Human Cytochrome P450 Activities Research Articles

Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat, rabbit, dog, micropig, monkey and man

1. In the present study, nine cytochrome P450 enzyme activities in seven species were characterized to allow a practical means of comparing this important metabolic step between various test animals and man. 2. Enzyme activities and kinetic parameters were first determined towards marker substrates for human cytochrome P450 enzymes. Inhibition profiles were then determined with both antibodies directed against various cytochrome P450 enzymes and with chemical inhibitors. 3. Both the enzyme kinetic parameters/enzyme activities, and the inhibition profiles obtained for the animal species were compared with those obtained for human liver microsomes in order to postulate the animal species most similar to man with regard to each individual cytochrome P450 enzyme activity. 4. It was found that, as expected, none of the tested species was similar to man for all the measured P450 enzyme activities, but that in each species only some of the P450 enzyme activities could be considered as similar to man. 5. When it is known which human cytochrome P450 enzymes are involved in the metabolism of a compound, the comparative data presented here can be used for selecting the most suitable species for in vitro and in vivo experiments.

Isoform selective inhibition and inactivation of human cytochrome P450s by methylenedioxyphenyl compounds

1. A series of methylenedioxyphenyl compounds were evaluated for their inhibitory and inactivation effects on nine human cytochrome P450 (CYP) activities using microsomes from human B-lymphoblast cells expressing specific human CYP isoforms. 2. Methylenedioxyphenyl compounds which possess a bulky structure such as 1,4- benzothiazine showed substantial inhibition of S-warfarin 7-hydroxylation catalysed by CYP2C9, S-mephenytoin 4'-hydroxylation by CYP2C19, bufuralol 1-hydroxylation by CYP2D6, and testosterone 6β-hydroxylation by CYP3A4. Regarding ethoxyresorufin O-deethylation catalysed by CYP1A1 and benzyloxyresorufin O-dealkylation by CYP2B6, the subtle change of a substitution of the 1,4-benzothiazine structure affected the inhibition selectivity. Ethoxyresorufin O-deethylation by CYP1A2, coumarin 7-hydroxylation by CYP2A6, and chlorzoxazone 6-hydroxylation by CYP2E1 were not inhibited by almost any of the methylenedioxyphenyl compounds. The inhibitory effects of methylenedioxyphenyl compounds that possess a short chain amino group on the human CYP isoforms were not significant. 3. The methylenedioxyphenyl compounds inactivated CYP1A1 (kinact=0.034 min-1 and Ki=0.81 μM), CYP2C9 (kinact=0.041 and 0.042 min-1 and Ki=0.56 and 0.15 μM), CYP2D6 (kinact=0.044-0.339 min-1 and Ki=0.21-19.88 μM), and CYP3A4 (kinact=0.076-0.251 min-1 and Ki=0.25-0.69 μM). These results suggested that the methylenedioxyphenyl compounds investigated in this study would be potent mechanismbased inactivators of these human CYP isoforms. In contrast, CYP2B6 and CYP2C19 were not inactivated. 4. The present study suggested that the selectivity of inhibition or inactivation of human CYP isoforms by methylenedioxyphenyl compounds may vary according to the structure of the side chain.

Effects of propofol on human hepatic microsomal cytochrome P450 activities

1. The potential of propofol to inhibit the activity of major human cytochrome P450 enzymes has been examined in vitro using human liver microsomes. Propofol produced inhibition of CYP1A2 (phenacetin O -deethylation), CYP2C9 (tolbutamide 4-hydroxylation), CYP2D6 (dextromethorphan O -demethylation) and CYP3A4 (testosterone 6 beta hydroxylation) activities with IC = 40, 49, 213 and 32 mu M respectively. K for propofol against all of these enzymes with the exception of CYP2D6, where propofolishowed little inhibitory activity, was 30, 30 and 19 mu M respectively for CYPs 1A2, 2C9 and 3A4. 2. Furafylline, sulphaphenazole, quinidine and ketoconazole, known selective inhibitors of CYPs 1A2, 2C9, 2D6 and 3A4 respectively, were much more potent than propofol having IC = 0.8, 0.5, 0.2 and 0.1 mu M; furafylline and sulphaphenazole yielded K = 0.6 and 0.7 mu M respectively. i 3. The therapeutic blood concentration of propofol (20 mu M; 3-4 mu g ml) together with the in vitro K estimates for each of the major human P450 enzymes have been used to i estimate the extent of cytochrome P450 inhibition, which may be produced in vivo by propofol. This in vitro - in vivo extrapolation indicates that the degree of inhibition of CYP1A2, 2C9 and 3A4 activity which could theoretically be produced in vivo by propofol is relatively low (40-51%); this is considered unlikely to have any pronounced clinical significance. 4. Although propofol has now been used in 190 million people since its launch in 1986,thereare onlysinglereportsofpossible druginteractions between propofoland either alfentanil or warfarin. Consequently, it is difficult to conclude from either the published literature or the ZENECA safety database whether there is any evidence to indicate that propofol produces clinically significant drug interactions through inhibition of cytochrome P450-related drug metabolism.

S-31-5 - Implications of comorbidity for the treatment of depression

It is known that many genotoxic chemicals require oxidative metabolism to elicit genotoxicity. Induced rat liver S9 fraction has been employed as a ‘metabolite factory’ in in vitro genotoxicity testing. However, the relevance of the induced rat liver S9 fraction has been called into question due to the differences in the rat and human cytochrome P450 (CYP) activities. In the present study, we used a series of ten transformants expressing major human CYP isoforms such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 in HepG2 cells. To elucidate the usefulness and feasibility of these transformants, genotoxicity was tested without using rat S9. Among these transformants, benzo(a)pyrene-induced or cyclophosphamide-produced micronucleus (MN) frequency was markedly increased in transformants expressing CYP1A2 or CYP2C9, respectively. To explore the possibility that these transformants can be used for screening the possible genotoxicity of newly developed drugs, a chemical which is known to enhance genotoxicity in the presence of external metabolic activation system, okadaic acid (OA), was investigated. OA-induced MN frequency was significantly induced in transformants expressing CYP1A2 compared with the other CYP isoforms. The induced MN frequency was suppressed by treatment with a CYP1A2 specific inhibitor and CYP1A2 to siRNA. In control HepG2 cells harboring an empty vector, OA was treated with microsomes expressing CYP1A2 to induce MN. These results demonstrated that this screening system worked well and OA was found to be metabolically activated by CYP1A2 to induce MN. Based on the results obtained in the present study, this system of transformants is useful to elucidate the genotoxicity involving human CYP metabolism in the process of drug discovery.

Comparison of human fetal hepatic and adrenal cytochrome P450 activities with some major gestational steroids and ethylmorphine as substrates

The immunoidentified human fetal liver and adrenal microsomal contents of cytochromes P450IIIA and P450VIIA1 were compared to the metabolism of steroids and ethylmorphine. In fetal liver microsomes, 16α-hydroxylation of dehydroepiandrosterone (DHA) was catalyzed at a high rate in almost all investigated specimens and accompanied by a high ethylmorphine N-demethylase activity. Progesterone 16α- and 17α-hydroxylation was found only in the livers with the highest DHA 16α-hydroxylation activities, while 21-hydroxylation of progesterone was catalyzed only occasionally in these samples. In fetal adrenal microsomes, 21-hydroxylation of progesterone to 11-desoxycorticosterone (DOC) and 11-desoxycortisol (DOCOL) was catalyzed. In contrast to fetal liver, the adrenals also catalyzed the 17α-hydroxylation of pregnenolone and the formation of DHA from 17α-OH-pregnenolone. 16α-hydroxylation of DHA and ethylmorphine N-demethylation were modest in the adrenals. P450IIIA/HLp was immunoidentified in all investigated liver specimens except two ( 18 20 ) in which no ethylmorphine N-demethylation or 16α-hydroxylation of DHA was found. P450XVIIA1 bands were observed in 8 20 blots of liver specimens, but there was no correlation between the density of these bands and the 17α-hydroxylation of progesterone. All 11 fetal adrenal samples catalyzed DHA 16α-hydroxylation, although only 8 were positive for P450IIIA/HLp. All investigated adrenals were positive in regard of the P450XVIIA1 band, except one ( 8 9 ) with a low 17α-hydroxylation of progesterone. All adrenal specimens catalyzed 21-hydroxylation of progesterone and contained P450C21 bands in immunoblots and all samples catalyzed the formation of DOC and DOCOL from progesterone. Our findings in the fetal livers show a correlation between the DHA 16α-hydroxylation and immunoidentified P450IIIA/HLp bands. In adrenals, there was a correlation between the immunoidentified P450XVIIIA1 bands and the 17α-hydroxylation of progesterone.

Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast

A 1.57kb BamH1 fragment containing a full-length human debrisoquine 4-hydroxylase cytochrome P450 ( CYP2D6) cDNA was inserted into the BglII site of the yeast expression plasmid pMA91 and the resulting recombinant plasmid, pELT1, introduced into Saccharomyces cerevisiae strain AH22. Microsomes prepared from AH22/pELT1 cells gave an absorption maximum at 448 nm and a P450 content of 67 ± 31 pmol/mg of microsomal protein. No P450 was detectable in microsomes prepared from AH22/pMA91 control cells. A western blot of microsomes prepared from yeast transformed with pELT1 were probed with a monoclonal antibody to CYP2D6 and revealed a strong band with a molecular mass consistent with that of CYP2D6 from human liver microsomes. No corresponding band was observed with microsomes from control yeast transformed with pMA91 alone. Microsomes from AH22/pELT cells showed catalytic activity towards metoprolol (α-hydroxylation and O-demethylation, 0.17 and 0.78 nmol/mg protein/h, respectively); and towards sparteine (2-and 5-dehydrogenation, 1.82 and 0.59 nmol/mg protein/h, respectively). The inhibition of metoprolol metabolism by quinidine (Qd) was 200 times more potent than that of quinine (Qn), both for α-hydroxylation (Qd IC 50) = 0.05 μM; Qn IC 50 = 4 μM) and O-demethylation (Qd IC 50 = 0.05 μM; Qn IC 50 = 4 μM). Negligible metabolism of tolbutamide and S-mephenytoin, substrates of the 2C sub-family, and of p-nitrophenol, a substrate of CYP2E1, was detected, although a trace of the N-deethylated metabolite of lignocaine, thought to be metabolised by CYP3A4, was detected with microsomes from CYP2D6-expressing yeast cells. The results indicate that yeast cells containing human CYP2D6 cDNA express a functionally active form of the enzyme, the immunochemical and catalytic properties of which are consistent with those of human liver.

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of ( S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [ Methyl- 14C]mephenytoin was synthesized by alkylation of S-nirvanol with 14CH 3I and used as a substrate. After incubation of [ methyl- 14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The K m and V max rates obtained with a human liver microsomal preparation were 0.1 m m and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basis system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.

Induction of drug metabolizing enzymes in human liver cell line Hep G2

Human cytochrome P-450, UDP-glucuronosyltransferase and sulphotransferase activities have been measured in the cell line Hep G2 following treatment of cells with 3-methylcholanthrene or phenobarbital. 3-Methylcholanthrene treatment caused a 20–30-fold increase in the O-deethylation of 7-ethoxycoumarin. The glucuronidation and sulphation of the product 7-hydroxycoumarin were increased 36 and 7 fold, respectively. In comparison, phenobarbital treatment did not increase these activities significantly. However, phenobarbital-inducible proteins were identified on ‘Western blots’ using antibodies to a rat liver phenobarbital inducible P-450 form. The molecular masses of the proteins did not coincide with those expected for cytochromes P-450. However, characteristic of P-450 forms, the synthesis of these proteins was suppressed by 3-methylcholanthrene treatment. The Hep G2 cell line represents a potentially useful model for studying the regulation of human P-450 genes.