Inhibition of 17ß-estradiol activation by CYP1A1: genotype- and regioselective inhibition by St. John's Wort and several natural polyphenols.

Several epidemiological studies associate certain CYP1A1 genotypes, alone or in combination, with an increased risk of estrogen-related cancers. Previously we demonstrated that metabolic activation of estrogens by CYP1A1 is a genotype-dependent reaction with the CYP1A1.2 (Ile462Val) variant being the most efficient catalyst (Kisselev et al.). To answer the question whether genotype-dependent inhibition of activation of estrogens by CYP1A1 could also contribute, we studied the inhibition of hydroxylation activity of the most common allelic variants of human CYP1A1 towards 17ß-estradiol. We expressed and purified CYP1A1.1 (wild-type), CYP1A1.2 (Ile462Val), and CYP1A1.4 (Thr461Asn) and performed inhibition assays by natural polyphenols of our diet and drugs of NADPH-dependent estradiol hydroxylation in reconstituted CYP1A1 systems. From the polyphenols studied, a St. John's Wort (Hypericum perforatum) extract, some of its main single constituents hypericin, pseudohypericin, and quercetin, as well as the flavonols kaempferol, myricetin and the phytoestrogens resveratrol and tetramethyl-stilbene exhibited strong inhibition. For the St. John's Wort extract and its single constituents hypericin, pseudohypericin, and quercetin, inhibition exhibited a remarkable dependency on the CYP1A1 genotype. Whereas (wild-type) CYP1A1.1 was most inhibited by the whole crude extract, the variant CYP1A1.2 (Ile462Val) was significantly stronger inhibited by the constituents in its pure form: IC50 values for 2-hydroxylation was more than two times lower compared with the wild-type enzyme and the variant CYP1A1.4 (Thr461Asn). Besides this, the inhibition exhibited a remarkable regioselectivity. The data suggest that risk of estrogen-mediated diseases might be not only influenced by CYP1A1 genotype-dependent activation but also its inhibition by natural polyphenols of our diet and drugs.

Recalling P446. P4501A1 (CYP1A1) opting for clinical application.

This review goes back to spectral studies [see Hildebrandt et al., 1968]. The findings of apparent absolute spectra of two interconvertible forms of microsomal mixed function oxidases are looked back on to recall whether their impact sustained scrutiny or are rather remembered as of sentimental value only. The second part summarizes studies on the clinical relevance of CYP1A1 with special reference to our investigations. The impact of genetic variability of CYP1A1 on cancer susceptibility, differential effects of polyphenols and hyperforin on toxification and detoxification pathways of benzo[a]pyrene, and differential metabolite patterns of 17 beta-estradiol, estrone, and eicosapentaenoic acid are presented.

Human CYP1A1 variants lead to differential eicosapentaenoic acid metabolite patterns.

To answer the question whether the most common allelic variants of human CYP1A1, namely CYP1A1.1 (wild type), CYP1A1.2 (Ile462Val), and CYP1A1.4 (Thr461Asn), differ in their catalytic activity towards eicosapentaenoic acid (EPA), in vitro enzymatic assays were performed in reconstituted CYP1A1 systems. All CYP1A1 variants catalyzed EPA epoxygenation and hydroxylation to 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EETeTr) and 19-OH-EPA, yet with varying catalytic efficiency and distinct regiospecificity. CYP1A1.1 and CYP1A1.4 formed 17(R),18(S)-EETeTr as main product (K(m)=53 and 50 microM; V(max)=0.60 and 0.50 pmol/min/pmol; V(max)/K(m)=0.11 and 0.10 microM(-1)min(-1), respectively), followed by 19-OH-EPA (K(m)=76 and 93 microM; V(max)=0.37 and 0.37 pmol/min/pmol; V(max)/K(m)=0.005 and 0.004 microM(-1)min(-1), respectively). The variant CYP1A1.2 produced almost equal amounts of both metabolites, but its catalytic efficiency for hydroxylation was five times higher (K(m)=66 microM; V(max)=1.7 pmol/min/pmol; V(max)/K(m)=0.026 microM(-1)min(-1)) and that for epoxygenation was twice higher (K(m)=66 microM; V(max)=1.5 pmol/min/pmol; V(max)/K(m)=0.023 microM(-1)min(-1)) than those of the wild-type enzyme. Thus, the Ile462Val polymorphism in human CYP1A1 affects EPA metabolism and may contribute to interindividual variance in the local production of physiologically active fatty acid metabolites in the cardiovascular system and other extrahepatic tissues, where CYP1A1 is expressed or induced by polycyclic aromatic hydrocarbons and other xenobiotics.

Association of CYP1A1 polymorphisms with differential metabolic activation of 17beta-estradiol and estrone.

Several epidemiologic studies associate certain CYP1A1 genotypes, alone or in combination, with an increased risk of estrogen-related cancers. To answer the question of whether genotype-dependent activation of estrogens by CYP1A1 could be the underlying mechanism, we studied the hydroxylation activity of the most common allelic variants of human CYP1A1 towards both endogenously occurring estrogens, 17beta-estradiol (E2) and estrone (E1). We expressed and purified CYP1A1.1 (wild-type), CYP1A1.2 (Ile(462)Val), and CYP1A1.4 (Thr(461)Asn) and did enzymatic assays of NADPH-dependent estrogen hydroxylation in reconstituted CYP1A1 systems. All CYP1A1 variants catalyzed the formation of 2-, 4-, 6alpha-, and 15alpha-hydroxylated estrogen metabolites from E2 and E1, yet with varying catalytic efficiency and distinct regiospecificity. Whereas the variant CYP1A1.2 (Ile(462)Val) had a significant higher catalytic activity for all hydroxylation sites and both substrates, it was most pronounced for 2-hydroxylation. Catalytic efficiencies for the formation of the major metabolites, 2-OH-E2 and 2-OH-E1, by CYP1A1.2 were 5.7- and 12-fold higher, respectively, compared with the wild-type enzyme. The catalytic efficiencies for hydroxylations catalyzed by CYP1A1.4 were roughly comparable with those of the wild-type enzyme. Enzyme kinetics showed that the superior activity of CYP1A1.2 (Ile(462)Val) is mainly caused by a higher V(max), whereas K(m) values of all variants were similar. The data suggest that risk of estrogen-induced cancers and cardiovascular diseases might be-at least partially-determined by the CYP1A1 genotype.

CYP1A1 genotype-selective inhibition of benzo[a]pyrene activation by quercetin.

Epidemiological studies suggest that food rich in quercetin and naringin may protect against certain types of lung cancer, and that genotype dependent inhibition of cytochrome P450 1A1 (CYP1A1)-mediated bioactivation of procarcinogens could be the underlying mechanism. We studied the inhibitory effects of quercetin and naringin on the terminal bioactivation step of benzo[a]pyrene (B[a]P), a member of the major class of lung carcinogens. This reaction (epoxidation of (+/-)-trans-7,8-dihydro-7,8-dihydroxy-B[a]P to the ultimate carcinogenic product, (+/-)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (diolepoxide 2)) was examined using three of the most common allelic variants of human CYP1A1, namely wild-type CYP1A1.1, CYP1A1.2, and CYP1A1.4. Quercetin potently inhibited diolepoxide 2 formation by all CYP1A1 types with IC(50) values between 1.6 and 7.0 microM. The differences between the wild-type enzyme and the variants were statistically highly significant (P < 0.01). Enzyme kinetics revealed quercetin as a mixed-type inhibitor of CYP1A1.1, CYP1A1.2, and CYP1A1.4 with K(i) values of 2.0, 6.4, and 9.3 microM, respectively. Naringin inhibited diolepoxide 2 formation only slightly. Our data support the hypothesis that quercetin may have a stronger chemopreventive effect in individuals carrying wild-type compared with variant CYP1A1 genes. Future studies should consider the influence of P450 polymorphisms on both procarcinogen activation and its inhibition to facilitate the development of genotype-specific chemoprevention regimes.

Arachidonic and eicosapentaenoic acid metabolism by human CYP1A1: highly stereoselective formation of 17(R),18(S)-epoxyeicosatetraenoic acid.

Human cytochrome P450 1A1 (CYP1A1) and human NADPH-cytochrome P450 reductase were expressed and purified from Spodoptera frugiperda insect cells. A reconstituted enzymatically active system metabolized polyunsaturated fatty acids such as arachidonic (AA) and eicosapentaenoic acid (EPA). CYP1A1 was an AA hydroxylase which oxidizes this substrate at a rate of 650+/-10 pmol/min/nmol CYP1A1, with over 90% of metabolites accounted for by hydroxylation products and with 19-OH-AA as major product. Epoxyeicosatrienoic acid (EET), mainly 14,15-EET, accounted for about 7% of total metabolites. Unlike rat CYP1A1, the human enzyme exhibited no 20-OH-AA as product. In contrast, with EPA as substrate CYP1A1 was mainly an epoxygenase, oxidizing with over 68% of total metabolites EPA to 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EETeTr). 19-OH-EPA accounted for about 31% of total metabolites. Significantly, the 17,18-olefinic bond of EPA was epoxidized to 17(R),18(S)-EETeTr with nearly absolute regio- and stereoselectivity. Molecular modeling analyses provided rationale for high efficiency of AA hydroxylation at C(19) and its gradual decrease down to C(14), as well as for the limited EPA 17(S),18(R) epoxidation due to unfavorable enzyme-substrate interactions. The absence of omega-hydroxylation for both substrates is not due to steric factors, but probably a consequence of different reactivities of omega and (omega-1) carbons for hydrogen abstraction. It is suggested that the capacity of human CYP1A1 to metabolize AA and EPA and its inducibility by polycyclic aromatic hydrocarbons may affect the production of physiologically active metabolites, in particular, in the cardiovascular system and other extrahepatic tissues including lung.

St. John's wort extracts and some of their constituents potently inhibit ultimate carcinogen formation from benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1.

Commercially available St. John's wort (Hypericum perforatum) preparations and some of their main constituents (hypericin, pseudohypericin, hyperforin, rutin, and quercetin) were examined for their potential to inhibit carcinogen activation by human cytochrome P450 1A1 (CYP1A1). We used a reconstituted system consisting of purified human CYP1A1, purified human NADPH-cytochrome P450 reductase, and dilaurylphosphatidylcholine as lipid component. St. John's wort extracts potently inhibited CYP1A1-catalyzed (+/-)-trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (7,8-diol-B[a]P) epoxidation, the terminal reaction leading to the ultimate carcinogenic product (+/-)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (diolepoxide 2). All constituents, except rutin, were shown to possess strong inhibitory potencies toward diolepoxide 2 formation from 7,8-diol-B[a]P, with IC(50) values of 0.5 microM (hypericin), 1.2 microM (hyperforin), 1.5 microM (quercetin), and 8 microM (pseudohypericin), respectively. Preincubation experiments revealed that their action was not mechanism based. Inhibition kinetics studies showed the anthrodianthrone compound hypericin to be a noncompetitive inhibitor, with a K(i) value of 0.6 microM, and the phloroglucinol hyperforin to be a competitive inhibitor, with a K(i) value of 1.1 microM. When the effects on NADPH-P450 reductase activity were investigated, all constituents of St. John's wort studied turned out to be rather ineffective inhibitors; quercetin was the only exception, with an IC(50) value of approximately 20 microM. These in vitro data indicate that St. John's wort extracts and some of their constituents potently inhibit the major human procarcinogen-activating enzyme CYP1A1.

In vitro assessment of inhibition by natural polyphenols of metabolic activation of procarcinogens by human CYP1A1.

Previously, inhibitors of CYP1A1 were rated as candidate chemopreventive agents against cancer mainly according to their effects on the 7-ethoxyresorufin O-deethylation (EROD) of diagnostic probe substrates. Surprisingly, several polyphenols including resveratrol, formerly identified as potent inhibitors by the EROD assay, exhibited no or weak inhibition of procarcinogen activation. We compared the effects of 11 representative natural polyphenols, which normally occur in food, on different activities of CYP1A1, namely epoxidation of 7,8-dihydrodiol-benzo[a]pyrene, the terminal step in the activation leading to the ultimate carcinogenic diolepoxides, hydroxylation of benzo[a]pyrene, and EROD. For the first time, a reconstituted system was used for the determination of IC(50) values, consisting of purified enzymes (human CYP1A1 and human NADPH-cytochrome P450 reductase) and dilaurylphosphatidylcholine. The results demonstrate that the inhibitory effects of dietary polyphenols on CYP1A1 activity depend on both the structure of the inhibitor and the type of the reaction and substrate used in the assay. Consequently, a potent EROD inhibition alone is insufficient to count a substance among the chemoprotective agents.

Human CYP1A1 allelic variants: baculovirus expression and purification, hydrodynamic, spectral, and catalytical properties and their potency in the formation of all-trans-retinoic acid.

Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CYP1A1.4 (T461N), were expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30-90 nmol (1.8-5.4 mg) spectrally active cytochrome P450 per one liter of culture and purified to electrophoretic homogeneity by Ni-agarose chromatography. The recombinant variants were structurally characterized by UV/Vis, ultracentrifugation, and EPR. Optical and EPR spectra showed all three variants predominantly in high spin state; moreover, EPR indicated changes in the electronic structure of the heme iron of the two mutant variants. Sedimentation equilibrium experiments demonstrated the purified variants in dimeric state in the presence of 0.2% emulgen+0.05% cholate. Higher detergent concentration, the presence of imidazole, and cleavage of the His-tag led to monomerization. Catalytic activity of all purified variants was reconstituted with purified human NADPH-P450 reductase and dilaurylphosphatidylcholine. Enzyme kinetics of ethoxyresorufin O-deethylation revealed similar K(m) ( approximately 0.4 microM) for all variants but slightly different V(max) values (CYP1A1.1: 4.2, CYP1A1.2: 7.0, and CYP1A1.4: 3.0 nmol/min/nmol CYP1A1). The extended C-terminus influenced the enzymatic activity only slightly. All three variants are able to produce significant amounts of all-trans-retinoic acid from all-trans-retinal with V(max) of 4.0, 3.3, and 5.6 nmol/min/nmol CYP1A1 and K(m) values of 111, 83, and 250 microM for CYP1A1.1, CYP1A1.2, and CYP1A1.4, respectively. Availability of the three purified human CYP1A1 variants should facilitate further characterization of their role in metabolism of endogenous and exogenous compounds as well as structural studies.

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane.

Human cytochrome P4501A1 (CYP1A1) is one of the key enzymes in the bioactivation of environmental pollutants such as benzo[a]pyrene (B[a]P) and other polycyclic aromatic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 purified insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. Conversion rates of up to 36 pmol x min(-1) x pmol(-1) CYP1A1 of the enantiomeric promutagens (-)- and (+)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P (7,8-diol) to the genotoxic diolepoxides were achieved. The highest rates were obtained when negatively charged lipids such as phosphatidylserine and phosphatidylinositol and/or nonbilayer phospholipids such as phosphatidylethanolamine were present in the membrane together with neutral lipids. Both Vmax and Km values were changed. This suggests a rather complex mechanism of stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase. Furthermore, the ratio of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE2) to r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE1) formed from (-)-7,8-diol was significantly increased by the introduction of anionic lipids, but not by that of nonbilayer lipids. Thus, charged lipids affect the stereoselectivity of the epoxidation by leading to the formation of a larger amount of the ultimate mutagen DE2 than of DE1, which is far less carcinogenic. These data suggest that membrane properties such as negative charge and nonbilayer phase propensity are important for the efficiency and selectivity of enzymatic function of human CYP1A1.