, respectively. These results indicate that (i) both P450IIE1 and P450IIC11/IIC6 are constitutive isozymes mainly responsible for the formation of BA and p-cresol from toluene as low- and high-Km isozymes, respectively; (ii) P450IIE1, but not P450IIC11/IIC6, is induced by one-day fasting and ethanol treatment; (iii) both P450IIE1 and P450IIC11/IIC6 are decreased by PB and MC treatments; (iv) P450IIE1 is inhibited by high concentration of toluene; (v) P450IIB1/IIB2 can contribute to the formation of BA, o- and p-cresol from toluene, while P450IAI/IA2 preferentially contributes to the formation of o-cresol.     

Metabolism of methyltertiary-butyl ether by rat hepatic microsomes

Exposure to methyltertiary-butyl ether (MTBE), a commonly used octane booster in gasoline, has previously been shown to alter various muscle, kidney, and liver metabolic activities. In the present study, the metabolism of MTBE by liver microsomes from acetoneor phenobarbital-treated Sprague-Dawley rats was studied at concentrations of up to 5 mM MTBE. Equimolar amounts oftertiary-butanol, as measured by head-space gas chromatography, and formaldehyde were formed. TheV _max for the demethylation increased by 4-fold and 5.5-fold after acetone and phenobarbital treatments, respectively. The apparentK _m value of 0.70 mM using control microsomes was decreased slightly after acetone treatment, but was increased by 2-fold after phenobarbital treatment. The metabolism of MTBE (1 mM) was inhibited by 35% by monoclonal antibodies against P450IIE1, the acetone/ethanol inducible form of cytochrome P450, suggesting a partial contribution by this isozyme. A single 18-h pretreatment of rats with 1 or 5 ml/kg MTBE (i. p.) resulted in a 50-fold induction of liver microsomal pentoxyresorufin dealkylase activity but no change inN-nitrosodimethylamine demethylase activity. These trends in activity agreed with immunoblot analysis which showed an elevation in P450IIB1 but no change in P450IIE1 levels.The nomenclature for the P450 isozymes follows the convention described by Nebert et al. (1987).Supported by Grant ES-03938 from the National Institutes of Health and Grant 88B18 from the American Institute for Cancer Research     

Comparative inhibition of inducible and constitutive CYPs in rat hepatic microsomes by parathion

1. In microsomal fractions, the phosphorothioate pesticide parathion inhibits cytochrome P450 (CYP) enzymes by reversible and irreversible mechanisms resulting in the long-term suppression of drug oxidation. The present study evaluated the relative susceptibilities of constitutive and inducible CYP2 and CYP3 steroid hydroxylases to inhibition by the pesticide.

2. Enzyme kinetic analysis indicated that constitutive and dexamethasone (DEX)-induced androst-4-ene-3,17-dione (AD) 6β-hydroxylations were similarly susceptible to inhibition by parathion (K_m/K_i ratios 1.5–1.6). However, preincubation of parathion with NADPH-fortified microsomes intensified the extent of inhibition of CYP3A-dependent 6β-hydroxylation. Comparison of K_m/K_i ratios indicated that 6β-hydroxylation activity in fractions from DEX-pretreated rats was about twice as susceptible as the control activity to inactivation by parathion metabolites (K_m/K_i ratio of 8.0 versus 4.0).

3. The time-dependent loss of AD 6β-hydroxylation by parathion occurred more efficiently in fractions from DEX-induced liver than in control. Thus, half-times of 1.3 and 6.1?min, respectively, were determined for the inactivation of DEX-inducible and constitutive activities. Parathion concentrations required for half-maximal inactivation were 32 and 67?μM in microsomes from DEX-induced and control rats.

4. In phenobarbital (PB)-induced fractions CYP2B1-mediated AD 16β-hydroxylation was inhibited potently in a reversible fashion by parathion (K_i?=?0.37?μM; K_m/K_i ratio about 73). Inhibition was not enhanced at parathion concentrations near the K_i by a preincubation step with NADPH.

5. In control microsomes parathion elicited a type I binding interaction with oxidized CYP (K_s?=?7.7?μM, ΔA_max?=?2.2?×?10^?2?a.u.?nmol CYP^?1; ΔA_max/K_s 2.86?×?10³?a.u. nmol?CYP^?1/mM). Ligand binding was 13- and 1.6-fold more efficient in PB and DEX microsomes, respectively.

6. These findings indicate that pretreatment of rats with enzyme-inducing drugs like DEX and PB alters the profile of CYPs and their susceptibility to inhibition by parathion. Potent reversible inhibition of CYP2B1 occurred in PB-induced fractions and DEX-inducible CYPs 3A were more susceptible to mechanism-based inactivation than the corresponding constitutive CYPs from the same subfamily.

     

Decreased effect of phenobarbital treatment on microsomal drug metabolizing enzyme activity during gestation

Summary Pregnant and nonpregnant rats were treated with 37.5 mg phenobarbital (PB) per kg body weight twice daily for 4 days. Pregnant rats were injected with PB from day 10 to day 13 and from day 17 to day 20 of gestation and were used for the experiments on day 14 or day 21, respectively. This treatment resulted in a 35% increase in liver weight in nonpregnant rats. The livers of pregnant rats weighed the same as the livers from PB-treated nonpregnant rats, and PB-treatment during gestation had only a small stimulatory effect on liver weight.—The duration of sleeping time after an i.p. injection of 125 mg hexobarbital per kg maternal body weight was identical in control nonpregnant and control pregnant rats. PB-treatment significantly reduced the sleeping time but PB-treated pregnant rats had a longer sleeping time than PB-treated nonpregnant rats.—In liver microsomes, the K _m and V _max values were measured for the ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase. For the two substrates the K _m values were identical in all groups. The V _max values for the two enzyme activities were not different in control pregnant and control nonpregnant rats when calculated per total liver. Treatment of the rats with PB increased the V _max values for the two enzyme activities, but these values were significantly lower in the microsomes obtained from PB-treated pregnant rats.—The content of cytochrome P-450 was higher in PB-treated nonpregnant rats than in control nonpregnant rats, but no increase in the amount of cytochrome P-450 was measured after treating pregnant rats with PB.—The electron microscopic examination of the livers of pregnant rats revealed that the smooth elements of the endoplasmic reticulum were predominant in the cytoplasm, and after PB-treatment the typical proliferation of the smooth ER was not detectable.The abbreviations used are PB Phenobarbital - BP Benzo(a)pyrene - ER endoplasmic reticulum; day 14 or day 21 refer to day 14 of gestation or to day 21 of gestation, respectively - N.P. nonpregnant - CO control This work was supported by a grant from the Deutsche Forschungsgemeinschaft given to the Sonderforschungsbereich 29, Embryonale Entwicklung und Differenzierung (Embryonal-Pharmakologie).Parts of this paper were presented at the Spring Meeting of the German Pharmacological Society held in Mainz, March 1973.     

A comparative study on the contribution of cytochrome P450 isozymes to metabolism of benzene, toluene and trichloroethylene in rat liver.

The contribution of P450IIE1, P450IIC11/6, P450IIB1/2 and P450IA1/2 to the formation of chloral hydrate (CH) from trichloroethylene (TRI) was investigated in microsomes from control, ethanol-, phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rats using monoclonal antibodies (MAbs) to the respective P450 isozymes, and compared with their roles in benzene and toluene metabolism. Anti-P450IIE1 inhibited the formation of CH from TRI more strongly in microsomes from ethanol-treated rats than in microsomes from control rats at low concentration of TRI when net inhibition was compared. Anti-P450IIC11/6 inhibited CH formation in microsomes from control and PB-treated rats at high, not low, concentration of TRI, but the net inhibition in control microsomes was less than that due to anti-P450IIE1. Anti-P450IIB1/2 and anti-P450IA1/2 also inhibited CH formation from TRI in microsomes from PB- and MC-treated rats, respectively, stronger at high substrate concentration than at low concentration. These results indicate that P450IIE1, P450IIC11/6, P450IIB1/2 and P450IA1/2 are involved in the metabolic step from TRI to CH, and the first isozyme may be a low-Km TRI oxidase and the others high-Km one. Comparing the contributions of four isozymes to benzene, toluene and TRI metabolism, all four acted in the metabolism of these compounds, but P450IIE1 did not catalyse o-cresol formation nor P450IA1/2 benzyl alcohol formation from toluene, suggesting regioselectivity of toluene metabolism in the action of these two isozymes. The contribution of P450IIE1 in benzene and TRI oxidation was greater than that of P450IIC11/6, but the reverse was seen with respect to benzyl alcohol formation from toluene, indicating that P450IIC11/6 is relatively inactive towards benzene and TRI oxidation, but is primarily involved in toluene metabolism. P450IIB1/2 and P450IIC11/6 attacked all the metabolic positions studied, but only in the side-chain metabolism of toluene was their contribution significant, suggesting that these two isozymes are quite similar in function.     

Differential Induction of Peroxygenase-Dependent Microsomal Aniline Hydroxylase by Chronic Ethanol Ingestion

ABSTRACT

The liver microsomal-mediated hydroxylation of aniline, which is selectively induced by chronic (EtOH) ingestion, has been studied as a function of NADPH plus dioxygen (O₂)- or hydroperoxide-dependent reactions. Consistent with the well-documented induction of aniline hydroxylase following chronic ethanol -ingestion, the results showed selectivity towards aniline hydroxylase by the NADPH plus 0₂- and tert-butyl hydroper-xide (t-BuOOH)-dependent reactions with microsomes from EtOH-fed rats. On the other hand, the cumene hydroperoxide (CumOOH)-dependent aniline hydroxylase activity was not discriminated between microsomes from EtOH-and pair-fed rats. In parallel experiments with positive controls, CumOOH did show selectivity for phenobarbital (PB)- induced microsomal aniline hydroxylase compared to chow-fed rats. The K_cat/K_m values, which indicate the efficiency of enzyme catalysis, for NADPH plus 0₂-, t-BuOOH, and CumOOH-dependent aniline hydroxylase from EtOH-fed rats were 102, 37, and 5 and from pair-fed rats were 68, 4, and 4 (nmol p-aminophenol/min/nmol cytochrome P-450)/mM aniline, respectively. The relative K_cat/K_m ratio for EtOH-fed to that of pair-fed microsomal aniline hydroxylase from NADPH plus 0₂-, t-BuOOH-, and CumOOH-dependent reactions were 1.5, 7.4. and 1.2, respectively. The present preliminary studies indicate that the catalytic efficiency of EtOH-induced aniline hydroxylase is significantly greater for the t-BuOOH-dependent reaction.     

Metabolic activation of halothane and its covalent binding to liver endoplasmic proteins in vitro

Summary Metyrapone inhibits the N-demethylation of aminopyrine and ethylmorphine in rat liver microsomes non-competitively with concentrations in the micromolar range, and competitively in the range of 10^–4 M. In the case of non-competitive inhibition, enzyme and inhibitor are present in similar amounts (mutual depletion system). Under these conditions Lineweaver-Burk kinetics do not apply, since the portion of inhibitor bound to the enzyme cannot be neglected. Inhibition of microsomal N-demethylating activity by increasing amounts of metyrapone and application of equations required for a mutual depletion system enable us to determine the following kinetic parameters: (1) the concentration of catalytically active and metyrapone sensitive centers (E _t ) of cytochrome P-450, (2) their turnover number (TN), and (3) the true dissociation constant of the enzyme inhibitor complex, K _i .—With aminopyrine as substrate and microsomes from phenobarbital (PB) pretreated rats, 1 mg of protein contains 4.7 nmoles of E _t , i.e. about 2.2 catalytically active sites per molecule of cytochrome P-450; TN=3.4 min^–1, and K _i =2.2·10^–6 M.The corresponding values for ethylmorphine and control rats are: E _t =3.1 nmolles per mg of protein (4.4 sites per molecule), TN=1.94 min^–1; K _i =2.37·10^–6M. PB-pretreated rats: E _t =7.3 nmoles per mg of protein (3.5 sites per molecule) TN=2.0 min^–1; K _i =2.3·10^–6 M.Comparing the values obtained for PB-pretreated rats and controls reveals that K _i and TN remain constant but E _t rises 2.3 fold. This is regarded to mean that PB pretreatment influences ethylmorphine N-demethylation only quantitatively but not qualitatively. The experiments described here provide data which improve the characterization of cytochrome P-450. They are derived from the overall rate of N-demethylation reactions in microsomes, independently from conventional spectrophotometric methods.     

Chlorzoxazone: a probe drug whose metabolism can be used to monitor toluene exposure in rats

In this study we investigated cytochrome P450 (CYP) 2E1 expression using a probe drug, chlorzoxazone (CZX), whose metabolism can be used to monitor toluene exposure in rats. The animals received an i.p. injection of toluene (0.25, 0.5 and 1 ml/kg) once a day for 3 days. The total CYP and CYP2E1 content and the aniline and CZX hydroxylase activity (V _max and CL_int) increased depending on the dose of toluene administered. At the highest concentration (128 mM) of diethyldithiocarbamate, a specific inhibitor of CYP2E1, the production of 6-hydroxychlorzoxazone (HCZX) in microsomes from toluene-treated rats was reduced by about 80%. The IC₅₀ values in microsomes from toluene-treated rats were between 3 and 5 μM. The production of HCZX and the activity of aniline hydroxylase in toluene-treated rats were correlated with the amount of rat CYP2E1 protein (r=0.88 and r=0.88, respectively). The elimination of CZX by toluene-treated rats was increased and the HCXZ production in the toluene-treated group was greater than that in the olive oil control group. The correlations between intrinsic clearance (CL_int: V _max/K _m) in vitro and total body clearance (CL_tot) of CZX hydroxylation and the elimination half-life (t _1/2) of CZX in vivo in toluene-treated rats were high (r=0.784, P < 0.001; r=−0.678, P < 0.001, respectively). In addition, the metabolic plasma HCZX/CZX ratio did not require multiple blood sampling and 2 h after CZX administration in vivo there was also a high correlation with CL_int (V _max/K _m) in vitro (r=−0.729, P < 0.001). In conclusion, these results demonstrate that CZX is a very good probe for monitoring induction in toluene-treated rats. Received: 28 September 1999 / Accepted: 10 January 2000     

Three forms of trichloroethylene-metabolizing enzymes in rat liver induced by ethanol, phenobarbital, and 3-methylcholanthrene

In vitro metabolism of trichloroethylene (TRI) and trichloroethanol (TCE) was investigated using liver microsomes from control and ethanol-, phenobarbital (PB)-, and 3-methylcholanthrene (MC)-treated rats. At least three forms of enzymes were involved in TRI metabolism. One was a low-Km type normally existing in microsomes from control rats. The ethanol-inducible enzyme was found to be catalytically identical to this low-Km isozyme. Another was a high-Km type which was induced exclusively by PB, and a third was an MC-inducible isozyme with a Km value between those of ethanol- and PB-inducible isozymes. Although MC treatment did not affect the rate of TRI metabolism in vitro, both ethanol and PB treatment markedly enhanced the metabolism. Ethanol-induced enhancement was different from PB-induced enhancement in that ethanol enhanced the metabolism predominantly at low substrate concentrations, whereas PB did so at high concentrations. In addition, TRI metabolism with enzymes from ethanol-treated rats was inhibited by the substrate itself at high concentrations. MC treatment of rats had little or no influence on the rate of TCE metabolism in vitro, whereas both ethanol and PB enhanced the microsomal conversion of TCE to chloral hydrate. As in the case of TRI metabolism, ethanol induced a microsomal TCE-metabolizing enzyme of low Km, whereas PB preferentially induced an enzyme of high Km.     

Metabolism of methamphetamine,amphetamine and p-hydroxymethamphetamine by rat-liver microsomal preparations in vitro

1. Methamphetamine N-demethylation and p-hydroxylation activities of rat liver were located mainly in the microsomal fraction.

2. The K_m values for methamphetamine and p-hydroxymethamphetamine demethyl-ations were 10 and 16mM, respectively. The K_m value for amphetamine p-hydroxylation was 10· μm; substrate inhibition occurred at high substrate concn. Two K_m values were obtained for the aromatic hydroxylation of methamphetamine (10·μM and 2·2mM).

3. N-Demethylation of methamphetamine and p-hydroxymethamphetamine were depressed in rats pretreated with 3-methylcholanthrene, CoCl₂ or SKF 525-A. In rats pretreated with phenobarbital, methamphetamine demethylase was induced and p-hydroxymethamphetamine demethylase was depressed.

4. The p-hydroxylation of methamphetamine and amphetamine in rats pretreated with phenobarbital, CoCl₂, SKF 525-A or iprindole were depressed.     

Kinetic studies on benzene metabolism in rat liver--possible presence of three forms of benzene metabolizing enzymes in the liver

The effects of food deprivation, ethanol consumption and phenobarbital (PB) administration on in vitro benzene metabolism in rat liver were studied by using benzene concentration ranging from 0.0055 to 6.25 mM. The kinetic analysis suggested that the liver of normally-fed rats contained two forms of benzene hydroxylases each with a Km value of 0.01 mM or 0.07 mM. The isozyme with a Km of 0.01 mM disappeared following one-day food deprivation, but the deprivation enhanced the activity of the other isozyme. Ethanol treatment markedly increased the activity of both normally-existing enzymes. On the other hand, PB treatment induced the synthesis of another benzene-metabolizing enzyme with a high Km value of 4.5 mM, the presence of which was indistinct in normal rats. The treatment had no influence on the activity of either of the normally-occurring low-Km isozymes. The combined effects of PB with food deprivation were additive, suggesting that the induction of low- and high-Km isozymes is each independent of the other.     

Inhibition of rat hepatic mitochondrial aldehyde dehydrogenase isozymes by repeated cyanamide administration: Pharmacokinetic-pharmacodynamic relationships

The inhibition of rat hepatic mitochondrial aldehyde dehydrogenase (ALDH) isozymes was studied in apparent steady-state conditions after repeated intra-peritoneal cyanamide administration. The low-K_m mitochondrial ALDH isozyme was more susceptible to cyanamide-induced inhibition (DI₅₀ = 0.104 mg kg^?1) than the high-K_m isozyme (DI₅₀ = 8.52 mg kg^?1), with almost complete inhibition occurring at 0.35 mg kg^?1 total cyanamide administered for the low-K_m isozyme. The relationships between plasma and liver cyanamide concentrations and the inhibition of high-K_m ALDH were established by means of the sigmoid I_max model. The effect of dosing rate on the plasma concentration of cyanamide at apparent steady-state showed non-linearity, indicating that clearance or first-pass metabolism of cyanamide during its absorption after intraperitoneal administration did not remain constant throughout the range of doses studied.     

Isolation of human hepatic microsomes and their inhibition by cimetidine and ranitidine

Summary Human hepatic microsomes were isolated from wedge biopsies of the liver from 13 patients undergoing abdominal surgery. Ultrasonic homogenisation was used to increase the yield of microsomal monooxygenase activity (7-ethoxycoumarin O-deethylase, NADPH-cytochrome c reductase), resulting in a 30% higher total enzyme activity per g liver than preparation by other techniques. In 4 individual microsomal preparations the influence of cimetidine and ranitidine on Michaelis-Menten kinetics of O-deethylation and of reductase activity were studied. Without the H₂-receptor blocking drugs, enzyme kinetics of O-deethylation with a K_m of 51.0±16.4 µM (n=3) were obtained using Lineweaver-Burke plots. Both, cimetidine and ranitidine inhibited the O-deethylation; cimetidine had a five-fold higher inhibitory affinity (K_i 1.01 and 3.94 mM) to the monooxygenase than ranitidine (K_i 4.96 and 17.70 mM) in the uninduced liver. However, in liver from a patient with induced enzyme activity (K_m=478.0 µM), the K_i of ranitidine was similar to that of cimetidine (K_i ran 3.57 versus K_i cim 2.49 mM). The reductase activity was not inhibited by ranitidine and only marginally so by cimetidine.The results suggest that in human hepatic microsomes oxidative drug metabolism is inhibited by both H₂-receptor antagonists. However, the inhibitory potency of the compounds seems to depend on the individual isozyme pattern of the hepatic microsomes. Thus, while cimetidine is an relatively nonspecific enzyme inhibitor, ranitidine might more selectively inhibit induced drug metabolizing enzymes.     

Glucuronidation of Entacapone, Nitecapone, Tolcapone, and Some Other Nitrocatechols by Rat Liver Microsomes

Purpose. Nitrocatechol COMT inhibitors are a new class of bioactive compounds, for which glucuronidation is the most important metabolic pathway. The objective was to characterize the enzyme kinetics of nitrocatechol glucuronidation to improve the understanding and predicting of the pharmacokinetic behavior of this class of compounds. Methods. The glucuronidation kinetics of seven nitrocatechols and 4-nitrophenol, the reference substrate for phenol UDP-glucuronosyltrans-ferase activity, was measured in liver microsomes from creosote-treated rats and determined by non-linear fitting of the experimental data to the Michaelis-Menten equation. A new method that combined densitometric and radioactivity measurement of the glucuronides separated by HPTLC was developed for the quantification. Results. Apparent K_m values for the nitrocatechols varied greatly depending on substitution pattern being comparable with 4-nitrophenol (0.11 mM) only in the case of 4-nitrocatechol (0.19 mM). Simple nitrocatechols showed two-fold V_max values compared with 4-nitrophenol (68.6 nmol min^–1 mg^–1), while all disubstituted catechols exhibited much lower glucuronidation rate. V_max/K_m values were about 10 times higher for monosubstituted catechols compared to disubstituted ones. The kinetic parameters for COMT inhibitors were in the following order: K_m nitecapone >> entacapone > tolcapone; V_max nitecapone > entacapone > tolcapone; V_max/K_m tolcapone > nitecapone > entacapone. Conclusions. Nitrocatechols can in principle be good substrates of UGTs. However, substituents may have a remarkable effect on the enzyme kinetic parameters. The different behaviour of nitecapone compared to the other COMT inhibitors may be due to its hydrophilic 5-substituent. The longer elimination half-life of tolcapone in vivo compared to entacapone could not be explained by glucuronidation kinetics in vitro.     

氧氟沙星对映体在经不同诱导剂诱导的大鼠肝微粒体中的葡醛酸化代谢

为评价氧氟沙星（OFLX）对映体葡醛酸化代谢的立体选择性,采用手性HPLC法测定大鼠肝微粒体孵育液中OFLX对映体. 结果显示：经苯巴比妥（PB）和β-萘黄酮（BNF）诱导的不同葡醛酸转移酶（UDPGT）亚族对OFLX对映体葡醛酸化代谢有不同的影响. 在所试验的对照,PB或BNF诱导的微粒体中S-（-）-和R-（+）-OFLX之间,K_m和V_max无显著性差异;但PB组中S- （-）-和R-（+）-OFLX的K_m和V_max与对照组或BNF组相应的对映体比较有显著性差异;OFLX对映体之间的Cl_int在对照组与BNF组没有显著性差异;而在PB组则有显著性差异. 另外BNF组的Cl_int较对照组和PB组分别有显著性差异. 因此,经PB诱导的UDPGT亚族对S-和R-OFLX的Ⅱ相代谢存在立体选择性,并主要是由于其催化部位的差异引起了内在清除率的变化.     

Changes in the rat hepatic mixed function oxidase system associated with chronic ethanol vapor inhalation

Chronic ethanol vapor inhalation by rats increased hepatic microsomal aniline hydroxylase activity, increasing the turnover number and decreasing the K_m. Activity of ethanol-induced microsomes toward other substrates was also examined. The increase in aniline hydroxylase activity as a result of ethanol treatment is attributed to an increase in a form of cytochrome P-450 with a high specific activity toward aniline. Since the ethanol effect on aniline hydroxylation had disappeared 24 hr after treatment was discontinued, a high rate of turnover of this enzyme was deduced. Dimethylsulfoxide (56 mM) produced a reverse type I spectral change in ethanol-induced, but not in control, microsomes. This was interpreted as being due to a change in the spin state of the cytochrome P-450 in these microsomes. Acetone added to the incubation produced an increased rate of aniline hydroxylation by microsomes from control and ethanol-induced rats. The difference between the rate of aniline hydroxylation by control microsomes and the rate by ethanol-induced microsomes was, however, abolished at higher acetone concentrations.     

Metabolism of tetraorganolead compounds by rat-liver microsomal mono-oxygenase: II. Enzymic dealkylation of tetraethyl lead

1. The biological degradation of tetraethyl lead to the triethyl lead cation by rat-liver microsomes from untreated, phenobarbital-pretreated and methylcholanthrene-pretreated rats has been studied; NADPH and oxygen are essential.

2. The reaction is inhibited by CO and can be reactivated in the presence of O₂ by irradiation with u.v. light with a max. at 450nm.

3. Substrate binding to cytochrome P-450 is of type 1.

4. Apparent K_m values for triethyl lead formation in microsomes were determined. The highest activities (i.e. about 2 nmol triethyl lead per nmol cytochrome P-450 per min) and the lowest apparent K_m values (i.e. 7 × 10^-6 M) are found in microsomes from methylcholanthrene-pretreated rats.

5. In microsomes from control and phenobarbital-pretreated rats K_s values from substrate-binding studies (about 2 × 10^-6M) are one order of magnitude lower than the apparent K_m values (3 × 10^-5 M).