7-ethoxyresorufin O-dealkylation Research Articles

Prokaryotic functional expression and activity comparison of three CYP9A genes from the polyphagous pest Helicoverpa armigera.

Cytochrome P450s (CYPs or P450s) have been long recognized as very important enzymes in the metabolism of xenobiotic and endogenous compounds, but only a few CYPs have been functionally characterized in insects. The effort in functional characterization of insect P450s is heavily hindered by technical difficulties in preparing active, individual P450 enzymes directly from the target insect. In this paper, we describe the functional expression of two additional pyrethroid resistance-associated CYP9A genes (CYP9A12 and CYP9A17) from the polyphagous pest Helicoverpa armigera in the facile Escherichia coli. The functionality of E. coli produced CYP9A12, CYP9A14, and CYP9A17 was investigated and activities of these CYP9As were compared against three probe substrates after reconstitution with NADPH-dependent cytochrome P450 reductase. The results showed that active forms of CYP9A12 and CYP9A17 were expressed in E. coli with a content of about 1.0-1.5 nmol mg-1 protein in membrane preparations. In vitro assays showed that CYP9A14 was capable of catalyzing O-dealkylation of methoxyresorufin (MROD), ethoxyresorufin (EROD), and benzyloxyresorufin (BROD), while CYP9A12 and CYP9A17 exhibited only MROD and EROD activities. Kinetic studies demonstrated that CYP9A14 had the greatest k cat/K m value for MROD, and CYP9A17 for EROD, while the lowest k cat/K m values for both MROD and EROD were observed for CYP9A12. The distinct biochemical traits suggest that the three paralogous CYP9As may play different roles in xenobiotic metabolism in this important pest.

Biochemical characterization of variants of canine CYP1A1 using heterologous expression.

Cytochrome P450 1A1 (CYP1A1) is a heme-containing mono-oxygenase involved in metabolism of environmental contaminants. Two variants of dog CYP1A1 with a single residue difference were identified and designated Sap1 and Sap2. Compared with Sap1, Sap2 had a Trp50Leu substitution. The biochemical characteristics of the variants were comparatively analyzed using heterologous expression in Escherichia coli. The membrane fraction of E. coli expressing Sap2 exhibited higher CYP holoprotein and heme contents than the Sap1-containing membranes, although the level of total CYP1A1 protein (i.e., apoprotein + holoprotein) was comparable between the groups. As normalized to holo-CYP content, the Sap2-expressing membranes showed lower CYP1A1-specific enzyme activities, such as 7-ethoxyresorufin O-dealkylation (EROD), than the Sap1 group. In single substitution variants of residue 50, proteins with hydrophobic residues having mass similar to Leu exhibited lower EROD activities than those with hydrophobic residues having larger mass than Leu. In addition, variants with polar or charged residues having mass similar to Leu showed activities that were comparable to those of Sap2. Taken together, these findings suggest that the Trp50Leu substitution leads to an enhancement of holo-CYP1A1 formation, but diminishes the enzyme activity because of the small size of Leu compared with Trp.

Characterization of the Gly45Asp variant of human cytochrome P450 1A1 using recombinant expression.

Cytochrome P450 1A1 (CYP1A1) is a heme-containing enzyme involved in metabolism of xenobiotics. CYP1A1 containing a Gly45Asp substitution has not yet been characterized. Escherichia coli expressing the Gly45Asp variant, as well as the purified variant protein, had lower CYP (i.e., holoenzyme) contents than their wild-type (WT) equivalents. The purified variant protein had reduced heme contents compared with their WT equivalents. Enhanced supplementation of a heme precursor during culture did not increase CYP content in E. coli expressing the variant, but did for the WT. Substitution of Gly45 with other residues, especially those having large side chains, decreased CYP contents of E. coli expressing the variants to a considerable extent. A 3D structure of CYP1A1 indicates that Gly45, along with other residues of the PR region, interacts with Arg77 of β- strand 1-1, which indirectly interacts with heme. Substitution analyses suggest the importance of residues of the PR region and Arg77 in holoenzyme expression. E. coli membrane and mammalian microsomes expressing the Gly45Asp variant, as well as the purified variant protein, had reduced ethoxyresorufin O-dealkylation activities, compared with the WT equivalents. These findings suggest the Gly45Asp substitution results in a structural disturbance of CYP1A1, reducing its holoenzyme formation and catalytic activity.

Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located.

Dehydroepiandrosterone post-transcriptionally modifies CYP1A2 induction involving androgen receptor

The pharmacological dosage of dehydroepiandrosterone (DHEA) protects against chemically induced carcinogenesis. The chemoprotective activity of DHEA is attributed to its inhibitory potential for the expression of CYP1A enzymes, which are highly responsible for metabolic activation of several mutagenic and carcinogenic chemicals. The present work investigated whether the chemoprevention by DHEA was due to diminished transcriptional activation of CYP1A genes or to the post-transcriptional modulation of CYP1A expression.In primary human hepatocytes, DHEA diminished the increase in CYP1A activities (7-ethoxyresorufin O-dealkylation and phenacetin O-dealkylation) and in CYP1A2 mRNA level induced by 3-methylcholanthrene, but did not alter the amount of CYP1A1 and CYP1B1 mRNA. The androgen receptor seemed to be involved in DHEA-mediated diminishment of CYP1A2 induction, which was attenuated in the presence of bicalutamide, the androgen receptor antagonist. The potential role of the glucocorticoid receptor and estrogen receptor in DHEA-mediated decrease in CYP1A2 induction was excluded. The developed computational model of CYP1A2 induction kinetics and CYP1A2 mRNA degradation proposed that a post-transcriptional mechanism was likely to be the primary mechanism of the DHEA-mediated diminishment of CYP1A2 induction. The hypothesis was confirmed by the results of actinomycin D-chase experiments in MCF-7 and LNCaP cells, displaying that the degradation rates of CYP1A2 mRNA were significantly higher in the cells exposed to DHEA.The novel findings on DHEA-mediated modulation of CYP1A2 mRNA stability may account for the beneficial effects of DHEA by decreasing the metabolic activation of pro-carcinogenic compounds.

Characterization of the Temporal Induction of Hepatic Xenobiotic-Metabolizing Enzymes by Glucosinolates and Isothiocyanates: Requirement for at Least a 6 h Exposure To Elicit Complete Induction Profile

A mechanism of action of chemopreventive glucosinolates/isothiocyanates, established largely in vitro, is to modulate carcinogen-metabolizing enzymes. Extrapolation in vivo involves relating in vitro concentrations to plasma/tissue concentrations attained in vivo, thus assuming that even transient exposure modulates enzyme activity. To test this hypothesis, precision-cut rat liver slices were incubated with glucosinolates for up to 24 h, and the O-dealkylation of methoxyresorufin and ethoxyresorufin was determined; increased activities were observed only at incubations of at least 6 h. To evaluate phase II enzymes, isothiocyanates, namely, sulforaphane, erucin, and phenethyl isothiocyanate, were similarly incubated; quinone reductase increased after incubation for 6 h or longer. When glutathione S-transferase was monitored, the phenethyl isothiocyanate-manifested rise necessitated at least a 6 h incubation, whereas in the case of sulforaphane and erucin, the activity was elevated after only 2 h. It is inferred that a rise in carcinogen-metabolizing enzymes by glucosinolates/isothiocyanates necessitates tissue exposure of at least 6 h.

Different profiles of hepatic alkoxyresorufin O‐dealkylase activities in small rodents

The aims of the present study were to determine cytochrome P450 enzyme activity in six strains of experimental rodents (n = 5/sex/species): ICR, C57BL/6 and DBA/2 mice; Sprague Dawley and Wistar rats; and Dunkin Hartley guinea pigs. After animals were treated with the typical inducers β-naphthoflavone (BNF), dexamethasone (DEX) and phenobarbital (PB), the levels of O-dealkylation of ethoxyresorufin (EROD), methoxyresorufin (MROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) activity were determined using responsive catalytic reactions to study CYP1A1, CYP1A2 and CYP2B, respectively. A maximal induction of EROD and MROD was found in BNF-treated animals from all strains (2.4- to 15.1-fold) except DBA/2 (0.9- to 1.8-fold). C57BL/6 mice had the strongest BNF-induced EROD (15.1-fold) and MROD (8.3-fold) activities. No differences in BNF-induced EROD and MROD activities were observed between males and females. However, the EROD activity of Wistar rats and the MROD activity of Sprague Dawley rats were higher in males than females. DEX induced PROD activity only in mice (1.3- to 7.1-fold), but not in rats and guinea pigs (0.2- to 1.1-fold). However, induction of BROD activity was found in DEX-treated mice and rats (1.5 to 12.5-fold), but not in guinea pigs (0.3 to 0.4-fold). PB caused a significant elevation of PROD (1.7- to 10.4-fold) and BROD (31- to 13.2-fold) activities in all the animals. PB-induced BROD activity was higher in females than males in Sprague Dawley rats. These observations strongly suggest that the choice of experimental animal strain, species and inducer is of critical importance for studies of drug metabolism and interaction.

Modulatory effects of Kaempferia parviflora extract on mouse hepatic cytochrome P450 enzymes

Ethnopharmacological relevanceKaempferia parviflora is a herbal plant, the extracts of which are commonly used as alternative medicines. It widely uses as aphrodisiac, anti-inflammation, anti-microbacterial, and anti-peptic ulcer. Aim of the studyIn order to obtain an effective utilization and safety of the herb, the influence of Kaempferia parviflora on hepatic CYP450 metabolizing enzymes including CYP1A1, CYP1A2, CYP2B, CYP2E1, and CYP3A was investigated. Materials and methodsThe impact of Kaempferia parviflora on CYP450 both in vitro and in vivo was examined by using ethoxyresorufin O-dealkylation, methoxyresorufin O-dealkylation, pentoxyresorufin O-dealkylation, p-nitrophenol hydroxylation, and erythromycin N-demethylation assays, respectively. ResultsIn vitro studies using non-induced mouse hepatic microsomes in the presence or absence of Kaempferia parviflora extract showed that Kaempferia parviflora extract altered CYP1A1, CYP1A2, CYP2B, and CYP2E1 activities by non-competitive, mixed-competitive, competitive, and uncompetitive mechanisms, respectively. Among these enzymes, CYP1A2 was affected by Kaempferia parviflora based on the highest value of Vmax (15.276±0.206nmol/min) and lowest of Ki value (0.008±0.002μg/ml). In addition, the plant extract also modulated CYP2B activity based on the low Km value (1.599±0.147pmol). For in vivo studies, mice were orally treated with 250mg/kg of Kaempferia parviflora extract for 7, 14, and 21 days. The results demonstrated that Kaempferia parviflora extract significantly induced CYP1A1, CYP1A2 enzyme activities following short-term treatment. CYP2B enzyme activities were markedly increased all Kaempferia parviflora extract treatment timepoints, whereas Kaempferia parviflora extract significantly enhanced CYP2E1 activity only after long-term treatment. However, Kaempferia parviflora extract did not affect the CYP3A enzyme activity. ConclusionsKaempferia parviflora extract modulated several CYP450 enzyme activities, thus, its utilization with drugs or other herbs should raise concern for potential drug–herb interactions.

Verapamil does not modify catalytic activity of CYP450 in rainbow trout after long-term exposure

Little is known about the effects of the cardiovascular drug verapamil (VRP) on metabolic processes in fish. Most calcium channel blockers including VRP are metabolized by cytochrome P450 (CYP450) enzymes. In this study we investigated the in vivo effect of VRP on some CYP450-mediated reactions in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270μgl−1) for 0, 21, and 42 day. The following CYP450-mediated reactions were studied in hepatic microsomes: O-dealkylation of ethoxyresorufin, methoxyresorufin, and pentoxyresorufin, hydroxylation of coumarin, tolbutamide, and p-nitrophenol, and O-debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin. The amounts of products of these reactions did not differ among fish exposed to different levels of VRP and control fish. This suggests that the levels of VPR used did not alter catalytic activity of the selected CYP450 enzymes. In conclusion, none of the investigated CYP450-mediated reactions has potential as a biomarker to monitor VRP contamination of the aquatic environment.

Comparison of three fluorescent CYP3A substrates in two vertebrate models: pig and Atlantic salmon

We investigated in vitro inhibitory effects of ketoconazole (KTZ) on cytochrome P450 activity in microsomes from pigs and Atlantic salmon. The following enzymatic reactions were studied: 7-benzyloxyresorufin and 7-ethoxyresorufin O-dealkylation (BROD and EROD, respectively), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylation (BFCOD) and 7-benzyloxyquinoline O-debenzylation (BQOD). KTZ was a potent non-competitive inhibitor of BROD and BQOD in the microsomes from pigs, whereas in the microsomes from Atlantic salmon, these reactions were competitively inhibited by KTZ. BFCOD activity was inhibited by KTZ in a non-competitive manner in both species. KTZ non-competitively inhibited EROD in Atlantic salmon, but not in porcine microsomes. The activity of BROD and BQOD was higher in male than that in female pigs, but the activity of BFCOD showed no sex-related differences.

Modified expression of aryl hydrocarbon receptor-related genes by deoxymiroestrol, a phytoestrogen, in mouse hepatocytes in primary culture

Ethnopharmacological relevanceDeoxymiroestrol (DM), a strong phytoestrogen from Pueraria candollei Wall. ex Benth. var. mirifica (family Leguminosae). This plant has long been used in traditional medicine for rejuvenation. Materials and methodsThe expression of aryl hydrocarbon receptor-related genes in mouse hepatocytes in primary culture was quantified by real-time RT-PCR and hepatic microsomal P450 activity was assessed by using ethoxyresorufin O-dealkylation. ResultsThe mRNA expression of the aryl hydrocarbon receptor (AhR), AhR nuclear translocator, and CYP1A1 was suppressed, whereas that of CYP1B1, estrogen receptor α (ERα), CYP2B9, and glutathione-S-transferase a2 (GSTa2) was increased. The effects of DM on the gene expression depended on treatment period and concentrations, and were similar to those of β-estradiol (E2). DM and E2 at pharmacological concentrations had a marked synergistic effect on CYP1A1 expression after combined treatment with a typical CYP1 inducer, β-naphthoflavone (βNF), at the level of both transcription and enzymatic activity. DM enhanced the inducible mRNA expression of CYP1A1 and CYP1B1 similar to E2. Meanwhile, the expression of ERα mRNA was not affected by βNF, which, on the contrary, completely eliminated the DM-induced mRNA expression of ERα, CYP2B9, and GSTa2. ConclusionThe findings that DM modified the expression of several metabolism-related genes suggest the need for caution when using health supplements having phytoestrogenic activity.

QUANTITATIVE RELATIONSHIP OF ELECTRONIC STRUCTURE AND INHIBITION ACTIVITY OF CURCUMIN ANALOGS ON ETHOXYRESORUFIN o-DEALKYLATION (EROD) REACTION

Electronic structure and inhibition activity relationship study of curcumin analogs has been established for 29 curcumin analogs on Ethoxyresorufin O-Dealkylation (EROD) reaction using atomic net charge descriptor based on AM1 semiempirical calculations. The QSAR (Quantitative Structure and Activities Relationships) equation model was determined by statistical parameter from multiple regression analysis and leave-one-out cross validation method. The best QSAR equation was described: Keywords: curcumin, QSAR, descriptor, atomic net charge, semiempirical methods.

Microfluidic-based measurements of cytochrome P450 enzyme activity of primary mammalian hepatocytes

A microfluidic-based system was developed for the in situ monitoring of the 7-ethoxyresorufin O-dealkylation (EROD) activity of primary rat hepatocytes by measuring the fluorescent intensity of both cells and their surrounding media. The microfluidic chip was designed to allow the cell suspension and test reagent to be introduced in a layer-by-layer flow format, thereby resulting in a short mixing time by diffusion. A good linear relationship was obtained between the resorufin concentration up to 30 microM and fluorescent intensity over the chip's circular chamber area. The EROD activity was determined with 3-methylcholanthrene (3-MC)-induced hepatocytes. The inhibition effect of alpha-naphthoflavone was also examined on EROD activity resulting in an IC(50) value of 12.98 microM.

Modulation of carcinogen-metabolising cytochromes P450 in human liver by the chemopreventive phytochemical phenethyl isothiocyanate, a constituent of cruciferous vegetables

On the basis of studies conducted in animals, it has been established that isothiocyanates suppress cytochrome P450 activity, leading to impairment of the bioactivation of carcinogens, this being a principal mechanism of their chemopreventive activity. However, no studies have been carried out in human tissue to ascertain whether hepatic cytochrome P450 composition is similarly modulated, and this is the objective of the present studies. Precision-cut liver slices from four donors were incubated with a range of concentrations of phenethyl isothiocyanate (PEITC) for 24 h, and the expression and activity of cytochrome P450 enzymes were determined; similar studies were performed in rat slices for comparison. PEITC suppressed the O-dealkylation of methoxyresorufin in all human livers and this was accompanied by a parallel drop in CYP1A2 apoprotein levels; the same effect was noted in rat liver slices. The O-dealkylation of ethoxyresorufin was also impaired in the human livers, despite a rise in CYP1A1 apoprotein levels. The CYP3A-mediated benzyloxyquinoline dealkylation was inhibited by PEITC in only two of the four human donors, whereas a rise in CYP3A4 apoprotein levels was noted in all human livers, albeit to different extent. It is concluded that: (a) PEITC can modulate cytochrome P450 composition in human liver, and (b) PEITC, at concentrations that can be achieved by dietary intake, can antagonise the carcinogenicity of chemicals which rely on the CYP1 family for their bioactivation such as heterocyclic amines and polycyclic aromatic hydrocarbons, and this is likely to be a major contributory mechanism to its chemopreventive activity.

Inhibition of procarcinogen‐bioactivating human CYP1A1, CYP1A2 and CYP1B1 enzymes by melatonin

Administration of melatonin to rodents decreases the incidence of tumorigenesis initiated by benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene, which requires bioactivation by cytochrome P450 enzymes, such as CYP1A1, CYP1A2 and CYP1B1, to produce carcinogenic metabolites. The present study tested the hypothesis that melatonin is a modulator of human CYP1 catalytic activity and gene expression. As a comparison, we also investigated the effect of melatonin on the catalytic activity of CYP2A6, which is also a procarcinogen-bioactivating enzyme. Melatonin (3-300 microm) decreased 7-ethoxyresorufin O-dealkylation catalyzed by human hepatic microsomes and recombinant CYP1A1, CYP1A2 and CYP1B1, whereas it did not affect coumarin 7-hydroxylation catalyzed by hepatic microsomes or recombinant CYP2A6. Melatonin inhibited CYP1 enzymes by mixed inhibition, with apparent K(i) values (mean +/- S.E.M.) of 59 +/- 1 (CYP1A1), 12 +/- 1 (CYP1A2), 14 +/- 2 (CYP1B1) and 46 +/- 8 microm (hepatic microsomes). Additional experiments indicated that melatonin decreased benzo[a]pyrene hydroxylation catalyzed by hepatic microsomes and CYP1A2 but not by CYP1A1 or CYP1B1. Treatment of MCF-10A human mammary epithelial cells with melatonin (up to 300 microm) did not affect basal or benzo[a]pyrene-inducible CYP1A1 or CYP1B1 gene expression. Consistent with this finding, melatonin did not influence reporter activity in aryl hydrocarbon receptor-dependent pGudluc6.1-transfected MCF-10A cells treated with or without benzo[a]pyrene, as assessed in an in vitro cell-based luciferase reporter gene assay. Overall, melatonin is an in vitro inhibitor of human CYP1 catalytic activity, and it may be useful to develop potent analogues of melatonin as potential cancer chemopreventive agents that block CYP1-mediated chemical carcinogenesis.

Modulation ofCYP1B1andCYP1A1gene expression and activation of aryl hydrocarbon receptor byGinkgo bilobaextract in MCF-10A human mammary epithelial cells

The aryl hydrocarbon receptor (AhR) signaling pathway regulates the production of CYP1B1 and CYP1A1, which catalyze the bioactivation of various procarcinogens. In the present study, we investigated the effect of Ginkgo biloba extract and some of its chemical constituents on CYP1B1 and CYP1A1 gene expression and AhR activity in cultured MCF-10A human mammary epithelial cells. Treatment of MCF-10A cells with noncytotoxic concentrations of G. biloba extract (25-300 microg/mL for 24 or 48 h) increased CYP1B1 and CYP1A1 mRNA expression, which was accompanied by an increase in CYP1-mediated ethoxyresorufin O-dealkylation activity. The inductive effects of G. biloba extract were attenuated by an AhR antagonist (3',4'-dimethoxyflavone). G. biloba extract (25-300 microg/mL) increased AhR-dependent reporter activity, as determined in MCF-10A cells transfected with an AhR-regulated luciferase reporter plasmid (pGudluc6.1). Bilobalide and ginkgolides A, B, C, and J were not responsible for the modulation of CYP1B1 and CYP1A1 gene expression or AhR activation by G. biloba extract. In contrast, quercetin increased CYP1B1 and CYP1A1 gene expression and activated AhR, whereas kaempferol and isorhamnetin suppressed constitutive CYP1B1 expression and antagonized AhR activation by benzo[a]pyrene. Overall, our findings provide an impetus for future investigations on the effect of G. biloba extract in CYP1-mediated chemical carcinogenesis.

Purification of CYP2B‐like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.

A novel gender-related difference in the constitutive expression of hepatic cytochrome P4501A subfamily enzymes in Meishan pigs

Constitutive expression levels of hepatic CYP1A subfamily enzymes, CYP1A1 and CYP1A2, in male and female Meishan pigs were examined at levels of the mRNA, protein, and enzyme activity. In mature (5-month-old) pigs, levels of hepatic CYP1A1 and CYP1A2 mRNAs, as determined by RT-PCR, were much higher in females than in males, but those of castrated male pigs were equivalent to female pigs. The gender-related differences in the levels of CYP1A mRNAs closely correlated with those of the corresponding apoproteins determined by Western blotting. Hepatic enzyme activities not only for the O-dealkylation of ethoxyresorufin and methoxyresorufin (typical substrates for CYP1A1 and CYP1A2, respectively) but also for the mutagenic activation of benzo[ a]pyrene and 2-amino-6-methyl-dipyrido[1,2- a; 3′,2′- d]imidazole (typical substrates for CYP1A1 and CYP1A2, respectively) were also much greater in female and castrated male pigs than in male pigs. In immature (1-month-old) pigs, no such gender-related differences were observed, and their gene expression levels of the CYP1A subfamily enzymes were almost the same as those of mature female pigs. Furthermore, treatment of immature pigs with testosterone resulted in a drastic decrease in the levels of the CYP1A1 and CYP1A2 mRNAs in both sexes. The present findings demonstrate a gender-related difference in the constitutive expression of hepatic CYP1A subfamily enzymes in Meishan pigs and further indicate that androgen down-regulates the constitutive gene expression of the enzymes.

Mitochondrial targeting of intact CYP2B1 and CYP2E1 and N‐terminal truncated CYP1A1 proteins in Saccharomyces cerevisiae − role of protein kinase A in the mitochondrial targeting of CYP2E1

Previously we showed that intact rat cytochrome P450 2E1, cytochrome P450 2B1 and truncated cytochrome P450 1A1 are targeted to mitochondria in rat tissues and COS cells. However, some reports suggest that truncated cytochrome P450 2E1 is targeted to mitochondria. In this study, we used a heterologous yeast system to ascertain the conservation of targeting mechanisms and the nature of mitochondria-targeted proteins. Mitochondrial integrity and purity were established using electron microscopy, and treatment with digitonin and protease. Full-length cytochrome P450 2E1 and cytochrome P450 2B1 were targeted both to microsomes and mitochondria, whereas truncated cytochrome P450 1A1 (+ 5 and + 33/cytochrome P450 1A1) were targeted to mitochondria. Inability to target intact cytochrome P450 1A1 was probably due to lack of cytosolic endoprotease activity in yeast cells. Mitochondrial targeting of cytochrome P450 2E1 was severely impaired in protein kinase A-deficient cells. Similarly, a phosphorylation site mutant cytochrome P450 2E1 (Ser129A) was poorly targeted to the mitochondria, thus confirming the importance of protein kinase A-mediated protein phosphorylation in mitochondrial targeting. Mitochondria-targeted proteins were localized in the matrix compartment peripherally associated with the inner membrane and their ethoxyresorufin O-dealkylation, erythromycin N-demethylase, benzoxyresorufin O-dealkylation and nitrosodimethylamine N-demethylase activities were fully supported by yeast mitochondrial ferredoxin and ferredoxin reductase.

Induction of CYP1A1 and CYP2E1 in rat liver by histamine: binding and kinetic studies

Histamine (HA) may bind to cytochrome P450 (CYP450) in rat liver microsomes. The CYP450-HA complex seems to regulate some cellular processes such as proliferation. In the present work, it is shown that HA increases the activity and protein level of CYP1A1 and CYP2E1, in vivo. CYP1A1 is associated with polycyclic aromatic hydrocarbon-mediated carcinogenesis and CYP2E1 with liver damage by oxidative stress. Studies of enzyme kinetics and binding with rat liver microsomes and supersomes were carried out to determine whether HA is a substrate of CYP1A1 and/or CYP2E1. The lack of NADPH oxidation in the presence of HA showed that it is not a substrate for CYP1A1. Activity measurements using the O-dealkylation of ethoxyresorufin indicated that HA is a mixed-type inhibitor of CYP1A1 in both microsomes and supersomes. On the other hand, HA induced a significant NADPH oxidation catalyzed by CYP2E1 supersomes, strongly suggesting that HA is a substrate for this isoform. Furthermore, HA is consumed in the presence of CYP2E1-induced microsomes and supersomes, as determined by o-phtalaldehyde complexes with HA by HPLC. The present findings may contribute to understand better the physiological function of CYP450 in relation with inflammation and other physiological processes in which HA may have a relevant role.