In vitro sulfoxidation of thioether compounds by human cytochrome P450 and flavin-containing monooxygenase isoforms with particular reference to the CYP2C subfamily.

Cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes are major catalysts involved in the metabolism of xenobiotics. The sulfoxidation of the thioether pesticides, phorate, disulfoton, sulprofos, and methiocarb, was investigated. Using pooled human liver microsomes (HLMs), thioether compounds displayed similar affinities; however, phorate and disulfoton displayed higher intrinsic clearance rates than either sulprofos or methiocarb. The sulfoxidation of thioethers by HLMs was found to be predominantly P450-driven (85-90%) compared with FMO (10-15%). Among 16 cDNA-expressed human P450 isoforms and 3 human FMO isoforms examined, the following isoforms and their polymorphisms had the highest rates for sulfoxidation, as follows: phorate, CYP1A2, 3A4, 2B6, 2C9*1, 2C18, 2C19, 2D6*1, and FMO1; disulfoton, CYP1A2, 3A4, 2B6, 2C9*1, 2C9*2, 2C18, 2C19, 2D6*1, and FMO1; sulprofos, CYP1A1, 1A2, 3A4, 2C9*1, 2C9*2, 2C9*3, 2C18, 2C19, 2D6*1, and FMO1; methiocarb, CYP1A1, 1A2, 3A4, 2B6, 2C9*1, 2C19, 2D6*1, and FMO1. Among these isoforms, members of the CYP2C subfamily often had the highest affinities and clearance rates. Moreover, sulfaphenazole, a CYP2C9 competitive inhibitor, inhibited disulfoton sulfoxidation by CYP2C9 (IC50 0.84 microM) as well as in HLMs. Ticlopidine, a CYP2C19 mechanism-based inhibitor, inhibited disulfoton sulfoxidation by CYP2C19 (IC50 after coincubation, 43.5 microM; IC50 after preincubation, 4.3 microM) and also in HLMs. Our results indicate that current models of the substrate binding site of the CYP2C subfamily would not effectively predict thioether pesticide metabolism. Thus, the substrate specificity of CYP2Cs is more extensive than is currently believed, and some reevaluation of structure-activity relationships may be required.     

Sulfoxidation of thioether-containing pesticides by the flavin-adenine dinucleotide-dependent monooxygenase of pig liver microsomes

In the presence of NADPH and under aerobic conditions, thioether-containing organophosphorus and carbamate pesticides were oxidized by the FAD-dependent monooxygenase (EC 1.14.13.8) purified from pig liver microsomes. The stoichiometric relationship between NADPH and substrate during the course of the reaction was 1:1, and the product, in the case of disulfoton and phorate, was the sulfoxide. The product was optically active and further oxidation to the sulfone was not apparent. Furthermore, the sulfoxides of disulfoton, phorate and croneton were not substrates for this enzyme. n-Octylamine, a known cytochrome P-450 inhibitor, increased the rate of sulfoxidation reactions catalyzed by the FAD-dependent monooxygenase. Structure-activity relationships were investigated using thirty-nine possible substrates. Structural changes around the thioether sulfur that affect nucleophilicity or that cause steric hindrance tended to decrease the sulfoxidation rate. With phosphorodithioates, changes around the phosphorus atom also affected oxidation of the thioether sulfur. Although neither the thiono nor the thiol sulfur atoms were attacked, substitution of either sulfur by oxygen decreased sulfoxidation. Thioether-containing O,O-dimethyl phosphorodithioates were not oxidized as readily as their O,O-diethyl analogs. Tetram and its analogs, which contain a tertiary amine group, were also substrates for this enzyme, presumably forming the N-oxide.     

Stereospecificity in the oxidation of phorate and phorate sulphoxide by purified FAD-containing mono-oxygenase and cytochrome P-450 isozymes

1. Both the cytochrome P-450-dependent mono-oxygenase system and the FAD-containing mono-oxygenase catalyse the sulphoxidation of thioether-containing organophosphate insecticides. Using purified FAD-containing mono-oxygenase and purified cytochrome P-450 isozymes isolated from mouse liver microsomes, the stereospecificity of the oxidation of phorate to (+)-and (-)-phorate sulphoxide and the further oxidations of the (+)-and (-)-phorate sulphoxides to the sulphone, the oxon sulphoxide and the oxon sulphone were examined. 2. The FAD-containing mono-oxygenase catalysed the formation of (-)-phorate sulphoxide, while two cytochrome P-450 isozymes (cytochrome P-450-B2, a constitutive form, and cytochrome P-450-PB, the principal form induced by phenobarbital) produced (+)-phorate sulphoxide. The other three constitutive cytochrome P-450 isozymes examined yielded racemic mixtures. 3. The FAD-containing mono-oxygenase had the lowest Km for the sulphoxidation reaction, 32 microM, while the Km values for the cytochrome P-450 isozymes ranged from 67 microM to 250 microM. No additional oxidation of phorate sulphoxide by the FAD-containing monooxygenase was detected using either (+)-phorate sulphoxide or (-)-phorate sulphoxide as substrates. 4. In contrast, all five cytochrome P-450 isozymes tested formed additional oxidation products; the (+)-phorate sulphoxide was the preferred substrate for all cytochrome P-450 forms. 5. The final oxidation product, phorate oxon sulphone, was derived by desulphuration of phorate sulphone, with the formation of the oxon sulphoxide being a terminal pathway.     

Differences in FMO2*1 allelic frequency between Hispanics of Puerto Rican and Mexican descent.

A polymorphism for the phase I drug-metabolizing enzyme, flavin-containing monooxygenase isoform 2 (FMO2), encoding either truncated inactive protein, FMO2X472 (FMO2.2A), or full-length active enzyme, FMO2Q472 (FMO2.1), is known and exhibits significant interethnic differences in allelic frequency. FMO2 is the major or sole FMO isoform expressed in the lung of most mammals, including nonhuman primates. To date, FMO2.1 has been found only in African-American and Hispanic populations, rendering individuals with this allele subject to drug metabolism that is potentially different from that of the general population. Approximately 26% of African-Americans (n = 180) possess the FMO2*1 allele. In preliminary studies, we initially estimated that 5% of Hispanics (n = 40) have the FMO2*1 allele, but access to large cohorts of individuals of defined national origin has allowed us to determine the occurrence among Mexican-American and Puerto Rican-American groups. We used allele-specific genotyping to detect FMO2*1 from 632 Hispanic individuals, including 280 individuals of Mexican origin and 327 individuals of Puerto Rican origin. Statistical analysis indicated that results from Mexican (five sample sources) and Puerto Rican (three sample sources) samples were consistent with the hypothesis of homogeneity within each group from different sources. Data were subsequently pooled across sources to test for evidence of a difference in occurrence of FMO2*1 between ethnic groups. There was strong evidence (p = 0.0066) that FMO2*1 is more common among Puerto Ricans (7%) than among individuals of Mexican descent (2%). The overall occurrence of FMO2*1 among Hispanics of all origins is estimated to be between 2 and 7%.     

Genetic polymorphisms of flavin-containing monooxygenase (FMO)

Mammalian flavin-containing monooxygenase (FMO) exists as six gene families and metabolizes a plethora of drugs and xenobiotics. The major FMO in adult human liver, FMO3, is responsible for trimethylamine (TMA) N-oxygenation. A number of FMO3 mutant alleles have been described and associated with a disease termed trimethylaminuria (TMAU). The TMAU patient excretes large amounts of TMA in urine and sweat. A more recent ethnically related polymorphism in expression of the major FMO in lung, FMO2, has been described. All Caucasians and Asians genotyped to date are homozygous for a CAG --> TAG amber mutation resulting in a premature stop codon and a nonfunctional protein truncated at AA 472 (wildtype FMO2 is 535 AA). This allele has been designated hFMO2*2A. Twenty-six percent of individuals of African descent and 5% of Hispanics genotyped to date carry at least one allele coding for full-length FMO2 (hFMO2*1 allele). Preliminary evidence indicates that FMO2.1 is very active toward the S-oxygenation of low MW thioureas, including the lung toxicant ethylene thiourea. Polymorphic expression of functional FMO2 in the individuals of African and Hispanic descent may markedly influence drug metabolism and/or xenobiotic toxicity in the lung.     

Mechanisms of fenthion activation in rainbow trout (Oncorhynchus mykiss) acclimated to hypersaline environments

Previous studies in rainbow trout have shown that acclimation to hypersaline environments enhances the toxicity to thioether organophosphate and carbamate pesticides. In order to determine the role of biotransformation in this process, the metabolism of the thioether organophosphate biocide, fenthion was evaluated in microsomes from gills, liver and olfactory tissues in rainbow trout (Oncorhynchus mykiss) acclimated to freshwater and 17‰ salinity. Hypersalinity acclimation increased the formation of fenoxon and fenoxon sulfoxide from fenthion in liver microsomes from rainbow trout, but not in gills or in olfactory tissues. NADPH-dependent and independent hydrolysis was observed in all tissues, but only NADPH-dependent fenthion cleavage was differentially modulated by hypersalinity in liver (inhibited) and gills (induced). Enantiomers of fenthion sulfoxide (65% and 35% R- and S-fenthion sulfoxide, respectively) were formed in liver and gills. The predominant pathway of fenthion activation in freshwater appears to be initiated through initial formation of fenoxon which may be subsequently converted to the most toxic metabolite fenoxon R-sulfoxide. However, in hypersaline conditions both fenoxon and fenthion sulfoxide formation may precede fenoxon sulfoxide formation. Stereochemical evaluation of sulfoxide formation, cytochrome P450 inhibition studies with ketoconazole and immunoblots indicated that CYP3A27 was primarily involved in the enhancement of fenthion activation in hypersaline-acclimated fish with limited contribution of FMO to initial sulfoxidation.     

The effect of detergents on the purified flavin-containing monooxygenase of mouse liver, kidney and lungs.

1. The effect of various commonly used membrane solubilizing detergents on the activity of the microsomal xenobiotic metabolizing enzyme, the flavin-containing monooxygenase (FMO) purified from mouse liver, kidney and lungs was determined. 2. Regardless of the type of detergent used, the effect on the enzyme activity was variable depending on the type of substrate used. 3. Emulgen 911 concentrations of up to 10% had very little effect on thiobenzamide-S-oxidation by liver, kidney or lung FMO. 4. While Emulgen 911 increased substrate dependent NADPH oxidation rate by thiourea and thioacetamide, it drastically reduced the activity toward the organophosphorous compounds, disulfoton, fenthion, fonofos and phorate at low concentrations. 5. Activities of fenthion, phorate and fonofos were decreased by 80, 65 and 55% by the inclusion of 0.25% Emulgen 911 in the assay mixture. 6. This decline in FMO activity for phorate was evident regardless of the type of detergent used. In contrast, thiourea dependent NADPH oxidation rate in the presence of various detergents was variable. 7. Thiourea oxidation rate was decreased by cholate and Zwittergent 3-12, whereas it was increased in the presence of Emulgen 911, Triton X-100 and Tween 20. 8. This study shows that before FMO activity is determined in the presence of detergents their effects should be carefully evaluated.     

Identification of active flavin-containing monooxygenase isoform 2 in human lung and characterization of expressed protein.

Full-length human (hFMO2.1) and monkey (mFMO2) flavin-containing monooxygenase proteins, which share 97% sequence identity, were produced by baculovirus-mediated expression in insect cells and assayed for S-oxygenation under conditions known to affect FMO activity. Both enzymes demonstrated maximal activity at pH 9.5; but hFMO2.1 retained significantly more activity than mFMO2 did at pH 9.0 and higher. hFMO2.1 also retained significantly more activity than mFMO2 did in the presence of magnesium and all detergents tested. Although hFMO2.1 had more residual activity after heating at 45 degrees C than mFMO2, under some conditions, both had less than 10% of control activity, whereas expressed rabbit FMO2 retained over 50% activity. Screening for NADPH-oxygenation by hFMO2.1, indicated that substituted thioureas with a small cross-sectional area (2.4-4.3 A) are good substrates, whereas 1,3-diphenylthiourea (11.2 A) was not oxygenated. We confirmed the presence of hFMO2.1 in lung tissue from a heterozygous individual (hFMO2*1/hFMO2*2A) by Western analysis and confirmed activity by S-oxygenation. These microsomes also demonstrated a heat-associated loss of activity similar to expressed hFMO2.1. The heat sensitivity of hFMO2.1 may partially explain why activity in post mortem human lung samples has previously been unreported. Individuals that have the FMO2*1 allele-encoding full-length hFMO2.1 may exhibit altered drug metabolism in the lung.     

Design and Evaluation of 3-(Benzylthio)benzamide Derivatives as Potent and Selective SIRT2 Inhibitors

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Extrahepatic metabolism of carbamate and organophosphate thioether compounds by the flavin-containing monooxygenase and cytochrome P450 systems.

The cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes are the major oxidative enzymes in phase I metabolism. Many organophosphate and carbamate thioether compounds are excellent substrates for these enzymes. Stereoselective sulfoxidation of fenthion and methiocarb by human liver, kidney, and microsomes was investigated. A high level of stereoselectivity in the formation of fenthion +-sulfoxide was observed in kidney and intestinal microsomes. This activity was not inhibited by the P450 inhibitor 1-aminobenzotriazole but was dramatically reduced following mild heat treatment. In liver, fenthion was metabolized to its sulfoxide in a nonstereoselective manner, and the activity was sensitive to both 1-aminobenzotriazole and heat treatment. The carbamate pesticide methiocarb also was sulfoxidated with a high degree of stereoselectivity in human kidney microsomes. Human liver microsomes formed both stereoisomers in equal amounts. Sulfoxide formation in kidney was not inhibited by 1-aminobenzotriazole but was abolished in liver microsomes. Formation of methiocarb sulfoxides was not observed in intestinal microsomes. The relative contribution of FMO1 and FMO3 to the sulfoxidation of carbophenothion, demeton-O, ethiofencarb, fonofos, and methiocarb also was investigated by using baculovirus-expressed recombinant proteins. FMO1 showed the highest catalytic activity for all pesticides. This study indicates that FMO1 may have a bigger role in extrahepatic metabolism than previously thought.     

The developmental neurotoxicity of organophosphorus insecticides: a direct role for the oxon metabolites

Several extensively used organophosphorus ester (OP) insecticides are phosphorothionates. The oxon metabolites of phosphorothionates have long been known to be responsible for the acute cholinergic neurotoxicity associated with OP poisoning. In addition, there is now sufficient evidence to suggest that the oxon metabolites may also be directly responsible for the particular neurotoxicity that phosphorothionate insecticides, and especially chlorpyrifos (CP) and diazinon (DZ), are known to inflict on the developing organism. In vitro data reveal that the oxons, which are present at increased levels in the developing brain, have the ability to directly disrupt, at toxicologically relevant doses, separately a number of neurodevelopmental processes, including those of neuronal proliferation, neuronal differentiation, gliogenesis and apoptosis. In most cases, the effects of the oxons are very potent. Inhibition of neuronal and glial cell differentiation by the oxons in particular is up to 1000-times stronger than that caused by their parent phosphorothionates. The neurodevelopmental toxicity of the oxons is not related to the inhibition of the enzymatic activity of acetylcholinesterase (AChE), but may be due to direct oxon interference with the morphogenic activity that AChE normally shows during neurodevelopment. Other possible direct targets of the oxons include neurodevelopmentally important cell signaling molecules and cytoskeletal proteins which have been found to be affected by the oxons and to which covalent binding of the oxons has been recently shown. Future studies should aim at confirming the developmental neurotoxic capacity of the oxons under in vivo conditions and they must also be extended to include OP parent insecticides with a PO moiety.     

The oxidative metabolism of aldicarb in pigs: in vivo-in vitro comparison

Aldicarb was administered (1 mg/kg b.w.) to four female pigs and the kinetics of its major oxidized metabolites (sulfoxide and sulfone) was followed for 6 hours. The in vitro transformations of the carbamate pesticide into these two still active metabolites were also investigated in hepatocytes and in microsomes from pig livers. In all cases, aldicarb was quickly oxidized to the sulfoxide (major metabolite) and only a minor quantity of sulfone was produced. The in vivo toxic symptomatology was related to the peak serum concentration of sulfoxide, suggesting that this metabolite is principally responsible for the aldicarb toxicity. Selective in vitro inhibition of flavin-containing and cytochrome P-450 monooxygenases confirmed that the former enzymes catalyze mainly sulfoxide production whereas the latter that of sulfone.     

In vitro sulfoxidation of aldicarb by hepatic microsomes of channel catfish, Ictalurus punctatus.

The carbamate pesticide, aldicarb, demonstrates significant acute toxicity in mammals, birds, and fish, and is readily biotransformed by most organisms studied. Metabolic products of aldicarb include the more toxic sulfoxide and the less toxic sulfone as two of the major products. Both the cytochrome P450 (CYP) and the flavin monooxygenase systems (FMO) are involved in this process. This study examined the capacities of liver microsomes of male channel catfish (Ictalurus punctatus), which lack FMO, to biotransform aldicarb in vitro. In addition, the acetylcholinesterase inhibitory potencies of aldicarb and its sulfoxide and sulfone derivatives were determined. For metabolism studies, incubations of [14C]-aldicarb (0.1mM) were carried out for up to 15-90 min using 1.0 mg/mL of hepatic microsomal protein. Total NADPH- dependent biotransformation was low (< 3.0% conversion to polar metabolites), and was inhibited by carbon monoxide. The only metabolite detected was aldicarb sulfoxide (Kmapp = 53.8 +/- 25.3 microM; Vmaxapp = 0.040 +/- 0.007 nmol/min/mg). Treatment of fish with the CYP modulators beta-naphthoflavone (BNF, 50 mg/kg) and ethanol (EtOH, 1.0% aqueous) had no effect on sulfoxide production. No correlation existed between CYP isoform expression (determined by western blot) and aldicarb sulfoxidation rates, suggesting the involvement of an unmeasured CYP isoform or involvement of several isoforms with low specificity. This study indicates that a low rate of bioactivation of aldicarb to aldicarb sulfoxide may be responsible for the resistance of channel catfish to aldicarb toxicity relative to that of other piscine species.     

Biotransformation of pantoprazole by the fungus Cunninghamella blakesleeana

To investigate the biotransformation of pantoprazole, a proton-pump inhibitor, by filamentous fungus and further to compare the similarities between microbial transformation and mammalian metabolism of pantoprazole, four strains of Cunninghamella (C. blakesleeana AS 3.153, C. echinulata AS 3.2004, C. elegans AS 3.156, and AS 3.2028) were screened for the ability to catalyze the biotransformation of pantoprazole. Pantoprazole was partially metabolized by four strains of Cunninghamella, and C. blakesleeana AS 3.153 was selected for further investigation. Three metabolites produced by C. blakesleeana AS 3.153 were isolated using semi-preparative HPLC, and their structures were identified by a combination analysis of LC/MS(n) and NMR spectra. Two further metabolites were confirmed with the aid of synthetic reference compounds. The structure of a glucoside was tentatively assigned by its chromatographic behavior and mass spectroscopic data. These six metabolites were separated and quantitatively assayed by liquid chromatography-ion trap mass spectrometry. After 96h of incubation with C. blakesleeana AS 3.153, approximately 92.5% of pantoprazole was metabolized to six metabolites: pantoprazole sulfone (M1, 1.7%), pantoprazole thioether (M2, 12.4%), 6-hydroxy-pantoprazole thioether (M3, 1.3%), 4'-O-demethyl-pantoprazole thioether (M4, 48.1%), pantoprazole thioether-1-N-beta-glucoside (M5, 20.6%), and a glucoside conjugate of pantoprazole thioether (M6, 8.4%). Among them, M5 and M6 are novel metabolites. Four phase I metabolites of pantoprazole produced by C. blakesleeana were essentially similar to those obtained in mammals. C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of pantoprazole.     

Synthesis of fenthion sulfoxide and fenoxon sulfoxide enantiomers: effect of sulfur chirality on acetylcholinesterase activity

Earlier reports have demonstrated that recombinant flavin-containing monooxygenase 1 (FMO1) catalyzes the oxidation of the organophosphate pesticide fenthion to (+)-fenthion sulfoxide in a stereoselective fashion. In order to elucidate the absolute configuration of the sulfoxide metabolite produced, we established an efficient synthesis of both enantiomers of fenthion sulfoxide, which were transformed into chiral fenoxon sulfoxides using a two-step protocol. The use of chiral oxidants, namely, N-(phenylsulfonyl)(3,3-dichlorocamphoryl) oxaziridines, afforded enantioenriched fenthion sulfoxides with high ee (>82%) from the parent sulfide. Single recrystallizations afforded chiral fenthion sulfoxides with >99% ee, measured by chiral HPLC analysis. The absolute configuration of the (+)-sulfoxide generated from fenthion metabolism by FMO1 was determined to be (R)-(+)-fenthion sulfoxide, confirmed by X-ray crystallographic analysis of the (S)-(-)-antipode. Inhibition of human recombinant (hrAChE) and electric eel (eeAChE) acetylcholinesterase were assayed with fenthion, fenoxon, and the racemates and enantiomers of fenthion sulfoxide and fenoxon sulfoxide. Results revealed stereoselective inhibition with (R)-(+)-fenoxon sulfoxide when compared with that of (S)-(-)-fenoxon sulfoxide (IC50 of 6.9 and 6.5 microM vs 230 and 111 microM in hrAChE and eeAChE, respectively). Fenthion sulfoxide (R or S enantiomers) did not present anti-AChE properties. Although the stereoselective sulfoxidation of fenthion to (R)-(+)-fenthion sulfoxide by FMO represents a detoxification pathway, the results of this study support the notion that subsequent oxidative desulfuration of (R)-(+)-fenthion sulfoxide (in vivo) may represent a critical bioactivation pathway, resulting in the production of (R)-(+)-fenoxon sulfoxide, a potent AChE inhibitor.     

The potentially deleterious functional variant flavin-containing monooxygenase 2*1 is at high frequency throughout sub-Saharan Africa

BACKGROUND: The drug-metabolizing enzyme flavin-containing monooxygenase 2 (FMO2) is the predominant FMO isoform present in the lung of most mammals, including non-human primates. All Europeans and Asians tested have been shown to be homozygous for a non-functional variant, FMO2*2A, which contains a premature stop codon due to a single-nucleotide change in exon 9 (g.23238C>T). The ancestral allele, FMO2*1, encodes a functionally active protein and has been found in African-Americans (26%) and Hispanics (2% to 7%). Possessing this variant increases the risk of pulmonary toxicity when exposed to thioureas, a widely used class of industrial compounds. FMO2 may also be involved in the metabolism of drugs that are used to treat diseases that are prevalent in Africa. RESULTS AND CONCLUSION: We conducted a survey of g.23238C>T variation across Africa that revealed that the distribution of this SNP is relatively homogeneous across sub-Saharan Africa, with approximately one third of individuals possessing at least one FMO2*1 allele, though in some populations the incidence of these individuals approached 50%. Thus many sub-Saharan Africans may be at substantially increased health risk when encountering thiourea-containing substrates of FMO2. Analysis of HapMap data with the Long-Range Haplotype test found no evidence for positive selection of either 23238C>T allele and maximum-likelihood coalescent analysis indicated that this mutation occurred some 500,000 years before present. This study demonstrates the value of performing genetic surveys in Africa, a continent in which human genetic diversity is thought to be greatest, but where studies of the distribution of this diversity are few.     

Rat liver metabolism of benzo[b]naphtho[2,1-d]thiophene.

Thioarenes, sulfur-containing polycyclic aromatic hydrocarbons, have been detected in a number of environmental sources. The metabolism of one thioarene, benzo[b]naphtho[2,1,d]thiophene ([2,1]BNT), by F344 rat liver 9000g supernatant (S-9) was studied. [2,1]BNT which is structurally analogous and has similar carcinogenic potency to chrysene, was metabolized to six ethyl acetate-extractable metabolites when incubated with S-9 from Aroclor 1254-treated F344 rats. Each metabolite was collected from reverse-phase HPLC, and their identities were determined by analysis of MS and NMR data. In order of elution from HPLC they are as follows: (1) trans-1,2-dihydroxy-1,2-dihydrobenzo[b]naphtho[2,1-d]thiophene, (2) benzo[b]naphtho[2,1-d]-thiophene sulfone, (3) benzo[b]naphtho[2,1-d]thiophene sulfoxide, (4) trans-3,4-dihydroxy-3,4-dihydrobenzo[b]naphtho[2,1-d]thiophene, (5) 8- or 9-hydroxybenzo[b]naphtho[2,1-d]thiophene, and (6) 7-hydroxybenzo[b]naphtho[2,1-d]thiophene. In addition, the identities of metabolites 1, 2, 3, and 4 were confirmed by comparison to standards. The syntheses of the sulfone and sulfoxide of [2,1]BNT are reported here. The syntheses of the dihydrodiols were reported previously. Metabolite 5, a hydroxy[2,1]BNT, was the major metabolite formed by liver S-9 from untreated F344 rats. Microsomal preparations from these rats also produced significant amounts of the dihydrodiols, 1 and 4, and the sulfoxide, 3. Microsomes prepared from Wistar rats produced dihydrodiols and the sulfone and sulfoxide of [2,1]BNT. Therefore, [2,1]BNT is metabolized by both ring oxidation and sulfur oxidation in these two strains of rats.     

Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon

Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC₅₀ values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k_inact/K_I) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC₅₀ values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1 and CES2 respectively. Together, the results define the kinetics of inhibition of three important hydrolytic enzymes by activated metabolites of widely used agrochemicals.     

Concentration-dependent binding of chlorpyrifos oxon to acetylcholinesterase.

The organophosphorus insecticides have been known for many years to cause cholinergic crisis in humans as a result of the inhibition of the critical enzyme acetylcholinesterase. The interactions of the activated, toxic insecticide metabolites (termed oxons) with acetylcholinesterase have been studied extensively for decades. However, more recent studies have suggested that the interactions of certain anticholinesterase organophosphates with acetylcholinesterase are more complex than previously thought since their inhibitory capacity has been noted to change as a function of inhibitor concentration. In the present report, chlorpyrifos oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate) was incubated with human recombinant acetylcholinesterase in the presence of p-nitrophenyl acetate in order to better characterize kinetically the interactions of this oxon with enzyme. Determination of the dissociation constant, Kd, and the phophorylation rate constant, k2, for chlorpyrifos oxon with a range of oxon and p-nitrophenyl acetate concentrations revealed that Kd, but not k2, changed as a function of oxon concentration. Changes in p-nitrophenyl acetate concentrations did not alter these same kinetic parameters. The inhibitory capacity of chlorpyrifos oxon, as measured by ki (k2/Kd), was also affected as a result of the concentration-dependent alterations in binding affinity. These results suggest that the concentration-dependent interactions of chlorpyrifos oxon with acetylcholinesterase resulted from a different mechanism than the concentration-dependent interactions of acetylthiocholine. In the latter case, substrate bound to the peripheral anionic site of acetylcholinesterase has been shown to reduce enzyme activity by blocking the release of the product thiocholine from the active site gorge. With chlorpyrifos oxon, the rate of release of 3,5,6-trichloro-2-pyridinol is irrelevant since the active site is not available to interact with other oxon molecules after phosphorylation of Ser-203 has occurred.     

Cytochrome P450 2C8 and flavin-containing monooxygenases are involved in the metabolism of tazarotenic acid in humans.

Upon oral administration, tazarotene is rapidly converted to tazarotenic acid by esterases. The main circulating agent, tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite. Therefore, alterations in the metabolic clearance of tazarotenic acid may have significant effects on its systemic exposure. The objective of this study was to identify the human liver microsomal enzymes responsible for the in vitro metabolism of tazarotenic acid. Tazarotenic acid was incubated with 1 mg/ml pooled human liver microsomes, in 100 mM potassium phosphate buffer (pH 7.4), at 37 degrees C, over a period of 30 min. The microsomal enzymes that may be involved in tazarotenic acid metabolism were identified through incubation with microsomes containing cDNA-expressed human microsomal isozymes. Chemical inhibition studies were then conducted to confirm the identity of the enzymes potentially involved in tazarotenic acid metabolism. Reversed-phase high performance liquid chromatography was used to quantify the sulfoxide metabolite, the major metabolite of tazarotenic acid. Upon incubation of tazarotenic acid with microsomes expressing CYP2C8, flavin-containing monooxygenase 1 (FMO1), or FMO3, marked formation of the sulfoxide metabolite was observed. The involvement of these isozymes in tazarotenic acid metabolism was further confirmed by inhibition of metabolite formation in pooled human liver microsomes by specific inhibitors of CYP2C8 or FMO. In conclusion, the in vitro metabolism of tazarotenic acid to its sulfoxide metabolite in human liver microsomes is mediated by CYP2C8 and FMO.