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.     

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.     

Further immunochemical and biocatalytic characterization of CYP1A1 from feral leaping mullet liver (Liza saliens) microsomes

CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7-methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7-methoxyresorufin O-demethylase (MROD) activity were studied. 7-ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by alpha-naphthoflavone (ANF). At 0.5 and 2.5 microM. ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1.     

Roles of NADPH-P450 reductase in the O-deethylation of 7-ethoxycoumarin by recombinant human cytochrome P450 1B1 variants in Escherichia coli

Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes.     

Differential inhibition of hepatic microsomal alkoxyresorufin O-dealkylation activities by tetrachlorobiphenyls

Polychlorinated biphenyls (PCBs) elicit a spectrum of biochemical and toxic effects in exposed animals. In the present study, we assessed the effect of PCB structure, using four symmetrically-substituted PCBs, on cytochrome P450 (CYP)-mediated methoxy-, ethoxy- and benzyloxyresorufin O-dealkylase (MROD, EROD and BROD, respectively) activities. We found that 2,2',4,4'-tetrachlorobiphenyl (PCB 47), 2,2',5,5'-tetrachlorobiphenyl (PCB 52), 2,2',6,6'-tetrachlorobiphenyl (PCB 54) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) inhibited alkoxyresorufin O-dealkylase activities in hepatic microsomes from 3-methylcholanthrene (MC) or phenobarbital (PB)-treated rats. Measurement of the in vitro inhibitory potencies of the tetrachlorobiphenyls revealed that MROD, EROD and BROD activities were differentially inhibited and the degree of inhibition was determined by the chlorination pattern of the PCB. PCB 77 was more potent than PCB 47 or PCB 52 at inhibiting MROD and EROD activities in hepatic microsomes from MC-treated rats, while no inhibition of either activity was observed with PCB 54. In contrast, BROD activity measured in hepatic microsomes from PB-treated rats was inhibited by PCB 47, PCB 52 and PCB 54 but not by PCB 77. The mode of inhibition for each activity was also evaluated statistically. Inhibition of the alkoxyresorufin O-dealkylase activities could not be discerned in hepatic microsomes from corn oil-treated rats because the activities were inherently too low. No evidence for mechanism-based inhibition of MROD, EROD or BROD activities or an effect via CYP reductase was found. The results demonstrate that relatively coplanar PCBs such as PCB 77 preferentially inhibit EROD and MROD activities, whereas noncoplanar PCBs such as PCB 54 preferentially inhibit BROD activity.     

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.     

Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5.

Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10-CYC1 promoter and the phosphoglycerate kinase gene terminator [Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194, 889-896]. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72, 463-472] increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25-iron-metabolite complexes. 9-fold, 8-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6 beta-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25-iron-metabolite complex. A P450 Fe(II)-(RNO) complex was obtained upon oxidation of N-hydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effect of copper (II) on the cytochrome P450 enzymes and NADPH-cytochrome P450 reductase: inhibition of cytochrome P450-catalyzed reactions by copper (II) ion

Inhibitory effects of Cu(2+) on the cytochrome P450 (P450)-catalyzed reactions of liver microsomes and reconstituted systems containing purified P450 and NADPH-P450 reductase (NPR) were seen. However, Zn(2+), Mg(2+), Mn(2+), Ca(2+), and Co(2+) had no apparent effects on the activities of microsomal P450s. Cu(2+) inhibited the reactions catalyzed by purified P450s 1A2 and 3A4 with IC(50) values of 5.7 and 8.4 microM, respectively. Cu(2+) also inhibited reduction of cytochrome c by NPR (IC(50) value of 5.8 microM). Copper caused a decrease in semiquinone levels of NPR, although it did not disturb the rate of formation of semiquinone. P450 reactions supported by an oxygen surrogate, tert-butyl hydroperoxide, instead of NPR and NADPH, were inhibited by the presence of Cu(2+). The results indicate that Cu(2+) inhibits the P450-catalyzed reactions by affecting both P450s and NPR. It was also found that the inhibition of catalytic activities of P450s by Cu(2+) involves overall conformational changes of P450s and NPR, investigated by CD and intrinsic fluorescence spectroscopy. These results suggest that the inhibitory effect of Cu(2+) on the P450-catalyzed reactions may come from the inability of an efficient electron transfer from NPR to P450 and also the dysfunctional conformation of NPR and P450.     

Furocoumarins from grapefruit juice and their effect on human CYP 3A4 and CYP 1B1 isoenzymes

Bioactive compounds present in grapefruit juice are known to increase the bioavailability of certain medications by acting as potent CYP 3A4 inhibitors. An efficient technique has been developed for isolation and purification of three furocoumarins. The isolated compounds have been tested for the inhibition of human CYP 1B1 isoform using specific substrates. Grapefruit juice was extracted with ethyl acetate (EtOAc) and the dried extract was loaded onto silica gel column chromatography. Further, column fractions were subjected to preparative HPLC to obtain three compounds. The purity of these compounds was analyzed by HPLC and structures were determined by NMR studies. The identified compounds, bergamottin, 6',7'-dihydroxybergamottin (DHB), and paradisin-A, were tested for their inhibitory effects on hydroxylase and O-dealkylase activities of human cytochrome P450 isoenzymes CYP 3A4 and CYP 1B1. Paradisin-A was found to be a potent CYP 3A4 inhibitor with an IC50 of 1.2 microM followed by DHB and bergamottin. All three compounds showed a substantial inhibitory effect on CYP 3A4 below 10 microM. Inhibitory effects on CYP 1B1 exhibited a greater variation due to the specificity of substrates. Paradisin A showed an IC50 of 3.56+/-0.12 microM for the ethoxy resorufin O-dealkylase (EROD) activity and 33.56+/-0.72 microM for the benzyloxy resorufin (BROD). DHB and bergamottin showed considerable variations for EROD and BROD activities with an IC50 of 7.17 microM and 13.86 microM, respectively.     

Electrostatic interaction between cytochrome P450 and NADPH-P450 reductase: comparison of mixed and fused systems consisting of rat cytochrome P450 1A1 and yeast NADPH-P450 reductase

The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of Vmax were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains.     

Modulation of porcine cytochrome P450 enzyme activities by surgical castration and immunocastration

The present study aimed to evaluate some cytochrome P450 metabolic enzyme activities in hepatic microsomes prepared from entire male pigs (uncastrated pigs), surgically castrated pigs and pigs immunized against gonadotropin-releasing hormone (immunocastrated pigs). The activities of the following enzymes were measured: ethoxyresorufin O-deethylase (EROD, CYP1A1/1A2), methoxyresorufin O-deethylase (MROD, CYP1A2), pentoxyresorufin O-depentylase (PROD, CYP2B), coumarin hydroxylase (COH, CYP2A) and p-nitrophenol hydroxylase (PNPH, CYP2A/2E1). The total cytochrome P450 contents were not affected by either surgical or immunocastration. Hepatic microsomal activities for EROD, PROD, COH and PNPH were lower in entire male pigs compared with surgically castrated and immunocastrated pigs (P < 0.05). Surgically and immunocastrated male pigs were similar with respect to EROD, MROD, PROD and COH activities (P > 0.05), whereas surgically castrated pigs exhibited lower PNPH activity compared with immunocastrated pigs (P = 0.029). The effect of different concentrations of testicular steroids - testosterone, 17β-estradiol, free estrone and androstenone - on enzyme activities was evaluated by in vitro microsomal study. Testosterone at the concentration of 8 pmol/ml inhibited EROD activities and estradiol-17β at the concentration of 1.8 pmol/ml inhibited PROD activities in hepatic microsomes from surgically castrated pigs. The highest concentration of androstenone (7520 pmol/ml) inhibited COH activities, whereas a 42-fold lower concentration of androstenone (180 pmol/ml) stimulated COH activities in surgically castrated pigs. Both free estrone (3.5 pmol/ml) and androstenone (55 pmol/ml) inhibited EROD activities in microsomes from entire male pigs. Stimulation of COH activities by the highest dose of free estrone (18 pmol/ml) was recorded in microsomes from entire male pigs. However, these effects of steroids were not concentration-dependent and the maximum extent did not exceed ±15% variation compared with the controls. There was no inhibition of PNPH activities in the hepatic microsomes from either entire or castrated pigs. In conclusion, we showed that EROD, PROD, COH and PNPH activities were lower in entire male pigs compared with those in surgically and immunocastrated pigs. Direct inhibition by the testicular steroids - testosterone, 17β-estradiol, free estrone and androstenone - was not the primary cause of the reduced enzyme activities.     

Inhibitory effects of detergents on rat CYP1A1-dependent monooxygenase: comparison of mixed and fused systems consisting of rat CYP 1A1 and yeast NADPH-P450 reductase

Inhibitory effects of detergents Triton X-100 and Chaps on 7-ethoxycoumarin O-deethylation activity were examined in the recombinant microsomes containing both rat CYP1A1 and yeast NADPH-P450 reductase (the mixed system) and their fused enzyme (the fused system). Triton X-100 showed competitive inhibition in both mixed and fused systems with K(i) values of 24.6 and 21.5 microM, respectively. These results strongly suggest that Triton X-100 binds to the substrate-binding pocket of CYP1A1. These K(i) values are far below the critical micelle concentration of Triton X-100 (240 microM). Western blot analysis revealed no disruption of the microsomal membrane by Triton X-100 in the presence of 0-77 microM Triton X-100. On the other hand, Chaps gave distinct inhibitory effects to the mixed and fused systems. In the fused system, a mixed-type inhibition was observed with K(i) and K(i)' values of 1.2 and 5.4 mM of Chaps, respectively. However, in the mixed system, multiple inhibition modes by Chaps were observed. Western blot analysis revealed that the solubilized fused enzyme by Chaps preserved the activity whereas the solubilized CYP1A1 and NADPH-P450 reductase reductase showed no activity in the mixed system. Thus, the comparison of the mixed and fused systems appears quite useful to elucidate inhibition mechanism of detergents.     

Two cytochrome P-450 isoforms catalysing O-de-ethylation of ethoxycoumarin and ethoxyresorufin in higher plants.

The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.4 microM for 7-ethoxyresorufin were determined. O-De-ethylase activity was also detected in microsomes prepared from several other plant species, including wheat, maize, tulip, avocado and Vicia. ECOD and EROD were low or undetectable in uninduced plant tissues, and both activities were stimulated by wounding or by chemical inducers. Two distinct cytochrome P-450 isoforms are involved in ECOD and EROD activities since (1) they showed different distributions among plant species; (2) they showed contrasting inhibition and induction patterns; and (3) ECOD but not EROD activity was supported by cumene hydroperoxide.     

Resveratrol, a red wine constituent, is a mechanism-based inactivator of cytochrome P450 3A4

Resveratrol, a phytoalexin found in red wine, has been shown to possess antioxidant and antimutagenic properties. Incubation of resveratrol with Sf9 insect microsomes containing baculovirus-derived human cytochrome P450 3A4 (CYP3A4) and NADPH-cytochrome P450 reductase showed that resveratrol inactivated CYP3A4 in a time- and NADPH-dependent manner. Resveratrol, erythromycin and troleandomycin inactivated CYP3A4 at a similar rate (as reflected by k(inact)) whereas the binding affinity to CYP3A4 (as reflected by K(I)) was in the order of: troleandomycin > erythromycin > resveratrol. (K(I) and k(inact) for CYP3A4 inactivation by resveratrol, erythromycin and troleandomycin are 20 microM and 0.20 min(-1), 5.3 microM and 0.12 min(-1) and 0.18 microM and 0.15 min(-1), respectively.) Fractionation studies of red wine showed that fractions that did not contain resveratrol inactivated CYP3A4 significantly. In addition, the resveratrol content in red wine used in the study was too low to account for the degree of CYP3A4 inactivation observed after red wine treatment. Inactivation studies using a variety of red wine types showed that the CYP3A4 inactivation did not correlate to their resveratrol content. In summary, data here showed that resveratrol is an effective mechanism-based inactivator of CYP3A4; however, it is not one of the main red wine constituents that are responsible for CYP3A4 inactivation by red wine. Nevertheless, inactivation of CYP3A4 by resveratrol may cause clinically relevant drug interactions with CYP3A4 substrates.     

Xenobiotic-metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 > 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.     

Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes

The involvement of cytochrome b₅ in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b₅. Anti-b₅ antiserum inhibited the reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethy-lase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b₅ is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b₅. The results suggest that cytochrome b₅ involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved. © 1994 Wiley-Liss, Inc.     

Influence of lipophilicity on the interactions of hydroxy stilbenes with cytochrome P450 3A4

Resveratrol, a polyphenol found in red wine, was recently suggested to act as an irreversible, mechanism-based inactivator of cytochrome P450 3A4 (CYP3A4). We found a significant inhibition of human CYP3A4-dependent transformation of cyclosporine by resveratrol, with IC50 = 4.5 microM. We studied the kinetics parameters of CYP3A4 transformation of resveratrol and structurally related, naturally occurring stilbenes. Resveratrol, piceid, resveratroloside, 5,4'-dihydroxy-3-O-methoxystilbene, and 5,3-dihydroxy-4'-O-methoxystilbene were all shown to inhibit hydroxylation of testosterone by CYP3A4. Both methoxy-stilbenes had lower IC50 values, ranging from 0.43 to 0.47 microM, suggesting that lipophilicity rather than number or positions of free hydroxyls (3,5 or 5,4') determines the CYP3A4 inhibition capacity of polyphenols. In line with these findings, both glucosyl-stilbenes were found to be weak inhibitors of CYP3A4. The affinity of the enzyme towards methoxy-stilbenes, expressed as apparent Km, was indeed higher than those for the parent resveratrol and its glucosides, in CYP3A4 reaction mixtures. Vmax values were similar, except for piceid. These results support the role of lipophilicity in the interaction of polyphenols with CYP3A4. It is suggested that selective structural modifications of substrates add significantly to knowledge acquired through molecular modifications of the enzyme.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Expression, Purification, and Characterization of a Catalytically Active Human Cytochrome P450 1A2:Rat NADPH-Cytochrome P450 Reductase Fusion Protein

An enzymatically active human cytochrome P450 (P450) 1A2:rat NADPH-P450 reductase fusion protein was purified and partially characterized following heterologous expression inEscherichia coli. A cDNA was engineered to include the coding sequence for human P450 1A2 at its 5′ end (up to but not including the stop codon) fused in-frame to the coding sequence for a truncated (soluble) rat NADPH-P450 reductase at its 3′ end via an oligonucleotide sequence encoding the hydrophilic dipeptide Ser–Thr. This fusion plasmid was expressed inE. coliand the recombinant protein was purified from the detergent-solubilized membrane fraction via sequential DEAE, ADP–agarose, and hydroxylapatite chromatographies. The purified protein has the spectral characteristics of human P450 1A2 and cytochromecreduction activity comparable to rabbit NADPH-P450 reductase. The fusion protein catalyzed 7-ethoxyresorufinO-deethylation and phenacetinO-deethylation to appreciable levels in the presence of NADPH and phospholipid. While these activities were comparable to those of other such P450:NADPH-P450 reductase fusion proteins, they were lower than those of the system reconstituted from its individual hemoprotein and flavoprotein components. Nevertheless, the production of a functional, catalytically self-sufficient monooxygenase inE. colienhances the prospect of using bacterial systems for production and characterization of human P450 drug metabolites as well as for biodegradation of chemicals in the environment.     

Triphenyltin and Tributyltin inhibit pig testicular 17beta-hydroxysteroid dehydrogenase activity and suppress testicular testosterone biosynthesis

We previously reported that tributyltin chloride (TBT) and triphenyltin chloride (TPT) powerfully suppressed human chorionic gonadotropin- and 8-bromo-cAMP-stimulated testosterone production in pig Leydig cells at concentrations that were not cytotoxic [Nakajima Y, Sato Q, Ohno S, Nakajin S. Organotin compounds suppress testosterone production in Leydig cells from neonatal pig testes. J Health Sci 2003;49:514-9]. This study investigated the effects of these organotin compounds on the activity of enzymes involved in testosterone biosynthesis in pig testis. At relatively low concentrations of TPT, 17beta-hydroxysteroid dehydrogenase (17beta-HSD; IC(50)=2.6microM) and cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (IC(50)=117microM) activities were inhibited, whereas cholesterol side-chain cleavage cytochrome P450 and 3beta-HSD/Delta(4)-Delta(5) isomerase activities were less sensitive. Overall, TPT was more effective than TBT. TPT also inhibited both ferredoxin reductase and P450 reductase activities at concentrations over 30microM; however, TBT had no effect, even at 100microM. The IC(50) values of TPT were estimated to be 25.7 and 22.8microM for ferredoxin reductase and P450 reductase, respectively. The inhibitory effect of TPT (30microM) on microsomal 17beta-HSD activity from pig testis was eliminated by pretreatment with the reducing agents dithiothreitol (1mM) and dithioerythritol (1mM). On the other hand, TPT (0.03microM) or TBT (0.1microM) exposure suppressed the testosterone production from androstenedione in pig Leydig cells indicating that these organotins inhibit 17beta-HSD activity in vivo as well as in vitro, and the IC(50) values of TPT and TBT for 17beta-HSD activity were estimated to be 48 and 114nM, respectively. Based on these results, it appears possible that the effects of TBT and TPT are largely due to direct inhibition of 17beta-HSD activity in vivo.