Microsomal Oxidation Research Articles

The microsomal dealkylation of N, N-dialkylbenzamides

The in vitro metabolism of N, N-dialkylamides by phenobarbital-induced rat liver microsomes yields an N-alkylamide and the corresponding aldehyde. Although, N-hydroxymethyl- N-alkylamide intermediates can be detected from N-methyl- N-alkylamides, no N-hydroxyalkyl- N-alkylamide inter- mediates are detected from the N, N-dialkylamide substrates. V max values were independent of amide structure, whereas V max K m values were dependent on the lipophilidty of the N, N-dialkylbenazamide studied. These results suggest that diffusion of substrate into the membrane-bound enzyme active site limits the rate of the microsomal oxidation of the amides. Metabolism of N-alkyl- N-methylamides reveals identical values of V max for demethylation and dealkylation. Values of V max K m for demethylation depend upon the lipophilicity of the N-alkyl group, whereas V max K m values for dealkylation appear to be dependent upon the steric bulk of the alkyl group, particularly around the α-carbon. Moreover, V max K m values for demethylation are larger than for dealkylation, implying the reactions are under kinetic control. Comparison of the kinetic data with theoretical AMI semi-empirical molecular orbital calculations suggests a mechanism involving formation of a carbon-centred radical. Use of an N-cyclo-propylmethylbenzamide substrate to trap such a radical failed, presumably because oxygen rebound is faster than radical rearrangement. An N-cyclopropylamide substrate did not undergo metabolism of the cyclopropyl ring, consistent with carbon-centred radical, but not nitrogen radical cation, formation.

The invertor mechanism of changes in the plasma membranes of hepatocytes during induction of microsomal monooxygenases in adult and old rats

Age-specific changes of some parameters of the state of the plasma membrane are studied for genetic induction of enzymes of microsomal oxidation. Changes of the state of plasma membrane of hepatocytes are shown to be associated with the synthesis of a specific intracellular regulator (invertor).

Oxidation of Multiple Substrates by Corn Shoot Microsomes

Microsomes isolated from excised shoots of 3-day-old, dark-grown, corn (Zea mays L., Pioneer Hybrid 3245) seedlings metabolized two endogenous substrates (cinnamic acid and lauric acid), one insecticide (diazinon), and six herbicides (metolachlor, bentazon, CGA-152005, triasulfuron, primisulfuron, and nicosulfuron). Pretreatment of the seed with the safener naphthalic anhydride resulted in enhanced metabolism of all substrates except cinnamic acid. Metabolism required NADPH and was affected by several cytochrome P450 monooxygenase inhibitors (tetcyclacis, piperonyl butoxide, 1-aminobenzolriazole, SKF-525A, and tridiphane). The inhibitors differentially affected metabolism of the substrates. Tetcyclacis and piperonyl butoxide strongly inhibited the metabolism of all substrates except cinnamic acid. Microsomal oxidations from both unsafened and safener-treated tissue responded similarly to the inhibitors. The differential inhibitory responses suggest that metabolism may involve several monooxygenase isoforms.

Stimulation by paraquat of microsomal and cytochrome P-450-dependent oxidation of glycerol to formaldehyde.

Glycerol can be oxidized to formaldehyde by microsomes in a reaction that is dependent on cytochrome P-450. An oxidant derived from the interaction of H2O2 with iron was responsible for oxidizing the glycerol, with P-450 suggested to be necessary to produce H2O2 and reduce non-haem iron. The effect of paraquat on formaldehyde production from glycerol and whether paraquat could replace P-450 in supporting this reaction were studied. Paraquat increased NADPH-dependent microsomal oxidation of glycerol; the stimulation was inhibited by glutathione, catalase, EDTA and desferrioxamine, but not by superoxide dismutase or hydroxyl-radical scavengers. The paraquat stimulation was also inhibited by inhibitors, substrate and ligand for P-4502E1 (pyrazole-induced P-450 isozyme), as well as by anti-(P-4502E1) IgG. These results suggest that P-450 still played an important role in glycerol oxidation, even in the presence of paraquat. Purified NADPH-cytochrome P-450 reductase did not oxidize glycerol to formaldehyde; some oxidation, however, did occur in the presence of paraquat. Reductase plus P-4502E1 oxidized glycerol, and a large stimulation was observed in the presence of paraquat. Rates in the presence of P-450, reductase and paraquat were more than additive than the sums from the reductase plus P-450 and reductase plus paraquat rates, suggesting synergistic interactions between paraquat and P-450. These results indicate that paraquat increases oxidation of glycerol to formaldehyde by microsomes and reconstituted systems, that H2O2 and iron play a role in the overall reaction, and that paraquat can substitute, in part, for P-450 in supporting oxidation of glycerol. However, cytochrome P-450 is required for elevated rates of formaldehyde production even in the presence of paraquat.

Two pathways of iron-catalyzed oxidation of bilirubin: Effect of desferrioxamine and trolox, and comparison with microsomal oxidation

The bilirubin-degrading activity of liver microsomes from rats induced with 3-methylcholanthrene has been shown to be markedly stimulated by addition of 3,3′,4,4′,5,5′-hexabromobiphenyl, a polyhalogenated chemical which resembles in size and shape the most effective inducers of cytochrome P450IA1, but lacks the structural features necessary for it to be metabolised. The degradation of bilirubin by this microsomal system has been compared to oxidation by a chemical model system involving H 2O 2 and Fe-EDTA (ethylenediaminetetraacetic acid). In both systems bilirubin disappearance was accompanied by bleaching. However, when either desferrioxamine or Trolox were present in the chemical model system, the rate of bilirubin oxidation was greatly enhanced and, at the same time, bilirubin was largely or entirely converted to biliverdin, a pathway of oxidation which proceeds by dehydrogenation. In the presence of desferrioxamine, biliverdin was also further oxidised to an unidentified red pigment.

Identification of multi-S-substituted conjugates of hydroquinone by HPLC-coulometric electrode array analysis and mass spectroscopy.

Chemical reaction of 1,4-benzoquinone with GSH gives rise to several multisubstituted hydroquinone (HQ)-GSH conjugates, each of which causes renal proximal tubular necrosis when administered to male Sprague-Dawley rats. In addition, HQ has recently been reported to be nephrocarcinogenic following long-term exposure in male rats. Since neither the mechanism nor the extent of HQ oxidation and thioether formation in vivo is known, we have assessed both the qualitative and quantitative significance of HQ-thioether formation in vivo and in vitro. HQ (1.8 mmol/kg, ip) was administered to AT-125-pretreated male Sprague-Dawley rats, and bile and urine samples were analyzed with a HPLC-coulometric electrode array system (CEAS) and by liquid chromatography (LC)/continuous-flow fast atom bombardment (CF-FAB) mass spectroscopy. Five S-conjugates of hydroquinone were identified in bile, and one S-conjugate was identified in urine. The major biliary S-conjugate identified was 2-glutathion-S-ylhydroquinone [2-(GSyl)HQ] (18.9 +/- 2.7 mumol). Additional biliary metabolites were 2,5-diglutathion-S-ylhydroquinone [2,5-(diGSyl)HQ] (2.2 +/- 0.6 mumol), 2,6-diglutathion-S-ylhydroquinone [2,6-(diGSyl)HQ] (0.7 +/- 0.3 mumol),2,3,5-triglutathion-S-ylhydroquinone [2,3,5-(triGSyl)HQ] (1.2 +/- 0.1 mumol), and 2-(cystein-S-ylglycyl)hydroquinone. 2-(N-Acetylcystein-S-yl)HQ was the only urinary thioether metabolite (11.4 +/- 3.6 mumol) identified. The quantity of S-conjugates excreted in urine and bile within 4 h of HQ administration [34.3 +/- 4.5 mumol (4.3 +/- 1.1% of dose)] appears sufficient to propose a role for such metabolites in HQ-mediated nephrotoxicity and nephrocarcinogenicity. Rat liver microsomes catalyzed the NADPH-dependent oxidation of HQ (300 microM), in the presence of GSH, to form 2-(GSyl)HQ,2,5-(diGSyl)-HQ, and 2,6-(diGSyl)HQ. A fraction of the microsomal oxidation of HQ appears to be catalyzed by cytochrome(s) P450, although the exact amount remains unclear. 2-(GSyl)HQ,2,5-(diGSyl)-HQ, and 2,6-(diGSyl)HQ (300 microM) also underwent NADPH-dependent oxidation and GSH conjugation in liver microsomes. The extent of the nonenzymatic oxidation of HQ and its GSH conjugates correlated, approximately, with their half-wave oxidation potentials.

Clinical pharmacokinetics of alprazolam. Therapeutic implications.

Alprazolam is a triazolobenzodiazepine that is extensively prescribed in the Western world for the treatment of anxiety and panic disorders. Its benzodiazepine receptor binding characteristics are qualitatively similar to those of other benzodiazepines. The drug is metabolised primarily by hepatic microsomal oxidation, yielding alpha-hydroxy- and 4-hydroxy-alprazolam as principal initial metabolites. Both have lower intrinsic benzodiazepine receptor affinity than alprazolam and appear in human plasma at less than 10% of the concentrations of the parent drug. Plasma concentrations of the 4-hydroxy metabolite exceed those of the alpha-hydroxy derivative, but urinary recovery of alpha-hydroxy-alprazolam greatly exceeds that of 4-hydroxy-alprazolam. This may be explained by chemical instability of 4-hydroxy-alprazolam in vitro. After single 1 mg oral doses in humans, typical pharmacokinetic variables for alprazolam are: a peak plasma concentration 12 to 22 micrograms/L occurring 0.7 to 1.8h postdose, a volume of distribution of 0.8 to 1.3 L/kg, elimination half-life of 9 to 16h and clearance of 0.7 to 1.5 ml/min/kg. Absolute bioavailability of oral alprazolam averages 80 to 100%. Pharmacokinetics are dose-independent and are unchanged during multiple-dose treatment. On average, mean steady-state plasma alprazolam concentrations change by 10 to 12 micrograms/L for each daily dosage change of 1 mg/day. Most studies show that alprazolam pharmacokinetics are not significantly influenced by gender. Clearance of alprazolam is reduced in many elderly individuals, even those who are apparently healthy. Clearance is significantly reduced in patients with cirrhosis. Renal disease causes reduced plasma protein binding of alprazolam (increased free fraction) and some data suggest reduced free clearance of alprazolam in such patients. Pharmacokinetics of alprazolam are not significantly altered in abstinent alcoholics or patients with panic disorder, and are not influenced by the phase of the menstrual cycle in women. Coadministration of cimetidine, fluoxetine, fluvoxamine or propoxyphene significantly impairs alprazolam clearance. However, alprazolam clearance is not altered by coadministration of propranolol, metronidazole, disulfiram, oral contraceptives or ethanol. Imipramine clearance may be impaired if alprazolam is coadministered. Alprazolam does not alter the pharmacokinetics of digoxin. Although a therapeutic concentration range is not clearly established, some studies indicate that optimal reduction of anxiety associated with panic disorder occurs at steady-state plasma alprazolam concentrations of 20 to 40 micrograms/L. Concentrations higher than this may be needed for suppression of the actual panic attacks. Side effects associated with alprazolam (drowsiness, sedation, etc.) are consistent with its primary benzodiazepine agonist action and increase in frequency with higher steady-state plasma concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of di-, tri- and tetrabromobiphenyls by hepatic microsomes isolated from control animals and animals treated with aroclor 1254, a commercial mixture of polychlorinated biphenyls (PCBs)

1. The metabolism of di-, tri- and tetrabromobiphenyls (PBBs) by hepatic microsomes isolated from control animals and animals treated with Arochlor 1254 was studied. 2. Hepatic microsomes isolated fom control rats expressed higher rates of oxidations than avians 3. Treatment of rats and pigeons with Arochlor 1254 induced cytochrome P450 dependent monooxygenases leading to an increased regioselective metabolism of PBB isomer and congeneres. 4. There was an inverse relationship between the degree of halosubstitution and microsomal oxidation. Meta-para carbon atoms free of halosubstitution were the preferred side for oxidation.

Metabolism of di-, tri-, tetra-, penta- and hexachlorobiphenyls by hepatic microsomes isolated from control animals and animals treated with aroclor 1254, a commercial mixture of polychlorinated biphenyls (pcbs)

1. The metabolism of a wide range of di-, tri-, tetra-, penta- and hexachlorobiphenyls by hepatic microsomes isolated from control animals and animals treated with Aroclor 1254 was studied. 2. Hepatic microsomes isolated from control rats expressed higher rates of oxidations than avians. 3. Treatment of rats and pigeons with Aroclor 1254 induced cytochrome P450 dependent mono-oxygenases leading to an increased regioselective metabolism of PCB isomer and congeneres. 4. There was an inverse relationship between the degree of halosubstitution and microsomal oxidation. Meta-para carbon atoms free of halosubstitution were the preferred side for oxidation. 5. A good correlation was found between the in vitro metabolism of PCBs and their relative abundance in tissue extracts, thus suggesting oxidative metabolism to be the major route of metabolic disposal.

Estradiol-induced effects on glutathione metabolism in rat hepatocytes.

Glutathione plays an important role in the intracellular protection against oxidative stress and damage in the liver. It is generally assumed that the toxic potential of estrogens is linked to reactive metabolites generated during their enzymatic oxidation in hepatic microsomes. In the present study, the effects of pharmacological doses of estradiol on glutathione metabolism using isolated rat hepatocytes are described. Estradiol (0.1-1.5 mM) produced a dose-dependent depletion of cellular reduced glutathione (GSH), whereas it did not alter the glutathione disulfide (GSSG) excretion into the medium. The viability of cells exposed to the estrogen did not change even when conditions of exacerbated toxicity (addition of either 1 mM diethylmaleimide or 30 microM dicoumarol) were used. In addition, estradiol was shown to exert protective effects against the spontaneous lipid peroxidation in liver cells. In rat liver microsomes, estradiol (5-50 microM) significantly interacted with GSH only when an NADPH-regenerating system was incorporated into the medium.

Correlations between the molecular structures of polyhalogenated biphenyls and their metabolism by hepatic microsomal monooxygenases

1. Collation of the data presented in the preceding papers (references 4,5) showed a significant correlation ( r = 0.83; P < 0.001) between the molecular mass (and hence the extent of halosubstitution) of halogenated biphenyls and their rate of hydroxylation by hepatic microsomal monooxygenases. 2. There was no relationship between the extent of poly ortho halosubstitution of biphenyl and the rate of metabolism. 3. A marginal correlation ( r = 0.33; P < 0.001) was found when the number of adjacent unsubstituted meta-para positions were linked to the rate of metabolism of PCBs. This structural feature facilitates microsomal oxidation. 4. The results support the proposal that PCBs with meta-para hydrogen atoms are less enriched in tissues of animals and humans as this structural feature favours their metabolism by P450 isoenzymes.

Exposure to various benzene derivatives differently induces cytochromes P450 2B1 and P450 2E1 in rat liver.

Benzene (B), toluene (T), ethylbenzene (EB), styrene (S) and xylene isomers (oX, mX, pX) are important environmental pollutants and B is a proved human carcinogen. Their inhalation by male Wistar rats (4 mg/l, 20 h/day, 4 days) caused cytochrome P450 (P450) induction. The degree of P450 2B1 induction increased and that of 2E1 decreased in the series B, T, EB, S, oX, mX and pX, as estimated by Western blots, while neither solvent was as effective for 2B1 induction as phenobarbital and B was more effective for 2E1 than ethanol. The levels of several other P450s decreased after exposure to these solvents, B being most effective. Exposure to these solvents increased in vitro hepatic microsomal oxidation of B and aniline (AN) (2E1 substrates) 3 to 6-fold, indicating induction of this P450. T oxidation was increased 2 to 4-fold and chlorobenzene (ClB) oxidation 3-fold. Sodium phenobarbital (PB, 80 mg/kg/day, 4 days, i.p.) did not increase ethylmorphine (EM) and benzphetamine (BZP) demethylation (2B1 substrates), neither of the B derivatives did so, and oX decreased it; however, pentoxyresorufin O-dealkylation was well related to the immunochemically detected 2B1 levels in control, PB and B microsomes. PB did not increase B, but increased T and ClB oxidation 2-4 and 3-fold, respectively, indicating possible 2B1 role in their oxidation. B oxidation after various inducers was related to immunochemical 2E1 levels, T and ClB oxidation to both 2B1 and 2E1 and AN oxidation to 2E1 and 1A2 levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-activity relationships of arylalkyl isothiocyanates for the inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism and the modulation of xenobiotic-metabolizing enzymes in rats and mice.

Many arylalkyl isothiocyanates are potent inhibitors of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in rats and mice. In the mouse, 4-phenylbutyl isothiocyanate (PBITC) and 6-phenylhexyl isothiocyanate (PHITC) exhibited greater inhibition than benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC). The present study was conducted to investigate the structure-activity relationships of these four arylalkyl isothiocyanates for their inhibition of NNK oxidation and effects on xenobiotic-metabolizing enzymes in rats and mice. A single dose (0.25 or 1.00 mmol/kg) of each isothiocyanate was given to F344 rats 6 or 24 h before death. The rates of NNK oxidation were decreased in microsomes from the liver, lung and nasal mucosa of rats. Generally, PEITC was more potent than BITC but less potent than PBITC and PHITC. The rates in rat liver microsomes were decreased at 6 h but recovered or increased at 24 h; the rates in rat lung microsomes were markedly decreased at both 6 and 24 h; and the rates in rat nasal mucosa microsomes were also significantly decreased. The same treatment decreased the rat liver N-nitrosodimethylamine demethylase activity dramatically and ethoxyresorufin O-dealkylase and erythromycin N-demethylase activities moderately. However, the rat liver microsomal pentoxy-resorufin O-dealkylase activity was decreased at 6 h but increased at 24 h, with PEITC showing the most marked induction. The rat liver NAD(P)H:quinone oxidoreductase activity was increased 1.4- to 3.3-fold, with PEITC being most effective; and the glutathione S-transferase activity was increased slightly. Similarly, at a single dose of 0.25 mmol/kg (5 mumol/mouse) 24 h before death, PEITC, PBITC, PHITC but not BITC, decreased NNK oxidation in mouse lung microsomes by 40-85%, with PBITC and PHITC showing greater inhibition. Furthermore, all four isothiocyanates extensively inhibited NNK oxidation in rat lung and nasal mucosa microsomes as well as mouse lung microsomes in vitro, with PEITC (IC50 of 120-300 nM) being more potent than BITC (IC50 of 500-1400 nM) but less potent than PBITC and PHITC (IC50 of 15-180 nM). PHITC was a very potent competitive inhibitor of NNK oxidation in mouse lung microsomes with apparent K(i) values of 11-16 nM. These results indicate that PBITC and PHITC are more potent inhibitors of NNK bioactivation in rats and mice than PEITC. In addition, these arylalkyl isothiocyanates could be effective in protecting against the actions of a broad spectrum of carcinogenic or toxic compounds.

Effects of Safeners on the Oxidation of Multiple Substrates by Grain Sorghum Microsomes

Microsomes isolated from excised shoots of 3-day-old, dark-grown, grain sorghum [ Sorghum bicolor (L.) Moench. Funk G522DR, and DK 41Y] seedlings metabolized cinnamic acid, lauric acid, metolachlor, bentazon, and diazinon. but did not metabolize triasulfuron or primisulfuron. Pretreatment of G522DR seed with safeners (naphthalic anhydride, dichlormid, flurazole, BAS 145138, oxabetrinil, fluxofenim, and benoxacor) resulted in enhanced metabolism of lauric acid, bentazon, and diazinon. However, metabolism of cinnamic acid was not affected and that of metolachlor was depressed by safener treatments. Microsomes isolated from DK 41Y seedlings had higher endogenous levels of oxidative activity for lauric acid and bentazon than microsomes isolated from G522DR seedlings. Metabolism required NADPH and was affected by CO and other cytochrome P450 monooxygenase inhibitors (tetcyclacis, piperonyl butoxide, 1-aminobenzotriazole, SKF-525A, and tridiphane). The inhibitors differentially affected metabolism of the substrates. Only tetcyclacis strongly inhibited the metabolism of all substrates except cinnamic acid. Microsomal oxidations from both unsafened and safener-treated tissue responded similarly to the inhibitors. The differential inhibitory responses suggest that each substrate was probably metabolized by a different monooxygenase isoform.

Immunohistochemical Demonstration of Ethanol-Inducible

The microsomal ethanol-oxidizing system (MEOS) is a P450-dependent pathway for ethanol oxidation in hepatic microsomes. The bulk of MEOS activity is catalyzed by P450 2E1 (an ethanol-inducible isozyme of P450) in animal livers treated with ethanol. Rat brains also metabolize certain drugs, and it is theorized that a mechanism for drug metabolism exists within the brain which acts on P450. We compared immunohistochemical localization of P450 2E1 between ethanol-treated rats and pair-fed control rats. In control rats, immunoreactive P450 2E1 was detected in minute amounts in both basal ganglia and cerebellar cortices. After ethanol treatment, the content of P450 2E1 increased in the basal ganglia, and the enzyme was also induced in the cerebellar cortices, substantia nigra and hippocampus. We speculate that the rat brain metabolizes ethanol by P450 2E1.

Effect of age and gender on in vivo ethanol elimination, hepatic alcohol dehydrogenase activity, and NAD+ availability in F344 rats.

It has been reported that aging strikingly decreases in vivo ethanol metabolism in F344 rats without major effects on hepatic alcohol dehydrogenase (ADH) activity. Because hepatic ADH activity is not always rate limiting in the oxidation of ethanol, we measured in vivo ethanol elimination rate (EER), hepatic ADH activity, and the hepatic-cytoplasmic and mitochondrial redox states after acute ethanol application in 2- and 12-month-old F344 rats of both sexes. In male, but not in female, animals EER decreased with age significantly, by 28% (P less than 0.01). The body-to-liver weight ratio was significantly increased in male (39.4 +/- 1.5 vs 46.5 +/- 2.0; P less than 0.05), but not in female, animals with age. Specific activity of ADH was not significantly changed by age, while the activity was significantly reduced with age in male, but not female, rats when related to body weight (5.1 +/- 0.4 vs 3.9 +/- 0.3 mumoles/100 g b.wt./min; P less than 0.05). The cytoplasmic, but not the mitochondrial (NAD+) to (NADH), ratio was significantly decreased with age in male livers (317 +/- 48 vs 793 +/- 128, P less than 0.05), while this was not the case in female livers. In summary, the data show a sex dependence of the effect of age on ethanol metabolism. The observed reduction in in vivo EER with age in male animals is due at least in part to an increased body-to-liver weight ratio, decreased hepatic ADH activity, and reduced availability of NAD+, the cofactor of the ADH reaction. The cause of this may be decreased transport of reducing equivalents through the mitochondrial membrane due to a lack of shuttle systems or a change in the physicochemical properties of the mitochondrial membrane, or decreased reutilization of NADH as NADPH resulting from a reduction of microsomal ethanol oxidation with age.

The effect of enzyme induction on the cytochrome P450-mediated bioactivation of carbamazepine by mouse liver microsomes

Predisposition to idiosyncratic toxicity with carbamazepine is thought to be due to a deficiency of the detoxication enzyme, microsomal epoxide hydrolase, although in some cases, concurrent administration of enzyme inducers might be a contributory risk factor, by altering the critical balance between bioactivation and detoxication. In this study, a mouse model has been used to determine the factors affecting carbamazepine bioactivation, using covalent binding and cytotoxicity as markers of bioactivation in vitro. Microsomes prepared from mice pre-treated with phenobarbitone increased (relative to the control microsomes) the formation of cytotoxic (12.3% vs 3.2%), protein-reactive (3.0% vs 2.0%) and stable (33.8% vs 18.1%) metabolites of carbamazepine. Similarly, pre-treatment with dexamethasone also increased the formation of the cytotoxic (24.8% vs 6.7%), protein-reactive (2.8% vs 1.5%) and stable (38% vs 19.8%) metabolites of carbamazepine, while β-naphthoflavone pretreatment did not increase the formation of either the toxic or stable metabolites of carbamazepine when compared with its control microsomes. Co-incubation with gestodene (10–250 μM) resulted in a dose-dependent inhibition of both the bioactivation of carbamazepine and the formation of its stable 10,11-epoxide. SDS-PAGE and immunoblotting of the microsomes with anti-CYP3A antibody revealed the presence of a 52 kDa protein band in each preparation of microsomes, but the relative intensities of the bands, as measured by laser densitometry, were highest with the phenobarbitone and dexamethasone microsomes. The microsomal oxidation of cortisol to 6β-hydroxycortisol was also enhanced by pretreatment of mice with phenobarbitone (6.5% vs 2.7%) and dexamethasone (8.2% vs 4.3%) but not β-naphthoflavone (2.2% vs 1.6%), when compared with their respective control microsomes, and was inhibited (range 25–68% inhibition), with all the microsomes by gestodene (50 μM). Taken collectively, the data in this study demonstrate that in the mouse, induction of the CYP3A subfamily significantly increases carbamazepine bioactivation. It is likely that in humans inducers of the orthologous form of this enzyme, most notably anticonvulsants, may increase the bioactivation of carbamazepine.

Tandem mass spectrometric identification of eicosanoids: Leukotrienes and hydroxyeicosatetraenoic acids

The product ion spectra of the fast atom bombardment (FAB) desorbed [M - H]− ions of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) have been obtained on a tandem magnetic sector mass spectrometer with introduction by coaxial continuous flow FAB. Upon collisional activation, the product ion spectra of the [M - H]− ions of these derivatives of arachidonic acid reveal structurally informative product ions. The location of the hydroxy groups on the fatty acid chain, and the position and type of the cysteine-containing moiety of the LTs, can be readily determined from the product ion spectra. Tandem mass spectrometry was used to identify 11-and 15-HETE produced in a low-temperature microsomal enzymatic oxidation of arachidonic acid.

The involvement of primary and secondary metabolism in the covalent binding of 1,2- and 1,4-dichlorobenzenes

The microsomal oxidation of 1,2-[ 14C]- and 1,4-[ 14C]dichlorobenzene (DICB) was investigated with special attention for possible differences in biotransformation that might contribute to the isomer-specific hepatotoxicity. Major metabolites of both isomers were dichlorophenols (2,5-DICP for 1,4-DICB and 2,3- and 3,4-DICP for 1,2-DICB, respectively) and dichlorohydroquinones. The formation of polar dihydrodiols appeared to be a major route for 1,2-DICB but not 1,4-DICB. Both the hepatotoxic 1,2-DICB and the non-hepatotoxic 1,4-DICB were oxidized to metabolites that covalently interacted with protein and only to a small extent with DNA. Protein binding could be inhibited by the addition of the reducing agent ascorbic acid with a concomitant increase in the formation of hydroquinones and catechols, indicating the involvement of reactive benzo-quinone metabolites in protein binding. However, in the presence of ascorbic acid, a substantial amount of protein-bound metabolites of 1,2-DICB was still observed, in contrast to 1,4-DICB where binding was nearly completely inhibited. This latter effect was ascribed to the direct formation of reactive benzo-quinone metabolites in a single P450-mediated oxidation of para-substituted dichlorophenols (such as 3,4-DICP) in the case of 1,2-DICB. In contrast, the major phenol isomer derived from 1,4-DICB (i.e. 2,5-DICP) is oxidized to its hydroquinone derivative, which needs prior oxidation in order to generate the reactive benzoquinone species. Residual protein binding in the presence of ascorbic acid could also indicate the involvement of reactive arene oxides in the protein binding of 1,2-DICB, but not of 1,4-DICB. However, MO computer calculations did not provide indications for differences in chemical reactivity and/or stability of the various arene oxide/oxepin tautomers that can be formed from either 1,2-DICB or 1,4-DICB. In conclusion, reactive intermediates in the secondary metabolism of 1,2-DICB lead to more covalent binding than those derived from 1,4-DICB, which correlates very well with their reported hepatotoxic potency.

Fluoxetine impairs clearance of alprazolam but not of clonazepam

Volunteer male subjects received single 1.0 mg oral doses of alprazolam and of clonazepam on two occasions, during coadministration of 40 mg/day fluoxetine or of placebo. When the sequence of trials was placebo first and fluoxetine second, fluoxetine coadministration significantly prolonged alprazolam half-life (20 versus 17 hours) and reduced clearance (48 versus 61 ml/min). No effect of fluoxetine was seen when fluoxetine was given first and placebo second, because norfluoxetine persisted into the placebo phase even though fluoxetine had been discontinued 2 weeks earlier. Fluoxetine had no significant effects on clonazepam elimination half-life or clearance regardless of the sequence of fluoxetine and placebo administration. In the fluoxetine-placebo sequence, fluoxetine significantly increased the rate of clonazepam absorption. Thus fluoxetine appears to impair clearance of alprazolam by way of microsomal oxidation but does not alter clearance of clonazepam by way of nitroreduction. The very slow elimination of norfluoxetine should be considered in the design of clinical or pharmacokinetic studies that involve fluoxetine.