P450 2A Research Articles

Molecular response and developmental speculations in metamorphosis of the veined rapa whelk, Rapana venosa.

Metamorphosis is the short developmental stage characterized by dramatic ontogenetic changes that occurs in most animals. However, this important process remains largely unclear in marine invertebrates. In this study, we performed the sequential RNA sequencing of a representative mollusk, the rapa whelk (Rapana venosa), that is undergoing metamorphosis and conducted differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) to investigate the overall and dynamic transcriptome responses. The results revealed that the expression of cytochrome P450 2A and 3A were upregulated during metamorphosis, while the expression of H/ACA ribonucleoproteins increased 4 h after metamorphosis induction (M4 stage), indicating that R. venosa mainly responded to the pelagobenthic changes. At the M24 stage, the enrichment of V-type proton ATPase and insulin indicated the complete development of secretory organs and initiation of hormone secretion. Furthermore, at the M48 stage, the enrichment of zinc metalloproteinase and conotoxin indicated a well-developed predation system that requires exogenous nutrition. Finally, during the PL stage, the genes associated with growth control were highly enriched, implying that R. venosa had completed metamorphosis and has entered the period of rapid growth. Therefore, our study provides useful transcriptomic resources for R. venosa and contributes new insights that may assist in elucidating the mechanisms underlying metamorphosis in marine invertebrates.

Genetic diversity of cytochrome P450 2A with different metabolic activities in domestic cats.

Knowledge of genetic polymorphisms of cytochrome P450 (CYP), the most important xenobiotic metabolizing enzyme, is very limited in cats. Preliminarily, we investigated genetic polymorphisms in CYP2A13, one of the major CYP isoforms in the liver and lung. Four synonymous and three non-synonymous polymorphic variants were identified in feline CYP2A13 in domestic cats in Japan, without an obvious major type. Metabolic parameters, Km and Vmax, of coumarin hydroxylation of CYP2A13 were shown to range within two times for the identified non-synonymous polymorphic variants by using heterologous coexpression system in Escherichia coli. The results confirmed the polymorphic nature of CYP2A13 as a basis for effective application of medicines and prevention of adverse reactions in treatment of domestic cats.

Electronic cigarettes: The nicotyrine hypothesis

There are conflicting reports about the efficacy of electronic cigarettes (e-cigs) as nicotine delivery devices and smoking cessation products. In addition, smokers’ responses to some nicotine dependence questions often change as they transition to exclusive e-cig use. Nicotyrine may explain these observations. Nicotyrine forms by the gradual oxidation of nicotine in e-liquids exposed to air. E-cigs aerosolize nicotyrine along with nicotine. Nicotyrine inhibits the cytochrome P450 2A family of enzymes (CYP2A) in airways and liver. These enzymes metabolize nicotine to cotinine, and then cotinine to trans 3-hydroxycotinine. In humans, nicotine is metabolized primarily by hepatic CYP2A6.We propose that e-cig users (vapers) achieve measurable serum nicotine levels when they inhale nicotine and nicotyrine together, because nicotyrine reversibly inhibits nicotine metabolism by CYP2A13 in airways. Consuming nicotyrine by any route should irreversibly inhibit hepatic CYP2A6. When CYP2A6 is substantially inhibited, nicotine clearance is delayed and nicotine withdrawal symptoms are attenuated. Small, relatively infrequent nicotine doses can then sustain satisfying nicotine levels.This theory has numerous implications for e-cig research and tobacco control. Behavioral and pharmacokinetic e-cig studies should be interpreted with attention to likely levels of nicotyrine delivery: e-cig studies may need to routinely measure nicotyrine exposure, assess CYP2A6 activity, confirm nicotine delivery, or deliberately compare unoxidized and oxidized e-liquids. The risks of nicotyrine exposure include impaired clearance of all CYP2A substrates and any effects of the metabolic products of nicotyrine. CYP2A inhibitors like nicotyrine may be useful for future smoking cessation therapy.

Tu2053 Precise Replication of Human Liver Fetal Development with Human Bioengineered Liver Organoids: A Novel Model to Test the Teratogenesis of New Drug Candidates?

Introduction Bioengineering of a fully functional tissue requires precise recapitulation of normal tissue development. Specifically for the liver, one may use bipotent human liver progenitor cells (hFLCs) capable of differentiation into hepatocytes and cholangiocytes. The goal of the current study was to develop a system that would efficiently recapitulate embryonic development of hepatic parenchymal tissue and bile ducts, using decellularized liver extracellular matrix (ECM) as scaffolds.Materials andMethods hFLCs were seeded on decellularized liver ECM discs (300 μm thickness, 8 mm diameter) and were cultured for up to 3 weeks in presence of hepatic differentiation medium. Immunofluorescence microscopy was used to determine the extent of progenitor cell differentiation into hepatocytes and cholangiocytes. Urea, albumin and drug metabolism were quantified as paramaters of liver function. Furthermore, a γ-secretase inhibitor was added to the culture media and bile duct and hepatocyte development was monitored. Results hFLCs seeded on acellular liver ECM discs differentiated into hepatocytes and cholangiocytes. The cells showed predominant albumin expression along with loss of α-feto protein (AFP) expression at 3 weeks (Fig. 1D,E). The cells also expressed other mature hepatocyte markers like HNF-4α, α-1-antitrypsin and cytochrome P450 1A2, 2A and 3A (Fig. 1B-E). The cells in the ductular structures expressed bile duct specific markers like CK19, SOX9, EpCAM, ASBT, β-catenin and the presence of apical primary cilia (stained with α-acetylated tubulin), thus demonstrating differentiation towards cholangiocyte lineage along with maintaining apico-basal polarity (Fig. 1B-E). Urea and albumin secretion was higher in the liver disc organoids compared to control hFLCs cultured in petri dishes. Several metabolites of the drugs diazepam and 7-ethoxycoumarin were also detected by LC-MS/MS, showing broad cytochrome P450 activity in these organoids. The addition of a γ-secretase inhibitor severely impacted the number and maturation of bile ducts formed, mirroring a biliary atresia model. Discussion and Conclusions Our results demonstrate the efficient generation of bioengineered human liver tissue with hFLC that recapitulates stepwise development of hepatocyte and bile duct formation (Fig. 1A). Altogether, this study demonstrates the potential of this technology to study and mimic human liver development. These models provide novel approaches for liver bioengineering, drug discovery and toxicology (including drug teratogenesis evaluation in vitro), and ultimately for the treatment of liver disease.

Identification and functional characterization of novel feline cytochrome P450 2A

1. Cytochrome P450s are the major metabolizing enzymes for xenobiotics in humans and other mammals. Although the domestic cat Felis catus, an obligate carnivore, is the most common companion animal, the properties of cytochrome P450 subfamilies are largely unknown.2. We newly identified the feline CYP2A13, which consists of 494 deduced amino acids, showing the highest identity to CYP2As of dogs, followed by those of pigs, cattle and humans.3. The feline CYP2A13 transcript and protein were expressed almost exclusively in the liver without particular sex-dependent differences.4. The feline CYP2A13 protein heterogeneously expressed in Escherichia coli showed metabolic activity similar to those of human and canine CYP2As for coumarin, 7-ethoxycoumarin and nicotine.5. The results indicate the importance of CYP2A13 in systemic metabolism of xenobiotics in cats.

A Targeted Proteomics Approach for Profiling Murine Cytochrome P450 Expression

The cytochrome P450 (P450) superfamily of enzymes plays a prominent role in drug metabolism. Although mice are a widely used preclinical model in pharmacology, the expression of murine P450 enzymes at the protein level has yet to be fully defined. Twenty-seven proteins belonging to P450 subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 2F, 2J, 2U, 3A, 4A, 4B, 4F, and 4V were readily detectable in Balb/c mouse tissue using a global mass spectrometry-based proteomics approach. Subsequently, a targeted mass spectrometry-based assay was developed to simultaneously quantify these enzymes in ranges of femtomoles of P450 per microgram of total protein concentration range. This screen was applied to mouse liver microsomes and tissue lysates of kidney, lung, intestine, heart, and brain isolated from mixed-sex fetuses; male and female mice that were 3-4 weeks, 9-10 weeks, and 8-10 months of age; and pregnant mice. CYP1A2 was consistently more abundant in male mouse liver microsomes compared with age-matched females. Hepatic expression of CYP2B9 was more abundant in 3- to 4-week-old male and female mice than in mice of other ages; in addition, CYP2B9 was the only enzyme that was detectable at higher levels in pregnant mouse liver microsomes compared with age-matched females. Interestingly, sexually dimorphic expression of CYP2B9, 2D26, 2E1, and 4B1 was observed in kidney only. The targeted proteomics assay described here can be broadly used as a tool for investigating the expression patterns of P450 enzymes in mice.

Metabolic Activation of Polycyclic Aromatic Hydrocarbons and Aryl and Heterocyclic Amines by Human Cytochromes P450 2A13 and 2A6

Human cytochrome P450 (P450) 2A13 was found to interact with several polycyclic aromatic hydrocarbons (PAHs) to produce Type I binding spectra, including acenaphthene, acenaphthylene, benzo[c]phenanthrene, fluoranthene, fluoranthene-2,3-diol, and 1-nitropyrene. P450 2A6 also interacted with acenaphthene and acenaphthylene, but not with fluoranthene, fluoranthene-2,3-diol, or 1-nitropyrene. P450 1B1 is well-known to oxidize many carcinogenic PAHs, and we found that several PAHs (i.e., 7,12-dimethylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene-5,6-diol, benzo[c]phenanthrene, fluoranthene, fluoranthene-2,3-diol, 5-methylchrysene, benz[a]pyrene-4,5-diol, benzo[a]pyrene-7,8-diol, 1-nitropyrene, 2-aminoanthracene, 2-aminofluorene, and 2-acetylaminofluorene) interacted with P450 1B1, producing Reverse Type I binding spectra. Metabolic activation of PAHs and aryl- and heterocyclic amines to genotoxic products was examined in Salmonella typhimurium NM2009, and we found that P450 2A13 and 2A6 (as well as P450 1B1) were able to activate several of these procarcinogens. The former two enzymes were particularly active in catalyzing 2-aminofluorene and 2-aminoanthracene activation, and molecular docking simulations supported the results with these procarcinogens, in terms of binding in the active sites of P450 2A13 and 2A6. These results suggest that P450 2A enzymes, as well as P450 Family 1 enzymes including P450 1B1, are major enzymes involved in activating PAHs and aryl- and heterocyclic amines, as well as tobacco-related nitrosamines.

The enhancing effect of ethanol on the mutagenic activation of N-nitrosomethylbenzylamine by cytochrome P450 2A in the rat oesophagus

Alcohol consumption is frequently associated with various cancers and the enhancement of the metabolic activation of carcinogens has been proposed as a mechanism underlying this relationship. The ethanol-induced enhancement of N-nitrosodiethylamine (DEN)-mediated carcinogenesis can be attributed to an increase in hepatic activity. However, the mechanism of elevation of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis remains unclear. To elucidate the mechanism underlying the role of ethanol in the enhancement of NMBA-induced oesophageal carcinogenesis, we evaluated the hepatic and extrahepatic levels of the cytochrome P450 (CYP) and mutagenic activation of environmental carcinogens by immunoblot analyses and Ames preincubation test, respectively, in F344 rats treated with ethanol. Five weeks of treatment with 10% ethanol added to the drinking water or two intragastric treatments with 50% ethanol, both resulted in elevated levels of CYP2E1 (1.5- to 2.3-fold) and mutagenic activities of DEN, N-nitrosodimethylamine and N-nitrosopyrrolidine in the presence of rat liver S9 (1.5- to 2.4-fold). This was not the case with CYP1A1/2, CYP2A1/2, CYP2B1/2 or CYP3A2, nor with the activities of 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, aflatoxin B(1) or other N-nitroso compounds (NOCs), including NMBA. Ethanol-induced elevations of CYP2A and CYP2E1 were observed in the oesophagus (up to 1.7- and 2.3-fold) and kidney (up to 1.5- and 1.8-fold), but not in the lung or colon. In oesophagus and kidney, the mutagenic activities of NMBA and four NOCs were markedly increased (1.3- to 2.4-fold) in treated rats. The application of several CYP inhibitors revealed that CYP2A were likely to contribute to the enhancing effect of ethanol on NMBA activation in the rat oesophagus and kidney, but that CYP2E1 failed to do so. These results showed that the enhancing effect of ethanol on NMBA-induced oesophageal carcinogenesis could be attributed to an increase in the metabolic activation of NMBA by oesophageal CYP2A during the initiation phase, and that this occurred independently of CYP2E1.

Cytochrome P450 2A isoenzymes in freshly prepared blood lymphocytes isolated from rats and validation as a biomarker for clinical studies in humans

1. The present study aimed to identify the expression of carcinogen metabolizing cytochrome P4502A (CYP2A) isoenzymes in freshly prepared rat peripheral blood lymphocytes (PBL) isolated from adult rats and investigate similarities in the regulation of lymphocyte CYP2A-isoenzymes with the tissue enzyme.2. qRT-PCR studies demonstrated significant constitutive mRNA expression of CYP2A-isoenzymes in PBL isolated from male and female rats which further increases significantly after pretreatment with nicotine or 3-methylcholanthrene (MC) indicating responsiveness of CYP2A-isoenzymes in PBL. This increase in the CYP2A expression was associated with an increase in the protein expression and CYP2A3-dependent coumarin hydroxylase (COH) activity in PBL.3. Clinical studies further demonstrated significant increase in the expression of CYP2A6 and associated enzyme activity in PBL isolated from lung cancer patients. Our data thus provided evidence for similarities in the regulation of carcinogen metabolizing CYP2A-isoenzymes in PBL with the tissue enzymes. Further, responsiveness of blood CYP2A6 in human blood lymphocytes isolated from lung cancer patients has led us to suggest that associating expression profiles of CYP2A6 and other polycyclic aromatic hydrocarbons (PAH)-responsive CYPs in PBL with the genotyping data could lead to the development of a possible screen to monitor and predict environment-induced diseases and toxicity in humans.

Nicotine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Binding and Access Channel in Human Cytochrome P450 2A6 and 2A13 Enzymes

Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.

Simultaneous determination of cytochrome P450 1A, 2A and 3A activities in porcine liver microsomes

The aim of this study was to develop a robust method for the simultaneous determination of the activities of three porcine CYP450 enzymes in hepatic microsomes. A cocktail consisting of three selective CYP450 probe substrates, 7-ethoxyresorufin (CYP1A), coumarin (CYP2A) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC; CYP3A), was incubated with porcine liver microsomes. The presence of 7-ethoxyresorufin appears to significantly influence the kinetics of coumarin hydroxylation and BFC O-debenzylation. These results indicate that the use of 7-ethoxyresorufin in substrate cocktails together with coumarin and BFC should be avoided.

The use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) technique to detect and isolate cytochrome P450 2E1 and 2A genes

Cytochrome P450 (CYP, P450) enzymes are widely studied for their involvement in the metabolism of drugs as well as endogenous substrates. Assessing some forms of P450 such as P450 2E1 and 2A phenotype may be of great importance to predict organism’s susceptibility to xenobiotic and environmental pollutants which are metabolically activated by these isoenzymes. In the present study, we designed universal degenerate oligonucleotide primers sets based on the highly conserved nucleotide sequences at the 5’ and 3’ cDNA ends of P450 2E1 and 2A gene family from Camelus dromedaries (Arabian camel), Bos Taurus (cow), Capra hircus (domestic goat), Ovis aries (domestic sheep), Oryctolagus cuniculus (European rabbit), Xenopus tropicalis (Western clawed frog), Rattus norvegicus (Norway rat), Mus musculus (house mouse) and Mesocricetus auratus (golden hamster). We successfully demonstrated that these primers sets were able to detect the expression of P450 2E1 and 2A genes from these eukaryotic organisms. Moreover, we amplified 1070 and 1200 nucleotides of both P450 2E1 and 2A from these organisms and this represent two third of the actual size of these P450 isoforms. The nucleotide sequences of these genes showed percent identities ranging from 80 to 85%. The degenerate primers designed in this study allowed the detection and amplification of known and unknown P450s belonging to the gene families 2E1 and 2A from different organisms. Key words : Cytochrome P450, xenobiotics, degenerate primers, mixed function oxidases.

Expression and activities of hepatic cytochrome P450 (CYP1A, CYP2A and CYP2E1) in entire and castrated male pigs

This study aimed to provide further insights into the mechanism of in vivo regulation of cytochrome P450 (CYP450) 1A, 2A and 2E1 activities in pigs with different levels of testicular steroids. Hepatic mRNA and protein expression and enzymatic activity of CYP1A, CYP2A and CYP2E1 were compared between entire male and castrated pigs. Castration was performed either surgically or immunologically. The pigs were divided into four groups. In the first group, piglets were surgically castrated without anaesthesia. Immunological castration was performed by vaccination with Improvac® (Pfizer Ltd). Vaccinated pigs were subdivided into two groups according to the vaccination regimen: early and standard vaccination. Pigs in the early vaccination group were vaccinated when aged 11 and 15 weeks. Pigs in the standard vaccination group were vaccinated when aged 17 and 21 weeks. In the control group, pigs remained intact throughout the study. Hepatic CYP450 mRNA expression, measured by real-time RT-PCR, differed significantly between groups for all isoforms measured: CYP1A2 (P = 0.002), 2A (P = 0.000) and 2E1 (P = 0.002). Lower CYP450 mRNA in entire male pigs suggests suppression of CYP1A2, CYP2A and CYP2E1 by testicular steroids at the transcriptional level. However, this suppression was not always reflected in decreased protein expression and activities of these isoforms, suggesting that at least some CYP450s (e.g. CYP2E1) are regulated by a post-transcriptional mechanism.

In vitro metabolism of the anti-androgenic fungicide vinclozolin by rat liver microsomes

Vinclozolin (V) is a fungicide used in agricultural settings. V administered to rats is hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2). V, M1 and M2 have antiandrogenic properties by interacting with the androgen receptor. Data on V, M1 and M2 biotransformation are limited. Our objective was to characterize V metabolism by rat liver microsomes. V was incubated with non-treated adult male Long-Evans rat liver microsomes and NADPH. Several metabolites were detected following the extraction of incubate with acetonitrile and analysis by HPLC/DAD/MSD. One metabolite was identified as [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione] (M4), which was gradually converted to 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (M5). Both co-eluted in the same HPLC peak. Another metabolite ([M7]) was detected by UV but was unstable for mass spectral analysis. The K(M app) for co-eluted M4/M5 and [M7] was 53.7 and 135.4 μM, the V(max app) was 0.812 and 0.669 nmoles/min/mg protein, and CL(int) was 15.1 and 4.9 ml/min/g protein, respectively. Pilocarpine, orphenadrine and proadifen and anti-rat cytochrome P450 (CYP)2A, 2B and 3A antibodies inhibited M4/M5 and [M7] formation. These results indicate that V is efficiently metabolized by CYP. Determination of the metabolites of V will provide further insight into the relationship between toxicity and tissue dose of V and its metabolites.

Abstract LB-432: Effect of chronic nicotine consumption on NNK-induced lung tumors in the A/J mouse

Abstract Nicotine replacement therapy (NRT) is often used to maintain smoking cessation. Some individuals continue to use NRT for years after smoking cessation, and some NRT users continue to smoke. It is important to understand the impact of chronic nicotine use on lung cancer risk. Therefore, we determined the effect of nicotine administration on NNK-induced lung tumors in the A/J mice. P450 2A enzymes are key catalysts in NNK bioactivation, and nicotine is both an inhibitor and a mechanism based inactivator of P450 2A-catalyzed nicotine metabolism. These data suggest nicotine might protect from NNK-induced tumorigenesis. However, several investigators have reported that nicotine inhibits apoptosis, enhances angiogenesis and enhances proliferation. We hypothesized that nicotine would prevent or delay initiation of NNK-induced tumors, but accelerate tumor growth by reducing apoptosis and promoting inflammation. To test this, female A/J mice were administered a single dose of NNK (100 mg/kg i.p) and 200 ug/ml nicotine in the drinking water. Nicotine was administered 2 weeks prior to NNK, 44 weeks after NNK, throughout the experiment, or without NNK treatment. There were several key outcomes of this study: Chronic nicotine consumption did not affect P450 2A5-catalyzed metabolism in the lung of liver. Nicotine administration alone for 44 weeks did not increase lung tumor multiplicity. Nicotine given before or after NNK administration did not increase, or decrease, tumor number or tumor size. There was also no difference in the number of carcinomas per mouse or the number of adenomas with dysplasia. In conclusion, nicotine consumption in the drinking water had no effect on the tumorigenicity of NNK in the A/J mouse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-432. doi:10.1158/1538-7445.AM2011-LB-432

In Vitro Cytochrome P450 2E1 and 2A Activities in the Presence of Testicular Steroids

Cytochrome P450 2E1 (CYP2E1) and 2A (CYP2A) are the main enzymes involved in the metabolism of skatole in pigs. In this study, physiological concentrations of androstenone, 17β-oestradiol and testosterone were tested for their ability to regulate CYP2E1 and CYP2A activity in liver microsomes isolated from entire male and female pigs as well as in microsomes from Saccharomyces cerevisiae expressing either human recombinant CYP2E1 or CYP2A6. We found that physiological concentrations of androstenone and oestradiol had the ability to inhibit CYP2E1 activity. The magnitude of this inhibition (approximately 30%) was similar in recombinant human CYP2E1 and microsomes from entire male pigs. This inhibition was only seen when adding the steroid to the assay 15 min before the substrate. Interestingly, CYP2E1 activity in the microsomes from female pigs was not affected. None of the investigated steroids modified the activity of recombinant human CYP2A6. However, CYP2A activity was slightly increased in the microsomes from female pigs in the presence of oestradiol, but the magnitude of this increase was very low (below 10%) and probably irrelevant. Overall, these results indicate that physiological concentrations of androstenone and oestradiol have a potential to inhibit CYP2E1 activities in vitro, and that this inhibition is gender-specific. Further studies are needed to investigate the biochemical mechanisms underlying those differences between the genders.

Inhibition of cytochrome P450 2A participating in coumarin 7‐hydroxylation in pig liver microsomes

Five commonly used human cytochrome P450 (CYP) inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A) activity in pig liver microsomes. The K(m) and V(max) values for coumarin 7-hydroxylation in pig liver microsomes were estimated to be 1 μm and 0.26 nmol·mg/min, respectively. The following human CYP inhibitors caused little or no inhibition of CYP2A as defined by a K(i) > 200 μm: quinidine (CYP2D6), troleandomycin (CYP3A4), and sulfaphenazole (CYP2C9). The other two human CYP inhibitors were classified as strong inhibitors of CYP2A: 8-methoxypsoralen (CYP2A6) and α-naphthoflavone (CYP1A1/2). In the absence of a preincubation period, 8-MOP inhibited the 7-hydroxylation of coumarin with a K(i) value of 1.1 μm, which decreased to 0.1 μm when 8-MOP was preincubated with pig liver microsomes for 3 min. α-Naphthoflavone inhibited the 7-hydroxylation of coumarin with a K(i) value of 32 μm, which did not increase ability to inhibitor CYP2A when α-naphthoflavone was preincubated with pig liver microsomes for 3 min. These results of this study suggest that 8-MOP is a potent, mechanism-based inhibitor of pig CYP2A activity in pig liver microsomes.

Gender-related differences in cytochrome P450 in porcine liver--implication for activity, expression and inhibition by testicular steroids.

In pigs, the hepatic cytochrome P450 (CYP) 1A2, 2A and 2E1 activity is important in the regulation of skatole accumulation in adipose tissue. This study investigated gender-related differences in CYP1A2, 2A and 2E1 dependent activity, protein and mRNA expression. This study also investigated the gonadal steroid dependent inhibition of CYP activity in relation to gender and dietary composition. Microsomes were prepared from the liver of female and entire male pigs (Landrace × Yorkshire sire and Duroc boars) reared under similar conditions and slaughtered at an age of 164 days. A group of entire male pigs fed dried chicory root for 16 days prior to slaughter were included in the study. CYP activities were assessed by the use of probe substrates, whilst mRNA and protein expression were analysed by RT-PCR and Western blotting. Furthermore inhibition of CYP dependent activity by gonadal steroids was assessed in vitro. Microsomes from female pigs had greater CYP1A2 and 2A activity, as well as mRNA expression compared to entire male pigs. The antibodies used did not detected differences in protein expression. In vitro inhibition by 17β-oestradiol, oestrone, androstenone and 3β-OH androstenol of CYP2E1 activity in microsomes from entire male pigs as well as inhibition of CYP1A activity in chicory fed entire male pigs was observed. Apart from that no effect of steroids was shown. In conclusion, female pigs show greater CYP activity and mRNA expression. Including chicory in the diet for 16 days changed the gonadal steroid dependent inhibition of CYP activity in entire male pigs.

Human Cytochrome P450 2A7: Stability and Activity Relative to other Human CYP2A Enzymes

Humans have three cytochrome P450 (CYP) 2A genes: CYP2A6, CYP2A7, and CYP2A13. CYP2A6 and CYP2A13 proteins metabolize coumarin and other compounds. Although CYP2A7 is 94% identical to CYP2A6, it has been reported that CYP2A7 is an inactive protein. CYP2A7 expressed in vaccinia virus did not yield functional holoprotein as evaluated by spectral analysis, Western blot, and catalytic activity, likely failing to incorporate heme1. Western blot revealed that CYP2A7 protein was produced in COS cells, but this protein did not metabolize coumarin to 7‐hydroxycoumarin2. Our goal is to reevaluate if CYP2A7 is a stable holoprotein capable of metabolizing coumarin or other CYP2A substrates. We have expressed truncated, His‐tagged CYP2A7 in E. coli. This system produces CYP2A7 protein that does incorporate heme, as indicated by a spectral shift in the reduced carbon monoxide difference assay. Generation of purified CYP2A7 protein is enabling new comparisons of human CYP2A stability, ligands, and catalytic activity. Additionally, comparison of the CYP2A7 sequence with the CYP2A6 and CYP2A13 structures suggests substitutions that may be responsible for differences among the human CYP2A enzymes. Research supported by NIH GM76343 (EES).1Yamano, S.; Tatsuno, J.; Gonzalez, F. J. Biochemistry 1990, 29, 1322‐1329.2Ding, S.; Lake, B. G.; Friedberg, T.; Wolf, C. R. Biochem J 1995, 306, 161‐166.

Cancer Chemoprevention: Selective Inhibition of Cytochrome P450 2A13

4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) is one of the most prevalent and carcinogenic compounds in tobacco. NNK is selectively activated by human cytochrome P450 2A13 (CYP2A13) to form two carcinogens that generate DNA adducts. CYP2A13 is found in the respiratory tract where NNK‐derived adducts can result in lung cancer. CYP2A13 is 93.5% identical to the hepatic cytochrome P450 2A6 (CYP2A6), which does not efficiently metabolize NNK but does metabolize other xenobiotic compounds. Thus a selective inhibitor of CYP2A13 could be used as a chemopreventative for tobacco users. High throughput screening was used to identify small molecule scaffolds that selectively bound CYP2A13 over CYP2A6. One such scaffold was benzylmorpholine. A small library of benzylmorpholine analogs was generated and has been evaluated for differential ligand binding (Kd) and inhibition of human cytochrome P450 2A enzymes. Examination of these results is revealing selective compounds that could be used for the reduction of lung cancer related to DNA damage in tobacco users.