Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC"lacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B(1) exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta-Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrome P450 enzyme involved in bioactivation of HCAs.     

Analysis of the involvement of human N-acetyltransferase 1 in the genotoxic activation of bladder carcinogenic arylamines using a SOS/umu assay system

Human acetyltransferase genes NAT1 or NAT2 were expressed in a Salmonella typhimurium strain used to detect the genotoxicity of bladder carcinogens. To clarify whether the human and rodent bladder carcinogenic arylamines are activated via either NAT1 or NAT2 to cause genotoxicity, a SOS/umu genotoxicity assay was used, with the strains S. typhimurium NM6001 (NAT1-overexpressing strain), S. typhimurium NM6002 (NAT2-overexpressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain). Genotoxicity was measured by induction of SOS/umuC gene expression in the system, which contained both an umuC"lacZ fusion gene and NAT1 or NAT2 plasmids. 4-Aminobiphenyl, 2-acetylaminofluorene, beta-naphthylamine, o-tolidine, o-anisidine, and benzidine exhibited dose-dependent induction of the umuC gene in strain NM6001. Although the induction of umuC by these chemicals was observed in the NM6002 strain, the induction was considerably lower than in the NM6001 strain. In the parent strain, NM6000, none of these compounds induced umuC gene expression. We also determined activation of these chemicals by recombinant human cytochrome P450 (P450 or CYP) 1A2 enzyme in three S. typhimurium tester strains. The activation of the chemicals was stronger in the NM6001 strain than that in NM6002. The specific NAT1 inhibitor 5-iodosalicylic acid inhibited umuC gene expression induced by aromatic amines used. These results could provide evidence that the bladder carcinogenic aromatic amines are mainly activated by the NAT1 enzyme to produce DNA damage rather than NAT2. The NAT1-overexpressing strain can be used to determine the genotoxic activation of bladder carcinogenic arylamines in the umu test and could provide a tool for predicting the carcinogenic potential of arylamines.     

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant (Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. alpha-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.     

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.     

Development of a Salmonella tester strain sensitive to promutagenic N-nitrosamines: expression of recombinant CYP2A6 and human NADPH-cytochrome P450 reductase in S. typhimurium YG7108

We developed a new Salmonella tester strain highly sensitive to promutagenic N-nitrosamines by introducing a plasmid carrying human cytochrome P450 2A6 (CYP2A6) and NADPH-cytochrome P450 reductase (OR) cDNA into the ada- and ogt-deficient strain YG7108. The YG7108 2A6/OR cells expressed high levels of CYP2A6 (77+/-8nmol/l) and OR (470+/-20 micromol cytochrome c reduced/min/l). The expressed CYP2A6 efficiently catalyzed coumarin 7-hydroxylation. N-Nitrosodiethylamine (NDEA), N-nitrosomethylphenylamine (NMPhA), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were mutagenic in the new strain in the absence of any exogenous activation system. The concentrations of promutagen that caused a two-fold increase in revertants were 7.1, 0.14, and 1.4 microM for NDEA, NMPhA, and NNK, respectively. YG7108 2A6/OR cells showed about 10- and 100-fold higher sensitivity to NDEA and NNK, respectively, than parental YG7108 cells assayed in the presence of rat liver S9 (final concentration, 21% (v/v)). Parental YG7108 cells did not detect NMPhA mutagenicity even in the presence of rat liver S9. We believe that this is the first demonstration that CYP2A6 is responsible for the metabolic activation of NMPhA. The established tester strain may be useful to predict human activation of N-nitrosamine promutagens.     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

Genetic polymorphism of CYP2A6 in relation to cancer.

To clarify the roles of human cytochrome P450 (P450 or CYP) 2A6 and 2E1 on the metabolic activation of N-nitrosamines, we established genetically engineered Salmonella typhimurium strains harboring human CYP2A6 or CYP2E1 together with NADPH-P450 reductase (OR). The 5'-terminus of CYP cDNA was modified to achieve a high-level expression in S. typhimurium. Modified CYP2A6 or CYP2E1 cDNA and native OR cDNA were introduced into a pCW vector. S. typhimurium YG7108 cells were transformed with this vector. The mutagen producing ability of these enzymes for some N-nitrosamines were evaluated using the established S. typhimurium cells. We found that the substrate specificity of CYP2A6 and CYP2E1 was different among mutagens. CYP2A6 was responsible for the metabolic activation of N-nitrosamines possessing relatively long alkyl chains, whereas CYP2E1 was responsible for the metabolic activation of N-nitrosamines with relatively short alkyl chains. It is likely that CYP2A6 gene polymorphism is responsible for the interindividual variability on the cancer susceptibility. We found the whole deletion of CYP2A6 gene as a type of genetic polymorphism in Japanese. Thus, we developed a gene diagnosis method to detect the variant. We evaluated the relationship between the CYP2A6 gene whole deletion and the susceptibility to the lung cancer. The frequency of CYP2A6 gene whole deletion was significantly lower in the lung cancer patients than that of healthy volunteers.     

Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450: comparison of three types of expression systems

Currently three different methods have been taken to develop new mutagenicity tester strains containing human cytochrome P450s (CYPs). Each of these use a single expression vector. In this paper we describe a fourth approach, i.e., the coexpression of a CYP and its electron-transfer flavoprotein, NADPH CYP reductase (RED), encoded by two different expression vectors. The Escherichia coli mutagenicity tester strain BMX100 has been expanded to a strain, MTC which stably expresses human RED. This new tester strain permits the biplasmid coexpression of human CYP1A2 and RED (MTC1A2). This novel strain can be used for the determination of the mutagenicity of chemicals known to be procarcinogens and metabolized by CYP1A2. The mutagenicity tester strain MTC1A2 was compared with: (i) BMX100 using the post-mitochondrial rat liver fraction (S9); (ii) BMX100 with expressing CYP1A2 alone (iii) or with expressing CYP1A2 fused to rat RED or (iv) with expressing CYP1A2, bicistronically coexpressed with rat RED. The biplasmid RED/CYP coexpression system generated a strain with the highest methoxy- and ethoxy-resorufin dealkylase activities and the highest mutagenic activities for the procarcinogens 2-aminoanthracene (2AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). Furthermore, the metabolism of 2AA and IQ was detected more efficiently using the MTC1A2 strain than with the BMX100 strain plus the standard rodent liver S9 metabolic system.     

Biphasic effects of the flavonoids quercetin and naringenin on the metabolic activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline by Salmonella typhimurium TA1538 co-expressing human cytochrome P450 1A2, NADPH-cytochrome P450 reductase, and cytochrome b5

Heterocyclic amines (HCAs) produced by cooking meat products at high temperatures are promutagens that are activated by cytochrome P450 (CYP) lA2. Using a newly developed Salmonella typhimurium TA1538/1A2bc-b5 strain, we tested the effect of quercetin and naringenin on the mutagenicity of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). TA1538/1A2bc-b5 bears two plasmids, one expressing human CYP1A2 and NADPH-P450 reductase (NPR), and the other plasmid which expresses human cytochrome b5 (cyp b5). TA1538/1A2bc-b5 cells showed high activities of 7-ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) associated with CYP1A2 and are very sensitive to mutagenesis induced by several HCAs. MeIQ was found to be the strongest mutagen among the HCAs tested in this system. Mutagenicity of MeIQ was enhanced 50 and 42% by quercetin at 0.1 and 1 microM, respectively, but suppressed 82 and 96% at 50 and 100 microM. Naringenin also increased the MeIQ-induced mutation about 37 and 22% at 0.1 and 1 microM, but suppressed it 32 and 63% at 50 and 100 microM concentrations, respectively, in TA 1538/1A2bc-b5 cells. Thus, they stimulated the MeIQ induced mutation at low concentrations, but strongly suppressed it at high concentrations. This biphasic effect of flavonoids was due to the stimulation or the inhibition of CYP1A2 activity in a dose-dependent manner judging by the activities of EROD or MROD in the Salmonella cells. These results indicate that quercetin and naringenin can exhibit inhibitory or stimulating effects on CYP1A2 mediated mutagenesis by MeIQ, depending on their concentrations.     

Escherichia coli MTC, a human NADPH P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450 isoforms 1A1, 1A2, 2A6, 3A4, or 3A5: catalytic activities and mutagenicity studies

We report here on the genetic engineering of four new Escherichia coli tester bacteria, coexpressing human CYP1A1, CYP2A6, CYP3A4 or CYP3A5 with human NADPH cytochrome P450 reductase (RED) by a biplasmid coexpression system, recently developed to express human CYP1A2 in the tester strain MTC. The four new strains were compared for CYP- and RED-expression levels and CYP activities with the formerly developed CYP1A2 expressing strain. CYP1A2 and CYP2A6 were expressed at the highest, CYP1A1 at the lowest and CYP3A4 and CYP3A5 at intermediate expression levels. Membranes of all five tester bacteria demonstrated similar RED-expression levels, except for the two CYP3A-containing bacteria which demonstrated slightly increased RED-levels. CYP-activities were determined as ethoxyresorufin deethylase (CYP1A1 and CYP1A2), coumarin 7-hydroxylase (CYP2A6) and erythromycin N-demethylase (CYP3A4 and CYP3A5) activities. Reaction rates were comparable with those obtained previously for these CYP-enzymes, except for CYP3A5 which demonstrated a lower activity. Benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene demonstrated mutagenicity in the CYP1A1 expressing strain with mutagenic activities, respectively, approximately 10-fold and 100-fold higher as compared with those obtained with the use of rat liver S9 fraction. Aflatoxin B1 demonstrated a significant mutagenicity with all CYP expressing strains, albeit lower as compared to those obtained with the use of rat liver S9. CYP1A2 was approximately 3-fold more effective in generating a mutagenic response of AFB1 as compared to CYP3A4. CYP3A5 and CYP3A4 demonstrated comparable capacities in AFB1 bioactivation which was equal as found for CYP1A1. It is concluded that these four new strains contain stable CYP- and RED-expression, significant CYP-activities and demonstrated significant bioactivation activities with several diagnostic carcinogens.     

CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the omega-alcohol and to the corresponding diacid.

A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10-epoxystearic acid was converted into one major metabolite identified by GC/MS as 18-hydroxy-9,10-epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 +/- 0.2 microM and Vmax,app = 27 +/- 1 nmol x min(-1) x nmol(-1) P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10-epoxy-octadecan-1,18-dioic acid. The diacid was also produced in microsomal incubations of 18-hydroxy-9,10-epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the omega-hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.     

Plasmid-mediated expression of the UmuDC mutagenesis proteins in an Escherichia coli strain engineered for human cytochrome P450 1A2-catalyzed activation of aromatic amines.

The mutagenic actions of many chemicals depend on the activities of bacterial "mutagenesis proteins", which allow replicative bypass of DNA lesions. Genes encoding these proteins occur on bacterial chromosomes and plasmids, often in the form of an operon (such as umuDC or mucAB) encoding two proteins. Many bacterial strains used in mutagenicity testing carry mutagenesis protein genes borne on plasmids, such as pKM101. Our objective was to introduce mutagenesis protein function into Escherichia coli strain DJ4309. This strain expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase and carries out the metabolic conversion of aromatic and heterocyclic amines into DNA-reactive mutagens. We discovered that many mutagenesis-protein plasmids severely inhibit the response of strain DJ4309 to 2-amino-3,4-dimethylimid-azo[4,5-f]quinoline (MeIQ), a typical heterocyclic amine mutagen. Among many plasmids examined, one, pGY8294, a pSC101 derivative carrying the umuDC operon, did not inhibit MeIQ mutagenesis. Strain DJ4309 pGY8294 expresses active mutagenesis proteins, as shown by its response to mutagens such as 1-nitropyrene and 4-nitroquinoline 1-oxide (4-NQO), and is as sensitive as the parent strain DJ4309 to P450-dependent mutagens, such as MeIQ and 1-aminopyrene.     

Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/umu assay

Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3microM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3-30microM) was similar (approximately 300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10microM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.     

Inter-individual differences in the metabolism of environmental toxicants: cytochrome P450 1A2 as a prototype.

Cytochrome P450 (P450) 1A2 provides an interesting paradigm for inter-individual differences in the metabolism of pro-carcinogens. The enzyme is known to vary 40-fold among individuals and may contribute to cancers caused by heterocyclic amines and other chemicals. Rat and human P450 1A2 are known to be 75% identical and were compared for several catalytic activities. The human enzyme was an order of magnitude more efficient in the N-hydroxylation of two heterocyclic amines. Further, the levels of P450 1A2 expressed in human livers show a 40-fold variation, with some as high as 0.25 nmol P450 1A2 per milligram microsomal protein. Some human liver samples are more active (than those isolated from polychlorinated biphenyl-treated rats) in the activation of heterocyclic amines. A bacterial genotoxicity assay has been developed in which human P450 1A2 and NADPH-P450 reductase are expressed within Escherichia coli and bacterial mutants can be assayed using reversion to lac prototrophy. A random mutagenesis strategy for human P450 1A2 has been developed and used to examine the changes in catalytic activity seen with many single-amino acid substitutions. These results may be of relevance in consideration of genetic polymorphisms. Further, the findings pose a challenge to molecular epidemiology effort in that results with one substrate do not necessarily predict those for others. Some dinitropyrenes are P450 1A2 substrates but others are not. 6-Nitrochrysene can be activated by human P450 1A2 but the (mono) nitropyrenes examined were not; these were oxidized by P450 3A4 instead.     

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.     

Cytochrome b(5) coexpression increases the CYP2E1-dependent mutagenicity of dialkylnitrosamines in methyltransferase-deficient strains of Salmonella typhimurium.

Addition of cytochrome b₅ to recombinant cytochrome P450 2E1 systems has been shown to enhance the metabolism of dialkylnitrosamines in vitro. To determine if this effect could be observed with recombinant expression systems in vivo, we have constructed mutagenicity tester strains that coexpress full-length human cytochrome P450 2E1 (CYP2E1), rat cytochrome P450 reductase, and human cytochrome b₅ in Salmonella typhimurium lacking ogt and ada methyltransferases (YG7104, ogt⁻; and YG7108, ogt⁻, ada⁻). These new recombinant strains exhibit a four- to five-fold greater mutagenic response to dimethylnitrosamine, diethylnitrosamine, and dipropylnitrosamine than strains that contain only CYP2E1 and reductase, and are over 100-fold more sensitive to nitrosamines than the parental strains in the presence of an exogenous activating system (S9 fraction). The four-fold increase in mutagenicity in the presence of cytochrome b₅ was consistent with increasing alkyl chain length up to dibutylnitrosamine, which was poorly activated by CYP2E1. The greatest enhancement was obtained with a tricistronic construct in which the b₅ cDNA preceded the P450 and reductase cDNAs; placing the b₅ cDNA after the reductase cDNA was substantially less effective. These new, highly sensitive strains may prove useful in the detection of nitrosamine contamination of food and environmental samples.     

Characterization of Cupriavidus metallidurans CYP116B1--a thiocarbamate herbicide oxygenating P450-phthalate dioxygenase reductase fusion protein

The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus?metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently (K(m[NADPH]) = 0.9 ± 0.5 μM and K(m[NADH]) = 399.1 ± 52.1 μM). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multi-angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.     

P450 reductase and cytochrome b5 interactions with cytochrome P450: effects on house fly CYP6A1 catalysis

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b5. The effects of cytochrome b5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost.     

Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench

The two multifunctional cytochrome P450 enzymes, CYP79A1 and CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench have been characterized with respect to substrate specificity and cofactor requirements using reconstituted, recombinant enzymes and sorghum microsomes. CYP79A1 has a very high substrate specificity, tyrosine being the only substrate found. CYP71E1 has less stringent substrate requirements and metabolizes aromatic oximes efficiently, whereas aliphatic oximes are slowly metabolized. Neither CYP79A1 nor CYP71E1 catalyze the metabolism of a range of different herbicides. The reported resistance of sorghum to bentazon is therefore not linked to the presence of CYP79A1 or CYP71E1. NADPH is a much better cofactor than NADH although NADH does support the entire catalytic cycle of both P450 enzymes. Km and Vmax values for NADPH when supporting CYP71E1 activity are 0.013 mM and 111 nmol/mg protein/s. For NADH, the corresponding values are 0. 3 mM and 42 nmol/mg protein/s. CYP79A1 is a fairly stable enzyme. In contrast, CYP71E1 is labile and prone to rapid denaturation at room temperature. CYP71E1 is isolated in the low spin form. CYP71E1 catalyzes an unusual dehydration reaction of an oxime to the corresponding nitrile which subsequently is C-hydroxylated. The oxime forms a peculiar reverse Type I spectrum, whereas the nitrile forms a Type I spectrum. Several compounds which do not serve as substrates formed Type I substrate binding spectra with the two P450 enzymes.