Neonicotinoid insecticides: reduction and cleavage of imidacloprid nitroimine substituent by liver microsomal and cytosolic enzymes

The major insecticide imidacloprid (IMI) is known to be metabolized by human cytochrome P450 3A4 with NADPH by imidazolidine hydroxylation and dehydrogenation to give 5-hydroxy-imidacloprid and the olefin, respectively, and by nitroimine reduction and cleavage to yield the nitrosoimine, guanidine, and urea derivatives. More extensive metabolism by human or rabbit liver microsomes with NADPH or rabbit liver cytosol without added cofactor reduces the IMI N-nitro group to an N-amino substituent, i.e., the corresponding hydrazone. A major metabolite on incubation of IMI in the human microsome-NADPH system is tentatively assigned by LC/MS as a 1,2,4-triazol-3-one derived from the hydrazone; the same product is obtained on reaction of the hydrazone with ethyl chloroformate. The hydrazone and proposed triazolone are considered here together (referred to as the hydrazone) for quantitation. Only a portion of the microsomal reduction and cleavage of the nitroimine substituent is attributable to a CYP450 enzyme. The cytosolic enzyme conversion to the hydrazone is inhibited by added cofactors (NAD > NADH > NADP > NADPH) and enhanced by an argon instead of an air atmosphere. The responsible cytosolic enzyme(s) does not appear to be DT-diaphorase (which is inhibited by several neonicotinoids), aldose reductase, aldehyde reductase, or xanthine oxidase. However, the cytosolic metabolism of IMI is inhibited by several aldo-keto-reductase inhibitors (i.e., alrestatin, EBPC, Ponalrestat, phenobarbital, and quercetin). Other neonicotinoids with nitroimine, nitrosoimine, and nitromethylene substituents are probably also metabolized by "neonicotinoid nitro reductase(s)" since they serve as competitive substrates for [(3)H]IMI metabolism.     

硝克柳胺在大鼠和人肝脏的体外代谢研究

研究硝克柳胺在大鼠肝微粒体和胞浆中的代谢动力学，鉴定硝克柳胺在大鼠和人肝微粒体中的主要代谢产物及参与代谢的药物代谢酶。采用高效液相色谱-紫外检测(HPLC-UV)方法测定大鼠肝微粒体和胞浆中硝克柳胺浓度，应用选择性抑制剂鉴定参与硝克柳胺代谢的药物代谢酶类型，采用液相色谱-串联质谱联用(LC-MS/MS)法分离鉴定硝克柳胺在大鼠和人肝微粒体中的主要代谢产物。硝克柳胺在大鼠和人肝微粒体的主要代谢产物(M₁)为硝基还原产物［3-(3′-羧基-4′-羟基苯胺羰基)-6-氨基-7-羟基-8-甲基香豆素］，大鼠体内(血浆、尿液、胆汁及肝组织)主要代谢产物与M₁一致。硝克柳胺的体外代谢是依赖多个药物代谢酶参与的酶促反应，包括微粒体CYP450还原酶、细胞色素b₅还原酶和CYP2C6以及胞浆NAD(P)H脱氢酶和黄嘌呤氧化酶。     

Characterization of the enzymes involved in the in vitro metabolism of amrubicin hydrochloride

The in vitro metabolism of amrubicin by rat and human liver microsomes and cytosol was examined. The main metabolic routes in both species were reductive deglycosylation and carbonyl group reduction in the side-chain. In vitro metabolism of amrubicinol by rat and human liver microsomes and cytosol was also examined and the main metabolic route of this active metabolite was reductive deglycosylation. Metabolism of amrubicin in human liver microsomes was inhibited by TlCl(3) and that in human liver cytosol was inhibited by dicumarol and quercetin. Generation of amrubicinol was inhibited only by quercetin. The results indicate that metabolism of amrubicin is mediated by NADPH-cytochrome P450 reductase, NADPH:quinone oxidoreductase and carbonyl reductase. In addition, generation of amrubicinol is mediated by carbonyl reductase. Metabolism of amrubicinol in human liver microsomes was inhibited by TlCl(3) and that in human liver cytosol was inhibited by dicumarol. The results indicate that metabolism of amrubicinol is mediated by NADPH-cytochrome P450 reductase and NADPH:quinone oxidoreductase. To investigate the influence of cisplatin on the metabolism of amrubicin and amrubicinol, human liver microsomes and cytosol were pre-incubated with cisplatin. This did not change the rates of amrubicin and amrubicinol metabolism in either human liver microsomes or cytosol.     

Characterization of the enzymes involved in the in vitro metabolism of amrubicin hydrochloride

The in vitro metabolism of amrubicin by rat and human liver microsomes and cytosol was examined. The main metabolic routes in both species were reductive deglycosylation and carbonyl group reduction in the side-chain. In vitro metabolism of amrubicinol by rat and human liver microsomes and cytosol was also examined and the main metabolic route of this active metabolite was reductive deglycosylation. Metabolism of amrubicin in human liver microsomes was inhibited by TlCl₃ and that in human liver cytosol was inhibited by dicumarol and quercetin. Generation of amrubicinol was inhibited only by quercetin. The results indicate that metabolism of amrubicin is mediated by NADPH-cytochrome P450 reductase, NADPH:quinone oxidoreductase and carbonyl reductase. In addition, generation of amrubicinol is mediated by carbonyl reductase. Metabolism of amrubicinol in human liver microsomes was inhibited by TlCl₃ and that in human liver cytosol was inhibited by dicumarol. The results indicate that metabolism of amrubicinol is mediated by NADPH-cytochrome P450 reductase and NADPH:quinone oxidoreductase. To investigate the influence of cisplatin on the metabolism of amrubicin and amrubicinol, human liver microsomes and cytosol were pre-incubated with cisplatin. This did not change the rates of amrubicin and amrubicinol metabolism in either human liver microsomes or cytosol.     

Metabolism of the styrene metabolite 4-vinylphenol by rat and mouse liver and lung

Styrene is a widely used chemical in the reinforced plastics industry and in polystyrene production. Its primary metabolic pathway to styrene oxide and then to styrene glycol, which is further metabolized to mandelic acid and phenylglyoxylic acid, has been well studied. However, a few studies have reported finding a minor metabolite, 4-vinylphenol (4-VP), in rat and human urine. The present studies sought to determine if the formation and metabolism of 4-VP in rat and mouse liver and lung preparations could be measured. When styrene was incubated with hepatic and pulmonary microsomal preparations, 4-VP formation could not be measured in these preparations. However, considerable 4-VP metabolizing activity, as determined by the loss of 4-VP, was observed in both mouse and rat liver and lung microsomal preparations. 4-Vinylphenol metabolizing activity in mouse liver microsomes was three times greater than that in rat liver microsomes, and activity in mouse lung microsomes was eight times greater than that in rat lung microsomes. This activity was completely absent in the absence of NADPH. Studies with cytochrome P-450 inhibitors indicated the involvement of CYP2E1 and CYP2F2. Induction of CYP2E1 by pyridine resulted in an increase in 4-VP metabolism by mouse hepatic microsomes but not by pulmonary microsomes. The metabolite(s) formed as a result of this oxidative pathway remain to be identified. In additional studies, glutathione conjugation appeared to be involved in 4-VP metabolism with the highest activity being in mouse lung, with or without the addition of NADPH.     

Metabolism of 2,6-dinitro[3-3H]toluene by human and rat liver microsomal and cytosolic fractions.

1. 2,6-Dinitrotoluene (2,6-DNT) metabolism by human liver and male Fischer F344 rat liver subcellular fractions under aerobic (100% oxygen) and anaerobic (100% nitrogen) incubation conditions was examined. Under aerobic conditions the major 2,6-DNT metabolite formed by hepatic microsomes was 2,6-dinitrobenzyl alcohol (2,6-DNBalc); under anaerobic conditions 2-amino-6-nitrotoluene (2Am6NT) was the major metabolite. 2. Rates of 2,6-DNBalc formation by human and rat liver microsomes under aerobic conditions were 247 and 132 pmol/min per mg protein, respectively. Rates of 2Am6NT formation by human and rat liver microsomes under anaerobic conditions were 292 and 285 pmol/min per mg protein, respectively. Anaerobic reduction of 2,6-DNT to 2Am6NT by rat and human liver microsomes was inhibited by carbon monoxide and metyrapone, which indicates that microsomal metabolism of 2,6-DNT to 2Am6NT is mediated by cytochrome P-450. 3. Liver cytosolic fractions also metabolized 2,6-DNT to 2Am6NT under anaerobic conditions. Formation of 2Am6NT by human and rat liver cytosols was supported by hypoxanthine, NADPH and NADH. Allopurinol inhibited the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by rat, but not human, liver cytosol. Dicumarol inhibited the NADPH-supported anaerobic metabolism of 2,6-DNT by human, but not rat, liver cytosol. These results indicate that xanthine oxidase contributes to the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by human liver cytosol.     

Metabolism of 2,6-dinitro[3-3H]toluene by human and rat liver microsomal and cytosolic fractions

1. 2,6-Dinitrotoluene (2,6-DNT) metabolism by human liver and male Fischer F344 rat liver subcellular fractions under aerobic (100% oxygen) and anaerobic (100% nitrogen) incubation conditions was examined. Under aerobic conditions the major 2,6-DNT metabolite formed by hepatic microsomes was 2,6-dinitrobenzyl alcohol (2,6-DNBalc); under anaerobic conditions 2-amino-6-nitrotoluene (2Am6NT) was the major metabolite.

2. Rates of 2,6-DNBalc formation by human and rat liver microsomes under aerobic conditions were 247 and 132pmol/min per?mg protein, respectively. Rates of 2Am6NT formation by human and rat liver microsomes under anaerobic conditions were 292 and 285pmol/min per?mg protein, respectively. Anaerobic reduction of 2,6-DNT to 2Am6NT by rat and human liver microsomes was inhibited by carbon monoxide and metyrapone, which indicates that microsomal metabolism of 2,6-DNT to 2Am6NT is mediated by cytochrome P-450.

3. Liver cytosolic fractions also metabolized 2,6-DNT to 2Am6NT under anaerobic conditions. Formation of 2Am6NT by human and rat liver cytosols was supported by hypoxanthine, NADPH and NADH. Allopurinol inhibited the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by rat, but not human, liver cytosol. Dicumarol inhibited the NADPH-supported anaerobic metabolism of 2,6-DNT by human, but not rat, liver cytosol. These results indicate that xanthine oxidase contributes to the hypoxanthline-supported anaerobic metabolism of 2,6-DNT by human liver cytosol.     

Reductive metabolism of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one, by rat liver preparations.

The reduction of the triple bond and carbonyl group of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one (PBYO), by rat liver microsomes and cytosol was investigated. The triple-bond-reduced product trans-4-phenyl-3-buten-2-one (PBO) and the carbonyl-reduced product 4-phenyl-3-butyn-2-ol (PBYOL) were formed when PBYO was incubated with rat liver microsomes in the presence of NADPH. The triple bond of 1-phenyl-1-butyne, deprenyl, ethynylestradiol, ethinamate, and PBYOL, in which the triple bond is not adjacent to a carbonyl group, were not reduced by liver microsomes even in the presence of NADPH. PBO was further reduced to 4-phenyl-2-butanone (PBA) by liver cytosol with NADPH. PBYOL was also formed from PBYO by liver cytosol in the presence of NADPH or NADH. The microsomal triple-bond reductase activity was inhibited by disulfiram, 7-dehydrocholesterol, and 18 beta-glycyrrhetinic acid but not beta-diethylaminoethyldiphenylpropylacetate or carbon monoxide. The triple-bond reductase activity in liver microsomes was not enhanced by several inducers of the rat cytochrome P450 system. These results suggested that the triple-bond reduction is caused by a new type of reductase, not cytochrome P450. The microsomal and cytosolic carbonyl reductase activities were not inhibited by quercitrin, indomethacin, or phenobarbital. Only S-PBYOL was formed from PBYO by liver cytosol. In contrast, liver microsomes produced R-PBYOL together with the S-enantiomer to some extent. Ethoxyresorufin-O-dealkylase activity in rat liver microsomes was markedly inhibited by PBYO and PBO, partly by PBYOL, but not by PBA.     

Role of cytochromes P450 in the metabolism of methyl tert -butyl ether in human livers

Methyl tert-butyl ether (MTBE) is widely used as a gasoline oxygenate for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health is a major public concern. However, information on the metabolism of MTBE in human tissues is lacking. The present study demonstrates that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and a marker for exposure to MTBE. The activity is localized in the microsomal fraction (125 ± 11 pmol TBA/min per mg protein, n = 8) but not in the cytosol. This activity level in human liver microsomes is approximately one-half of the value in rat and mouse liver microsomes. Formation of TBA in human liver microsomes is NADPH-dependent, and is significantly inhibited by carbon monoxide (CO), an inhibitor of cytochrome P450 (CYP) enzymes, suggesting that CYP enzymes play a critical role in the metabolism of MTBE in human livers. Both CYP2A6 and 2E1 are known to be constitutively expressed in human livers. To examine their involvement in MTBE metabolism, human CYP2A6 and 2E1 cDNAs were individually co-expressed with human cytochrome P450 reductase by a baculovirus expression system and the expressed enzymes were used for MTBE metabolism. The turnover number for CYP2A6 and 2E1 was 6.1 and 0.7 nmol TBA/min per nmol P450, respectively. The heterologously expressed human CYP2A6 was also more active than 2E1 in the metabolism of two other gasoline ethers, ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME). Although the contributions of other human CYP forms to MTBE metabolism remain to be determined, these results strongly suggest that CYP enzymes play an important role in the metabolism of MTBE in human livers. Received: 2 July 1996 / Accepted: 7 November 1996     

Effects of propranolol on xenobiotic enzyme activities in rat type II pneumocytes and alveolar macrophages in vivo

The accumulation of basic drugs (cationic amphiphilic), such as beta-adrenergic antagonists, by pulmonary tissue is well known. Ring hydroxylation of nonselective beta-adrenergic blocking agent propranolol is mediated mainly by cytochrome P450 (CYP) 2D6 and N-desisopropylation by CYP1A2 in human and rat liver microsomes. In this study, the repeated administration of propranolol resulted in a marked inhibition of hepatic metabolism and an increase in its systemic availability, due to covalent binding of reactive metabolites (formed from 4-OH-propranolol) to liver microsomal P4502D enzymes. The absence of CYP1A2 and the presence of CYP2D in the lung suggest a different pulmonary metabolism of propranolol in comparison with those in the liver. In this study, we investigated its effects in vivo on some xenobiotic-metabolizing enzymes in rat type II pneumocytes (RTII) and rat alveolar macrophages (RAM). Twenty hours after the last multiple (7 days) oral administration, propranolol (100 mg/kg b.w.) decreased NADPH cytochrome c reductase activity and cytochrome P-450-dependent dealkylation of 7-benzyloxyresorufin (BROD) (CYP1A1, 2A1, 3A1) and 7-ethoxyresorufin (EROD) (CYP1A1) in RTII, while glutathione-S-transferase (GST), DT-diaphorase (QR), gamma-glutamyl transferase (gamma-GT) activities, intracellular reduced glutathione level and dealkylation of 7-pentoxyresorufin (PROD) (CYP2B1) were not changed. It was found that propranolol significantly increased NADPH cytochrome c reductase and BROD activities in RAM. The results suggest a different susceptibility of RTII and RAM to propranolol and its contrary effects on lung xenobiotic-metabolizing enzyme activities in both types of cells.     

Identification of catalase in human livers as a factor that enhances phenytoin dihydroxy metabolite formation by human liver microsomes

We have reported previously that the formation of a 3',4'-dihydroxylated metabolite of phenytoin (3',4'-diHPPH) by human liver microsomal cytochrome P450 (P450) is enhanced by the addition of human liver cytosol [Komatsu et al., Drug Metab Dispos 2000;28:1361-8]. The enhancing factor was determined in this study. The addition of cytosolic proteins precipitated by 50% ammonium sulfate to incubation mixtures increased the rate of microsomal 3',4'-diHPPH formation. This fraction was separated further by diethylaminoethyl-, carboxymethyl-, and hydroxyapatite-column chromatography. The amino acid sequence of the purified protein of approximately 55kDa by electrophoresis revealed this protein to be a catalase. The addition of purified or authentic catalase to the incubation mixtures increased the rates of microsomal 3',4'-diHPPH formation from 3'- and 4'-hydroxylated metabolites and from phenytoin in a concentration-dependent manner. In reconstituted systems containing CYP2C9, CYP2C19, and CYP3A4, the formation of 3',4'-diHPPH was also enhanced by catalase to different extents. This is the first report that catalase in livers enhances drug oxidation activities catalyzed by P450 in human liver microsomes.     

Stereo- and regioselectivity account for the diversity of dehydroepiandrosterone (DHEA) metabolites produced by liver microsomal cytochromes P450.

The purpose of this study was to quantify the oxidative metabolism of dehydroepiandrosterone (3beta-hydroxy-androst-5-ene-17-one; DHEA) by liver microsomal fractions from various species and identify the cytochrome P450 (P450) enzymes responsible for production of individual hydroxylated DHEA metabolites. A gas chromatography-mass spectrometry method was developed for identification and quantification of DHEA metabolites. 7alpha-Hydroxy-DHEA was the major oxidative metabolite formed by rat (4.6 nmol/min/mg), hamster (7.4 nmol/min/mg), and pig (0.70 nmol/min/mg) liver microsomal fractions. 16alpha-Hydroxy-DHEA was the next most prevalent metabolite formed by rat (2.6 nmol/min/mg), hamster (0.26 nmol/min/mg), and pig (0.16 nmol/min/mg). Several unidentified metabolites were formed by hamster liver microsomes, and androstenedione was produced only by pig microsomes. Liver microsomal fractions from one human demonstrated that DHEA was oxidatively metabolized at a total rate of 7.8 nmol/min/mg, forming 7alpha-hydroxy-DHEA, 16alpha-hydroxy-DHEA, and a previously unidentified hydroxylated metabolite, 7beta-hydroxy-DHEA. Other human microsomal fractions exhibited much lower rates of metabolism, but with similar metabolite profiles. Recombinant P450s were used to identify the cytochrome P450s responsible for DHEA metabolism in the rat and human. CYP3A4 and CYP3A5 were the cytochromes P450 responsible for production of 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, and 16alpha-hydroxy-DHEA in adult liver microsomes, whereas the fetal/neonatal form CYP3A7 produced 16alpha-hydroxy and 7beta-hydroxy-DHEA. CYP3A23 uniquely formed 7alpha-hydroxy-DHEA, whereas other P450s, CYP2B1, CYP2C11, and CYP2D1, were responsible for 16alpha-hydroxy-DHEA metabolite production in rat liver microsomal fractions. These results indicate that the stereo- and regioselectivity of hydroxylation by different P450s account for the diverse DHEA metabolites formed among various species.     

The enzymology of doxorubicin quinone reduction in tumour tissue.

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.     

Oxidative inactivation of cytochrome P-450 1A (CYP1A) stimulated by 3,3',4,4'-tetrachlorobiphenyl: production of reactive oxygen by vertebrate CYP1As.

Microsomal cytochrome P-450 1A (CYP1A) in a vertebrate model (the teleost fish scup) is inactivated by the aryl hydrocarbon receptor agonist 3,3',4,4'-tetrachlorobiphenyl (TCB). Here, the mechanism of CYP1A inactivation and its relationship to reactive oxygen species (ROS) formation were examined by using liver microsomes from scup and rat and expressed human CYP1As. In vitro inactivation of scup CYP1A activity 7-ethoxyresorufin O-deethylation by TCB was time dependent, NADPH dependent, oxygen dependent, and irreversible. TCB increased microsomal NADPH oxidation rates, and CYP1A inactivation was lessened by adding cytochrome c. CYP1A inactivation was accompanied by loss of spectral P-450, a variable loss of heme and a variable appearance of P-420. Rates of scup liver microsomal metabolism of TCB were < 0.5 pmol/min/mg, 25-fold less than the rate of P-450 loss. Non-heme iron chelators, antioxidant enzymes, and ROS scavengers had no influence on inactivation. Inactivation was accelerated by H(2)O(2) and azide but not by hydroxylamine or aminotriazole. TCB also inactivated rat liver microsomal CYP1A, apparently CYP1A1. Adding TCB to scup or rat liver microsomes containing induced levels of CYP1A, but not control microsomes, stimulated formation of ROS; formation rates correlated with native CYP1A1 content. TCB stimulated ROS formation by baculovirus-expressed human CYP1A1 but not CYP1A2. The results indicate that TCB uncouples the catalytic cycle of CYP1A, ostensibly CYP1A1, resulting in formation of ROS within the active site. These ROS may inactivate CYP1A or escape from the enzyme. ROS formed by CYP1A1 may contribute to the toxicity of planar halogenated aromatic hydrocarbons.     

Cyclosporin A-induced free radical generation is not mediated by cytochrome P-450

1. Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA). 2. The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS. 3. We show that CsA (1 -- 10 microM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7-dichlorofluorescin diacetate. 4. Using cytochrome c as substrate, we show that CsA (10 microM) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. 5. CsA (10 microM) did not uncouple the electron flow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. 6. CsA (10 microM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. 7. Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. 8. These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes.     

Nitroreduction of 5-nitrofuran derivatives by rat liver xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase

Rat liver cytosol and microsomes catalyzed the nitroreduction of N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide (NFTA), a potent carcinogen for the mouse, rat, hamster and dog, and formed a metabolite capable of binding to protein. The cytosol nitroreductase was NADH or hypoxanthine dependent and strongly inhibited by a low concentration of allopurinol. Partial purification of the cytosol nitroreductase resulted in the parallel purification of nitroreductase and xanthine oxidase. Furthermore, NFTA, as well as 11 other nitrofurans, was reduced by purified milk xanthine oxidase. The metabolite formed was capable of binding to protein. These observations suggested that the cytosol nitroreductase activity was due to xanthine oxidase. The microsomal nitroreductase, which is NADPH dependent, was probably NADPH-cytochrome c reductase. Microsomal nitroreductase activity paralleled NADPH-cytochrome c reductase activity in rats pretreated with allylisopropylacetamide (AIA), phenobarbital or 3-methylcholanthrene (3-MC), but did not parallel the level of microsomal cytochrome P-450. A partial purification of the microsomal nitroreductase resulted in the parallel purification of nitroreductase and NADPH-cytochrome c reductase. The NFTA metabolite formed by the partially purified enzyme was capable of binding to protein. Other nitrofurans also were reduced by the same enzyme preparation. Hence, microsomal nitroreductase activity may be due to NADPH-cytochrome c reductase.     

Dydrogesterone metabolism in human liver by aldo-keto reductases and cytochrome P450 enzymes

1.?The metabolism of dydrogesterone was investigated in human liver cytosol (HLC) and human liver microsomes (HLM). Enzymes involved in dydrogesterone metabolism were identified and their relative contributions were estimated.

2.?Dydrogesterone clearance was clearly higher in HLC compared to HLM. The major active metabolite 20α-dihydrodydrogesterone (20α-DHD) was only produced in HLC.

3.?The formation of 20α-DHD by cytosolic aldo-keto reductase 1C (AKR1C) was confirmed with isoenzyme-specific AKR inhibitors.

4.?Using recombinantly expressed human cytochrome P450 (CYP) isoenzymes, dydrogesterone was shown to be metabolically transformed by CYP3A4 and CYP2C19.

5.?A clear contribution of CYP3A4 to microsomal metabolism of dydrogesterone was demonstrated with HLM and isoenzyme-specific CYP inhibitors, and confirmed by a significant correlation between dydrogesterone clearance and CYP3A4 activity.

6.?Contribution of CYP2C19 was shown to be clearly less than CYP3A4 and restricted to a small group of human individuals with very high CYP2C19 activity. Therefore, it is expected that CYP2C19 genetic variations will not affect dydrogesterone pharmacokinetics in man.

7.?In conclusion, dydrogesterone metabolism in the liver is dominated primarily by cytosolic enzymes (particularly AKR1C) and secondarily by CYP3A4, with the former exclusively responsible for 20α-DHD formation.     

Enzymology of the reductive bioactivation of SR 4233. A novel benzotriazine di-N-oxide hypoxic cell cytotoxin

SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel benzotriazine di-N-oxide which shows unusually high selective toxicity towards hypoxic cells, probably as a result of reductive bioactivation. Using an HPLC assay for the parent drug and its 2- and 4-electron reduction products (SR 4317 and SR 4330, respectively), we have examined the enzymology of SR 4233 reductive metabolism in vitro using a variety of different enzyme preparations. SR 4233 was converted extremely rapidly to SR 4317 under N2 by mouse liver microsomes, and showed a marked preference for NADPH over NADH as a reduced cofactor. The reaction was inhibited completely in air and boiled preparations. It was also inhibited by 78-86% in carbon monoxide (CO), implicating cytochrome P-450 as the major microsomal SR 4233 reductase. The kinetics of reductive metabolism of SR 4233 to SR 4317 by mouse liver microsomes conformed to Michaelis-Menten kinetics, with a Km of 1.4 mM and a Vmax of 950 nmol/min/mg protein. SR 4233 reduction was also catalysed by mouse liver cytosol under N2. However, rates were markedly slower than for microsomes and showed an equal dependency on NADH and NADPH. The cytosolic enzymes aldehyde oxidase and xanthine oxidase both catalysed SR 4233 reduction to SR 4317 under N2. Purified buttermilk xanthine oxidase also catalysed this reaction. In contrast to other enzyme preparations, DT-diaphorase from Walker 256 tumour cells reduced SR 4233 predominantly to SR 4330, and this reaction occurred under aerobic conditions. These data illustrate that SR 4233 is reduced rapidly by a wide variety of reductases. We propose that the therapeutic selectivity of SR 4233 will be controlled by the relative expression of reductases in tumour versus normal tissues, and in particular by the differential participation of putative activating versus detoxifying enzymes.     

In vitro formation of selegiline-N-oxide as a metabolite of selegiline in human, hamster, mouse, rat, guinea-pig, rabbit and dog.

It is well established in the litrature, that selegiline is metabolised to its N-dealkylated metabolites, N-desmethylselegiline, methamphetamine and amphetamine. However, most studies on selegiline metabolism did not characterize the species differences in the formation of the metabolites. Therefore, in this study, we investigated the in vitro metabolism of selegiline in liver microsomes of different species. In addition, to the previously well-characterized metabolites, selegiline-N-oxide (selegiline-NO) was found to be formed as a metabolite of selegiline in rat liver microsomal preparation. The results of experiments with liver microsomes from other species indicated species differences in the rate and extent of formation of selegiline-NO. The dog and hamster liver microsomal preparations were the most active in terms of selegiline-NO production, whereas little selegiline was metabolized to its N-oxide in human liver microsomes. When selegiline-NO was incubated with rat liver microsomes, no metabolism occurred. When a short incubation time was applied in selegiline expriments no increase in the amount of selegiline-NO was detected. Accordingly, it was clear that selegiline was not metabolized to the N-dealkylated or N,N-bis-dealkylated compounds via selegiline-NO. Studies with different isoenzyme inhibitors indicated that the formation of selegiline-NO might be catalyzed at least partly by cytochrome P450 (CYP) 2D6 and CYP3A4. With the exception of hamster microsomes in the microsomal preparations in vitro, the formation of the R,S-stereoisomer of selegiline-NO was preferred.     

Identification of enzymes responsible for rifalazil metabolism in human liver microsomes

1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32-hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. Km and Vmax of the rifalazil-25-deacetylation in microsomes were 6.5 microM and 11.9 pmol/min/mg with NADPH, and 2.6 microM and 6.0 pmol/min/mg without NADPH, indicating that, although rifalazil-25-deacetylation did not require NADPH, NADPH activated it. Rifalazil-32-hydroxylation was NADPH dependent, and its Km and Vmax were 3.3 microM and 11.0 pmol/min/mg respectively. 3. Rifalazil-25-deacetylation in microsomes was completely inhibited by diisopropyl fluorophosphate, diethyl p-nitrophenyl phosphate and eserine, but not by p-chloromercuribenzoate or 5,5'-dithio-bis(2-nitrobenzoic acid), indicating that the enzyme responsible for the rifalazil-25-deacetylation is a B-esterase. 4. Rifalazil-32-hydroxylation in microsomes was completely inhibited by CYP3A4-specific inhibitors (fluconazole, ketoconazole, miconazole, troleandomycin) and drugs metabolized by CYP3A4 such as cyclosporin A and clarithromycin, indicating that the enzyme responsible for the rifalazil-32-hydroxylation is CYP3A4.