Formation In Liver Microsomes Research Articles

Propranolol is a mechanism-based inhibitor of CYP2D and CYP2D6 in humanized CYP2D6-transgenic mice: Effects on activity and drug responses.

Genetics and drug interactions contribute to large interindividual variation in human CYP2D6 activity. Here, we have characterized propranolol inhibition of human and mouse CYP2D using transgenic (TG) mice, which express both mouse CYP2D and human CYP2D6, and wild-type (WT) mice. Our purpose was to develop a method for in vivo manipulation of CYP2D6 enzyme activity which could be used to investigate the role of CYP2D6 in drug-induced behaviours. Dextromethorphan metabolism to dextrorphan was used to measure CYP2D activity and to characterize propranolol inhibition in vitro and in vivo. Effects of propranolol pretreatment (24 hr) on serum levels of the CYP2D6 substrate haloperidol and haloperidol-induced catalepsy were also studied. Dextrorphan formation velocity in vitro was threefold higher in liver microsomes of TG compared to WT mice. Propranolol acted as a mechanism-based inhibitor (MBI), inactivating CYP2D in liver microsomes from TG and WT mice, and humans. Pretreatment (24 hr) of TG and WT mice with 20 mg·kg-1 intraperitoneal propranolol reduced dextrorphan formation in vivo and by liver microsomes in vitro. Serum haloperidol levels and catalepsy were increased. Propranolol was a potent MBI of dextrorphan formation in liver microsomes from TG and WT mice, and humans. The inhibition parameters in TG overlapped with those in WT mice and in humans. Inhibition of CYP2D with propranolol in vivo in TG and WT mice altered drug responses, allowing further investigation of variations in CYP2D6 on drug interactions and drug responses.

Modulation of serum concentrations and hepatic metabolism of 17β-estradiol and testosterone by amitraz in rats

The present study has investigated the ability of amitraz, a widely used formamidine pesticide, to modulate serum concentrations and liver microsomal metabolism of 17beta-estradiol (E2) and testosterone in rats. Amitraz was administered intraperitoneally to male rats for 4 days and to intact female rats or ovariectomized (OVX) and 0.5 mg/kg E2-supplemented female rats for 7 days. E2 and metabolites were analyzed by gas chromatography-electron capture detection and testosterone and metabolites were analyzed by high-pressure liquid chromatography. In OVX and E2-supplemented females, 50 mg/kg amitraz caused an 85% decrease of serum E2 concentration and a marked increase of 2-OH-E2 concentration. Amitraz at 25 and 50 mg/kg produced 9.0-fold or greater increases of serum testosterone and 2beta-OH-testosterone levels in males. Amitraz at 25 mg/kg resulted in no or minimal increases of liver microsomal formation of E2 or testosterone metabolites. Amitraz at 50 mg/kg produced 1.4- to 3.6-fold increases of 2-OH-E2; estrone; 2beta-, 6beta-, and 16alpha-OH-testosterone; and androstenedione formation in males and intact females. Amitraz at 50 mg/kg preferentially increased intact female 16beta-OH-testosterone production by 8.6-fold. In OVX females, E2 supplement alone or cotreatment with E2 and 50 mg/kg amitraz produced 1.3- to several-fold increases of 2- and 4-OH-E2 formation and 2beta- and 16alpha-OH-testosterone production. The cotreatment increased 6beta- and 16beta-OH-testosterone formation by 1.8- and 1.6-fold, respectively. The present findings show that amitraz induces hepatic E2 and testosterone metabolism in male and female rats, decreases serum E2 concentration in OVX and E2-supplemented females, but increases serum testosterone in males.

Characterization of Diuron N-Demethylation by Mammalian Hepatic Microsomes and cDNA-Expressed Human Cytochrome P450 Enzymes

Diuron, a widely used herbicide and antifouling biocide, has been shown to persist in the environment and contaminate drinking water. It has been characterized as a "known/likely" human carcinogen. Whereas its environmental transformation and toxicity have been extensively examined, its metabolic characteristics in mammalian livers have not been reported. This study was designed to investigate diuron biotransformation and disposition because metabolic routes, metabolizing enzymes, interactions, interspecies differences, and interindividual variability are important for risk assessment purposes. The only metabolic pathway detected by liquid chromatography/mass spectometry in human liver homogenates and seven types of mammalian liver microsomes including human was demethylation at the terminal nitrogen atom. No other phase I or phase II metabolites were observed. The rank order of N-demethyldiuron formation in liver microsomes based on intrinsic clearance (V(max)/K(m)) was dog > monkey > rabbit > mouse > human > minipig > rat. All tested recombinant human cytochrome P450s (P450s) catalyzed diuron N-demethylation and the highest activities were possessed by CYP1A1, CYP1A2, CYP2C19, and CYP2D6. Relative contributions of human CYP1A2, CYP2C19, and CYP3A4 to hepatic diuron N-demethylation, based on average abundances of P450 enzymes in human liver microsomes, were approximately 60, 14, and 13%, respectively. Diuron inhibited relatively potently only CYP1A1/2 (IC(50) 4 microM). With human-derived and quantitative chemical-specific data, the uncertainty factors for animal to human differences and for human variability in toxicokinetics were within the range of the toxicokinetics default uncertainty/safety factors for chemical risk assessment.

In vitro metabolism and interaction of profenofos by human, mouse and rat liver preparations

Biotransformations of profenofos were studied in vitro. Two metabolites, desthiopropylprofenofos and hydroxyprofenofos, were detected by LC–MS after incubation of profenofos with human liver homogenates and different mammalian liver microsomes. The rank order of desthiopropylprofenofos formation in liver microsomes based on intrinsic clearance ( V max/ K m) was mouse > human > rat, while for profenofos hydroxylation it was mouse > rat > human. In view of the ratio between desthiopropylation and hydroxylation intrinsic clearance rates, human liver microsomes were most active in profenofos bioactivation. The interspecies differences and interindividual variation were within range of the default uncertainty/safety factors for chemical risk assessment. CYP3A4, CYP2B6 and CYP2C19 were identified as profenofos-oxidizing enzymes in human liver on the basis of recombinant expressed enzymes and correlation with CYP model activities. The rank order of CYPs in profenofos activation was CYP3A4 > CYP2B6 > CYP2C19, whereas it was the contrary for profenofos hydroxylation. Profenofos inhibited relatively potently several human liver microsomal activities: the lowest IC 50 values were about 3 μM for CYP1A1/2 and CYP2B-associated activities. Profenofos is extensively metabolized by liver microsomal CYP enzymes and its interaction potential with several CYP activities is considerable.

Characterization of hydroxyl radical formation by microsomal enzymes using a water-soluble trap, terephthalate

Using terephthalic acid as a water-soluble trap, we characterized hydroxyl radicals (HO ) formation by liver microsomal enzymes from isoniazid-treated rats. We found that HO formation was entirely dependent on intact microsomal enzymes, the presence of NADPH, and iron complexed with EDTA. In contrast to the other radical traps, we found no evidence that terephthalate is a substrate for cytochrome P450 . Cumene hydroperoxide, an artificial supporter of cytochrome P450-catalyzed oxidation, failed to maintain HO formation. HO formation in liver microsomes was inhibited by the HO radical scavengers: dimethyl sulfoxide (DMSO), mannitol, and citrulline. It was abolished by catalase, but not superoxide dismutase (SOD), indicating that hydrogen peroxide was the sole precursor of the HO . Therefore, the generation of hydroxyl radicals by microsomal enzymes appears to be dependent on two processes: (1) the rate of hydrogen peroxide production; and (2) the availability of iron ions or other transition metals for Fenton type reactions.

CYP2D6 and CYP3A4 involvement in the primary oxidative metabolism of hydrocodone by human liver microsomes.

To determine the Michaelis-Menten kinetics of hydrocodone metabolism to its O- and N-demethylated products, hydromorphone and norhydrocodone, to determine the individual cytochrome p450 enzymes involved, and to predict the in vivo hepatic intrinsic clearance of hydrocodone via these pathways. Liver microsomes from six CYP2D6 extensive metabolizers (EM) and one CYP2D6 poor metabolizer (PM) were used to determine the kinetics of hydromorphone and norhydrocodone formation. Chemical and antibody inhibitors were used to identify the cytochrome p450 isoforms catalyzing these pathways. Expressed recombinant cytochrome p450 enzymes were used to characterize further the metabolism of hydrocodone. Hydromorphone formation in liver microsomes from CYP2D6 EMs was dependent on a high affinity enzyme (Km = 26 microm) contributing 95%, and to a lesser degree a low affinity enzyme (Km = 3.4 mm). In contrast, only a low affinity enzyme (Km = 8.5 mm) formed this metabolite in the liver from the CYP2D6 PM, with significantly decreased hydromorphone formation compared with the livers from the EMs. Norhydrocodone was formed by a single low affinity enzyme (Km = 5.1 mm) in livers from both CYP2D6 EM and PM. Recombinant CYP2D6 and CYP3A4 formed only hydromorphone and only norhydrocodone, respectively. Hydromorphone formation was inhibited by quinidine (a selective inhibitor of CYP2D6 activity), and monoclonal antibodies specific to CYP2D6. Troleandomycin, ketoconazole (both CYP3A4 inhibitors) and monoclonal antibodies specific for CYP3A4 inhibited norhydrocodone formation. Extrapolation of in vitro to in vivo data resulted in a predicted total hepatic clearance of 227 ml x h-1 x kg-1 and 124 ml x h-1 x kg-1 for CYP2D6 EM and PM, respectively. The O-demethylation of hydrocodone is predominantly catalyzed by CYP2D6 and to a lesser extent by an unknown low affinity cytochrome p450 enzyme. Norhydrocodone formation was attributed to CYP3A4. Comparison of recalculated published clearance data for hydrocodone, with those predicted in the present work, indicate that about 40% of the clearance of hydrocodone is via non-CYP pathways. Our data also suggest that the genetic polymorphisms of CYP2D6 may influence hydrocodone metabolism and its therapeutic efficacy.

Cytochrome P-4503A1 Catalyzes the Formation of MA 1 from Territrem a in Liver Microsomes of 7-Week-Old Female Wistar Rats

Liver microsomal territrem A (TRA) metabolism was studied in 7-wk-old female Wistar rats. Pretreatment with phenobarbital (PB) or dexamethasone (DEX) resulted in a significant increase in 4 g -hydroxymethyl-4 g -demethylterritrem A (MA 1 ) production. SKF 525A (0.025 and 0.05 mM), a general cytochrome P-450 (CYP450) inhibitor, blocked MA 1 formation in liver microsomes from PB-pretreated female rats. Anti-CYP2B antibody had no marked effect on MA 1 formation, although orphenadrine (0.5 mM), which inhibits CYP2B, blocked MA 1 formation in liver microsomes from PB-treated female rats. An immunoinhibition study showed that anti-CYP3A2 antibody reduced MA 1 formation to nondetectable levels in liver microsomes from PB-treated female rats. Furthermore, immunoblotting showed that CYP3A1 protein was expressed in 7-wk-old female rat and only MA 1 was formed from TRA using supersomes from CYP3A1-expressing baculovirus-infected insect cells. Further, Western blot analysis indicated that CYP3A2 protein was expressed in 2-wk-old rats of both sexes and 7-wk-old male rats, and 3 metabolites of TRA, such as MA 1 , MAX, and MA 2 , were formed using supersomes from CYP3A2-expressing baculovirus-infected insect cells. These results suggest that MA 1 formation in liver microsomes of 7-wk-old female Wistar rats is mediated by CYP3A1.

METABOLISM OF TERRITREM A IN LIVER MICROSOMES FROM MALE WISTAR RATS: 3. CYTOCHROME P-450 ISOFORMS CATALYZING TRA METABOLISM

The cytochrome P-450 isoforms involved in territrem A (TRA) metabolism in liver microsomes of male Wistar rats have been characterized. Pretreatment with phenobarbital (PB) or dexamethasone (DEX) resulted in a similar significant increase in TRA metabolic activity. Although PB treatment resulted in a significant elevation in CYP2B, CYP2C11, and CYP3A levels, only CYP3A levels were significantly increased by DEX treatment. Cimetidine markedly reduced the formation of the TRA metabolites 4 g -hydroxymethyl-4 g -demethylterritrem A (MA 1 ), 4 g -oxo-4 g -demethylterritrem A (MAX) and 2-dihydro-4 g -demethylterritrem A (MA 2 ) in liver microsomes from 2-wk-old rats (mainly containing CYP3A2) and 7-wk-old rats (containing CYP2B, CYP2C11, and CYP3A2). SKF 525A, which inhibits CYP2B, CYP2C11, and CYP3A2, and orphenadrine, which inhibits CYP2B, also decreased MA 2 formation in liver microsomes from 7-wk-old phenobarbital-pretreated rats. The formation of MA 1 and MAX was not affected. Furthermore, an immunoinhibition study demonstrated that anti-CYP3A2 antibody reduced MA 1 , MAX, and MA 2 formation to nondetectable levels in liver microsomes from 2- and 7-wk-old rats, whereas anti-CYP2C11 or anti-CYP2B antibody, respectively, had no marked effect on MA 1 , MAX, and MA 2 formation in liver microsomes from 7-wk-old untreated or PB-treated rats. These results suggest that the CYP3A isoform is mainly responsible for MA 1 , MAX, and MA 2 formation in liver microsomes in male Wistar rats.

Catechol estrogen formation in liver microsomes from female ACI and Sprague-Dawley rats: comparison of 2- and 4-hydroxylation revisited.

Estradiol (E(2))-hydroxylation was studied in liver microsomes from ACI and Sprague-Dawley female rats, which differ markedly in their susceptibility to E(2)-induced formation of mammary tumors. NADPH-dependent oxidation of E(2) by liver microsomes from ACI and Sprague-Dawley rats produced several metabolites of which 2-hydroxyestradiol (2-OH-E(2)), estrone (E(1)), and 2-hydroxyestrone (2-OH-E(1)) were predominant. Incubations with either low (9 nM) or high (50 microM) concentrations of radiolabeled E(2) and with varying amounts of microsomal protein indicated the formation of only small amounts of 4-hydroxyestradiol (4-OH-E(2)). The ratio of 2-OH-E(2) to 4-OH-E(2) formed with the low concentration of E(2) was about 10:1 regardless of the amount of microsomal protein used, and about 20:1 using a high concentration of E(2). Thus, oxidation of E(2) by liver microsomes from female ACI and Sprague-Dawley rats occurs primarily via 2-hydroxylation, and 4-hydroxylation is only a minor pathway. These results are in disagreement with a recent report indicating substantial 4-hydroxylation of E(2) by liver microsomes from female ACI rats.

Antioxidative Activity of Natural Isorhapontigenin

Isorhapontigenin (ISOR), isolated from Belamcanda chinensis, is a derivative of stilbene. Its chemical structure is very similar to that of resveratrol, with a potent antioxidative effect. In the present study, we investigated the antioxidative activity of ISOR in vitro. Oxidative damage of rat liver microsomes, brain mitochondria and synaptosomes was induced by Fe2+-Cys, VitC-ADP-Fe2+ and H2O2, respectively. The formation of malondialdehyde (MDA), decrease of reduced glutathione (GSH) and increase of ultra-weak chemiluminescence during the lipid peroxidation process were determined. In addition, the characteristic ultra-weak chemiluminescence of oxidative DNA damage induced by CuSO4-Phen-VitC-H2O2 system was studied. The results showed that ISOR significantly inhibited MDA formation in liver microsomes, brain mitochondria and synaptosomes induced by Fe2+-Cys. Also, ISOR markedly prevented the decrease of GSH in mitochondria and synaptosomes induced by H2O2 and the increase of ultra-weak chemiluminescence during lipid peroxidation induced by VitC-ADP-Fe2+ as well as oxidative DNA damage induced by CuSO4-Phen-VitC-H2O2. The effects of ISOR at 10-5 and 10-6 mol/L on the MDA formation and decrease of GSH were similar to that of the classical antioxidant vitamin E (10-4 mol/L). It may be concluded that ISOR possessed potent antioxidative activity and was much more potent than vitamin E.

In vitro metabolism of trazodone by CYP3A: inhibition by ketoconazole and human immunodeficiency viral protease inhibitors

Background: Pharmacologic treatment of emotional disorders in HIV-infected patients can be more easily optimized by understanding of potential interactions of psychotropic drugs with medications used to treat HIV infection and its sequelae. Methods: Biotransformation of the antidepressant trazodone to its principal metabolite, meta-chlorophenylpiperazine (mCPP), was studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. Interactions of trazodone with the azole antifungal agent, ketoconazole, and with human immunodeficiency virus protease inhibitors (HIVPIs) were studied in the same system. Results: Formation of mCPP from trazodone in liver microsomes had a mean (± SE) K m value of 163 (± 21) μmol/L. Ketoconazole, a relatively specific CYP3A inhibitor, impaired mCPP formation consistent with a competitive mechanism, having an inhibition constant (K i) of 0.12 (± 0.01) μmol/L. Among heterologously expressed human cytochromes, only CYP3A4 mediated formation of mCPP from trazodone; the K m was 180 μmol/L, consistent with the value in microsomes. The HIVPI ritonavir was a potent inhibitor of mCPP formation in liver microsomes (K i = 0.14 ± 0.04 μmol/L). The HIVPI indinavir was also a strong inhibitor, whereas saquinavir and nelfinavir were weaker inhibitors. Conclusions: CYP3A-mediated clearance of trazodone is inhibited by ketoconazole, ritonavir and indinavir, and indicates the likelihood of pharmacokinetic interactions in vivo.

Arachidonic Acid Metabolism in the Marine FishStenotomus chrysops(Scup) and the Effects of Cytochrome P450 1A Inducers

Cytochrome P450-mediated arachidonic acid (AA) metabolism was investigated in the marine fish scup,Stenotomus chrysops.Liver microsomes incubated with AA and NADPH produced epoxyeicosatrienoic acids (EETs) and their hydration products (dihydroxyeicosatrienoic acids, DHETs), midchain conjugated dienols (midchain HETEs), and C16- through C20-alcohols of AA (ω-terminal HETEs), all identified by HPLC and GC/MS. Gravid females had 4-fold lower AA metabolism rates than males but identical metabolite profiles. The 5,6-EET (inferred from stable metabolites) was most abundant (47% of total EETs) followed by 14,15-, 11,12-, and 8,9-EET (27, 13, and 13%, respectively). The 12-HETE represented 25% of total HETEs followed in abundance by 16-, 15-, 11-, 19-, 20-, 8-, and 9-HETE. Antibodies against scup CYP1A and a scup CYP2B-like protein inhibited liver microsomal AA metabolism by 30 and 46%, respectively. GC/MS analysis revealed EETs and DHETs as endogenous constituents in scup liver; the predominant EETs were 8,9- and 14,15-EET, followed by a lesser amount of 11,12-EET. Chiral analysis showed a preference for the S,R-enantiomers of endogenous 8,9-, 11,12-, and 14,15-EET (optical purities 80, 64, and 64%, respectively). Treatment of scup with the CYP1A inducer benzo(a)pyrene (BP) increased liver microsomal formation of EETs and HETEs by 2.7-fold in spring and 1.7-fold in summer. BP treatment did not affect microsomal EET regioselectivity, but shifted hydroxylation in favor of 19-HETE and induced 17-HETE formation. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) treatment in summer did not induce liver microsomal AA metabolism rates, yet BP and TCDD both increased endogenous EET content of liver (5- and 3-fold, respectively), with a shift to 14,15-EET. BP treatment increased the selectivity for the S,R-enantiomers of endogenous 8,9-, 11,12-, and 14,15-EET (optical purities 91, 84, and 83%, respectively). Kidney, gill, and heart microsomes all metabolized AA, at rates 10- to 30-fold less than liver microsomes. Similar amounts of endogenous 8,9- and 14,15-EET and less 11,12-EET were detected in heart and kidney, and there was a strong enantioselectivity for 8(R),9(S)-EET in heart (optical purity 78%) but not in kidney. BP treatment did not alter the total EET content in these organs but did shift the regiochemical profile in heart to favor 14,15-EET. Thus, scup liver and extrahepatic organs metabolize AA via multiple cytochrome P450 (CYP) forms to eicosanoidsin vitroandin vivo.BP or TCDD induced endogenous AA metabolism in liver, altering EET regioselectivity and, with BP, stereoselectivity. While AhR agonists alter metabolism of AA in early diverging vertebrates expressing both CYP1A and AhR, the magnitude of effects may depend upon the type of inducer.

Glucuronidation of dihydrocodeine by human liver microsomes and the effect of inhibitors.

1. Glucuronidation is the major route of metabolism of dihydrocodeine (DHC) and accounts for 25-30% of an oral dose in urine. The kinetics of DHC-6-glucuronide formation in liver microsomes from five human donors and the effect of a number of potential inhibitor drugs were examined using a newly developed and validated HPLC assay. 2. The formation of DHC-6-glucuronide exhibited atypical kinetics that conformed to the Hill equation. The mean intrinsic dissociation constant (Ks) and maximum velocity (Vmax) values were 1566 micromol/L and 0.043 micromol/min per g, respectively. The Ks and Vmax values varied 1.5- and 3.5-fold, respectively. 3. Seven drugs were tested for inhibitory effects on DHC glucuronidation at low (50 micromol/L) and high (500 micromol/L) concentrations. At 50 micromol/L, only diclofenac produced greater than 50% inhibition, while at concentrations of 500 micromol/L inhibition was greater than 35% for diclofenac, amitriptyline, oxazepam, naproxen, chloramphenicol and probenecid, but not paracetamol. 4. The present study found little interindividual variation in the activity of human liver microsomes for glucuronidation of DHC. Comparison of the results from the inhibition studies with those reported previously for codeine and morphine suggest that the UDP-glucuronosyltransferase isoform UGT2B7 is involved in the glucuronidation of DHC.

Effect of propylthiouracil treatment on NADPH-cytochrome P450 reductase levels, oxygen consumption and hydroxyl radical formation in liver microsomes from rats fed ethanol or acetone chronically

The antithyroid drug propylthiouracil (PTU) has been shown previously to reduce hepatic oxygen utilization and to protect the liver from ethanol-induced injury. The present study examined the effect of PTU on hepatic microsomal oxygen consumption and on the activities of NADPH-cytochrome P450 reductase (CYP-reductase) and cytochrome P4502E1 (CYP2E1) in rats receiving ethanol or acetone chronically. Liver microsomes from rats treated with ethanol for 29 days displayed increases in (i) O 2 consumption (70%), (ii) hydroxyl radical (OH) production (49%) and (iii) ethanol oxidation (50%). Microsomal CYP2E1 levels were increased markedly by chronic ethanol administration, while CYP-reductase was affected marginally, but not significantly ( P = 0.06). Chronic treatment with acetone for 14 days, produced similar effects, except that OH production was not enhanced. Administration of PTU (25 mg/kg/day) to ethanol- or acetone-fed rats, for 10 and 14 days, respectively, led to a marked reduction in the levels and activity of CYP-reductase, and to a decrease in the rates of microsomal O 2 consumption, OH production and ethanol oxidation, but did not lower the levels of CYP2E1 or the metabolism of the CYP2E1 substrate N, N-nitrosodimethylamine. These data suggest that the ability of PTU to protect the liver from ethanol-induced injury may be due to a reduction in the levels of CYP-reductase, thereby minimizing the enhancement of microsomal oxygen consumption and free radical generation associated with ethanol-induced CYP2E1 activity.

Formation of 19(S)-, 19(R)-, and 18(R)-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1

When reconstituted with cytochrome b5 and NADPH cytochrome P450 oxidoreductase, cytochrome P450 2E1 metabolized lauric, stearic, oleic, linoleic, linolenic, and arachidonic acid to multiple metabolites. Two major metabolites, accounting for 78% of the total metabolism, were produced with arachidonic acid. The Vmax for total metabolite formation from arachidonic acid was 5 nmol/min/nmol P450 with an apparent Km of 62 microM. Gas chromatography-mass spectrometry analysis identified the two major metabolites as monohydroxylated eicosatetraenoic acids (HETEs). The major HETE was 19-hydroxyeicosatetraenoic acid (19-HETE) and comprised 46% of the total metabolite produced. The second metabolite was the omega-2 hydroxylated metabolite (18-HETE) and comprised 32% of the total product formed. Chiral analysis demonstrated that 19-HETE was 70% 19(S)-HETE and 30% 19(R)-HETE. In contrast, 18-HETE was essentially 100% R isomer. Approximately 18% of the total metabolite produced from arachidonic acid coeluted with epoxyeicosatrienoic acid (EET) standards. The EET metabolites were 56.4% 14,15-EET and 43.6% as a mixture of 11,12-EET and 8,9-EET. 5,6-EET was not detected. Anti-P450 2E1 IgG inhibited arachidonic acid metabolism by renal and hepatic microsomes prepared from acetone-treated rabbits. With renal cortex microsomes, the formation of 18-HETE and 19-HETE was inhibited 67 and 25%, respectively, by the antibody. Liver microsomal formation of 18-HETE was inhibited by 87% and 19-HETE by 70%. Thus, under conditions where cytochrome P450 2E1 is induced, the enzyme could contribute significantly to the formation of the omega-1 and omega-2 hydroxylated metabolites of arachidonic acid.

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by inducible and constitutive cytochrome P450 enzymes in rats

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces tumor formation in the liver, lung, nasal cavity, and pancreas of rats. Metabolic activation is required for the tumorigenicity of this compound. The involvement of cytochrome P450 enzymes in NNK bioactivation was investigated in rats by studies with chemical inducers and antibodies against P450s. Liver microsomal enzymes catalyzed the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), 4-(methylnitrosamino)-1-(3-pyridyl- N-oxide)-1-butanone (NNK- N-oxide), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) from NNK. When the activity was expressed on a per nanomole P450 basis, treatments of rats with 3-methylcholanthrene (MC), phenobarbital (PB), pregnenolone 16-α-carbonitrile (PCN), Aroclor 1254 (AR), safrole (SA), and isosafrole (ISA) increased the keto aldehyde formation in liver microsomes 2.0-, 2.4-, 3.8-, 2.5-, 2.1-, and 1.8-fold, respectively; PB, AR, SA, and ISA increased the keto alcohol formation 1.7-, 1.3-, 2.0-, and 1.3-fold, respectively. The extents of induction were more pronounced when expressed on a per milligram protein basis, due to the higher microsomal P450 contents in the induced microsomes. The formation of NNK- N-oxide was markedly increased by PB and PCN and slightly increased by AR, SA, and ISA. However, the formation of NNAL, the major metabolite due to carbonyl reduction, was not increased by the treatments but was decreased by AR, ISA, and acetone (AC). The kinetic parameters of NNK metabolism by control, MC-, PB-, and PCN-induced liver microsomes were obtained. A panel of monoclonal (anti-1A1, -2B1, -2C11, and -2E1) and polyclonal (anti-1A2, -2A1, and -3A) antibodies were used to assess the involvement of constitutive hepatic P450 enzymes in NNK metabolism. Keto aldehyde formation was inhibited by anti-1A2 and anti-3A (about 15%) but not by others; the formation of keto alcohol was inhibited by anti-1A2, anti-2A1, and anti-3A (by 13–26%). In incubations with lung microsomes, the formation of keto aldehyde, keto alcohol, NNK- N-oxide, and NNAL were observed. With nasal mucosa microsomes, however, only keto aldehyde and keto alcohol formation were appreciable. SA and AC significantly decreased NNK metabolism in lung and nasal mucosa microsomes. Furthermore, SA inhibited the NNK oxidative metabolism in lung and nasal mucosa microsomes in vitro. AC significantly inhibited the NNK oxidative metabolism in lung microsomes in vitro, but not in nasal mucosa microsomes. The present results demonstrate the important role of P450 enzymes in the metabolism of NNK. Several P450 isozymes can catalyze the oxidation of NNK with different but overlapping regioselectivities and P450 inducers affect the metabolism of NNK differently in the liver, lung, and nasal mucosa.

Modulation of aflatoxin B 1 biotransformation in rabbit pulmonary and hepatic microsomes

Aflatoxin B 1 (AFB 1) is a carcinogenic mycotoxin that requires activation to the corresponding 8,9-epoxide for activity. In addition to being present in foodstuffs, AFB 1 can contaminate respirable grain dusts and thus the respiratory system is a potential target for carcinogenesis. In the present study, we have investigated the role of polycyclic aromatic hydrocarbon-inducible forms of cytochrome P-450 in the pulmonary and hepatic microsomal activation ([ 3H]AFB 1-DNA binding) and detoxification ([ 3H]AFM 1 and [ 3H]AFQ 1 formation) of [ 3H]AFB 1. In rabbit lung microsomes, the apparent V max for [ 3H]AFM 1 formation was increased significantly when values were expressed per mg microsomal protein or per nmol P-450 present. In liver microsomes, the apparent V max for DNA binding and [ 3H]AFM 1 formation were increased by β-naphthoflavone (BNF) treatment (to 2.3 and 3.3 times control, respectively) when expressed per mg protein, but when expressed per nmol P-450, only AFM 1 formation was significantly increased. The apparent K m values for both these reactions were unaffected. The apparent V max for [ 3H]AFQ 1 formation was not affected by BNF treatment, but the apparent K m was increased to 4.5 times control. Boiling of microsomes or omitting the NADPH-generating system decreased DNA binding, AFM 1 formation and AFQ 1 formation by 89–97%, while addition of 1.0 mM SKF-525A inhibited these reactions by 46–57%. Addition of 1.0 mM α-naphthoflavone (ANF) had no effect on the biotransformation of [ 3H]AFB 1 in lung microsomes of control rabbits, but significantly decreased AFM 1 formation (by 31%) in lung microsomes from BNF-treated animals (other reactions were unaffected). In liver microsomes from BNF treated rabbits, 1.0 mM ANF inhibited DNA binding of [ 3H]AFB 1 by 68%, while there was no effect in control microsomes. ANF significantly inhibited AFM 1 formation in liver microsomes from both control and BNF-treated animals (by 87–97% and 67–78% at 1.0 mM and 2.0 μM, respectively), but had no effect on AFQ 1 formation in liver microsomes from animals in either treatment group. These results indicate an important role for the 1A subclass of P-450 isozymes in the biotransformation of AFB 1 to AFM 1 in rabbit lung and liver, and a minor role in AFB 1 activation in liver.

Formation of glutathionyl-spironolactone disulfide by rat liver cytochromes P450 or hog liver flavin-containing monooxygenases: a functional probe of two-electron oxidations of the thiosteroid?

We have previously reported that the diuretic thiosteroid spironolactone (SPL) inactivates rat liver microsomal cytochromes P450 [P450 (P450 3A and P450 2C11)] in a in a mechanism-based fashion, and we have identified two polar SPL metabolites (SPL-sulfinic acid and -sulfonic acid), formed in a partition ratio of approximately 20:1 in such rat liver microsomal incubations [Decker et al. (1989) Biochemistry 28, 5128-5136]. We proposed at the time that these metabolites were most likely derived from further enzymatic (or nonenzymatic) oxidations of the one-electron oxidation product [SPL-thiyl radical (SPL-S.)] and/or the two-electron-oxidized species [SPL-sulfenic acid (SPL-SOH)]. In those studies, glutathione (GSH) was found to attenuate both SPL-mediated P450 loss as well as polar metabolite formation by approximately 40%. We have now reexamined this in greater detail and report that it is due to GSH trapping of an electrophilic oxidized SPL species to form an adduct that we have isolated and unambiguously characterized by mass spectral analyses as the glutathionyl-SPL adduct (SPL-SSG). Moreover, we have found not only that rat liver microsomal formation of this adduct is enhanced at pH 9.0, the pH optimum for flavin-containing monooxygenase (FMO), but also that such adduct formation was indeed efficiently catalyzed by purified hog liver FMO. Because FMO oxidations of thiols are thought to entail a two-electron process to form the corresponding sulfenic acids, we infer that such a SPL-SSG adduct most likely reflects FMO-catalyzed oxidation of SPL to SPL-SOH, which on leaving the FMO active site is then trapped by GSH.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB 1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.

Biotransformation of aflatoxin B1 in rabbit lung and liver microsomes

Aflatoxin B1 (AFB1) is a potent hepatotoxic and hepatocarcinogenic mycotoxin that requires bioactivation to AFB1-2,3-oxide for activity. In addition to epoxidation, microsomal monooxygenases biotransform AFB1 to the less toxic metabolites, aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1). The lung is at risk from AFB1 both via inhalation and via the circulation. In the present study, we have characterized rabbit lung and liver microsomal AFB1-DNA binding (an index of AFB1-2,3-oxide formation), AFM1 formation and AFQ1 formation. Vmax values for AFB1-DNA binding were not different between lung and liver when expressed per mg microsomal protein (1.06 +/- 0.13 and 2.12 +/- 1.30 nmol/mg/h for lung and liver respectively), but lung values were greater than liver when expressed per nmol cytochrome P450 (3.64 +/- 0.31 and 1.29 +/- 0.70 nmol/nmol P450/h for lung and liver respectively). Km values for this reaction were not different between lung and liver. Vmax values for AFM1 formation in liver microsomes were greater than in lung when expressed per mg protein, but not when expressed per nmol P450. No differences were detected for the Km for AFM1 formation between lung and liver microsomes. For AFQ1 formation, no differences were detected between Vmax values of lung and liver, regardless of whether results were expressed per mg protein or per nmol P450, while the Km for AFQ1 formation was lower in liver. SKF-525A inhibited these reactions by 63-74% in lung microsomes and 90-96% in liver microsomes. These results indicate that the lung is capable of activating AFB1, and that rabbit lung microsomes contain high activity for this reaction. Furthermore, little AFM1 and AFQ1 are formed in lung microsomes, leading to minimal shunting of AFB1 from the activation pathway.