High-affinity Phenacetin O-deethylase Research Articles

Diazinon Is Activated by CYP2C19 in Human Liver

Phosphorothioate compounds are used throughout the world as agricultural and domestic pesticides. Here, the activation of the phosphorothioate diazinon to diazoxon in human liver is described. In an initial study using three human liver microsomal samples, Km for diazoxon formation varied markedly (31, 208, and 660 μM; Vmax 1125, 685, and 1028 pmol/min/mg protein, respectively), suggesting the involvement of more than one P450 enzyme. A wide variation in activity was found using 50 μM diazinon as substrate, (11–648 pmol/min/mg protein, n = 15), whereas, with 500 μM, variation was less (164–978 pmol/min/mg protein). Among eight P450-catalyzed reactions, the putative high-affinity component (50 μM diazinon) correlated with S-mephenytoin 4′-hydroxylase activity (r = 0.686, p < 0.01), suggesting the involvement of CYP2C19. The putative low-affinity component (500 μM diazinon) correlated with both S-mephenytoin 4′-hydroxylase (r = 0.714; p < 0.005) and high-affinity phenacetin O-deethylase activity (r = 0.625; p < 0.05). This activity was partially inhibited by furafylline, troleandomycin, and ketoconazole. These data suggest contributions from CYP2C19, CYP1A2, and CYP3A4. None of the inhibitors affected the high-affinity component. Of seven heterologously expressed human P450 enzymes, CYP2C19 activated diazinon (500 μM) at the fastest rate, followed by CYP3A4, CYP1A2, and CYP2C9. Both hepatic microsomal S-mephenytoin 4′-hydroxylase and high-affinity phenacetin O-deethylase activities were strongly inhibited by diazinon (IC50 < 2.5 μM), while no effect was seen on midazolam 1′-hydroxylase activity. These data indicate that CYP2C19 is the major enzyme involved in diazinon activation in human liver, while other enzymes including CYP1A2 may play a more minor role.

Inhibition of human CYP1A2 activity in vitro by methylxanthines: potent competitive inhibition by 8-phenyltheophylline

1. Humans are exposed in vivo to methylxanthines by dietary ingestion, as well as from their use as therapeutic agents. The inhibitory effect of a series of these compounds on high-affinity phenacetin O-deethylase activity in the human liver microsomal fraction, a measure of CYP1A2 activity, has been evaluated. 2. Paracetamol, the product of phenacetin O-deethylase activity, was analysed by gas chromatography/negative-ion mass spectrometry using a novel bistrifluoromethylbenzoyl/ trimethylsilyl derivative, and incubation conditions for assessing high-affinity phenacetin O-deethylase activity were examined and optimized. 3. 1-Methylxanthine, caffeine, theophylline, 8-methylxanthine, pentoxyfylline and 3- isobutyl-1-methylxanthine caused moderate inhibition with IC50 = 260, 140, 120, 100, 62 and 36 μM respectively. 4. 8-Phenyltheophylline was a potent competitive inhibitor of high-affinity phenacetin O-deethylase activity with an IC50 = 0.7 μM and Ki = 0.11 μM. 5. The specificity of inhibition by 8-phenyltheophylline was assessed by measuring its effect on debrisoquine 4-hydroxylase (CYP2D6), terfenadine hydroxylase (CYP3A4), chlorzoxazone 6-hydroxylase (CYP2E1) and tolbutamide 4-hydroxylase (CYP2C9) activities in human liver microsomal fraction. No inhibition of any of these activitieswas observed. 6. The potency and specificity of 8-phenyltheophylline as an inhibitor of human hepatic CYP1A2 indicate that the compound may be useful as a chemical inhibitor of this enzyme for further in vitro studies.

Specific inhibition of human CYPLA2 using a targeted antibody

The structural similarity of related forms of P450 makes selective immunoinhibition of individual forms notoriously difficult to achieve. We report the use of a targeted antibody to overcome this problem. An antibody was raised against the synthetic peptide, Ser-Lys-Lys-Gly-Pro-Arg-Ala-Ser-Gly-Asn-Leu-Ile, corresponding to residues 291–302 of human CYP1A2. This sequence of human CYP1A2 is located in a similar position to a proinhibitory region previously identified in rat CYP1A1 and CYP1A2. The antibody bound strongly and specifically to CYP1A2 in human hepatic microsomal fraction. Binding was unaffected by denaturation of the protein. The specificity of the antibody was demonstrated by immunoblotting of human hepatic microsomal fraction where a single immunoreactive band was identified at M, 54,000. The intensity of this band correlated strongly with high-affinity phenacetin O-deethylase activity of the microsomal fractions. In addition, the antibody bound to a single protein at M, 54,000 in the microsomal fraction of lymphoblastoid cells expressing human CYP1A2, but not to any other recombinant P450 enzyme. CYPlA2-dependent activity (high-affinity phenacetin O-deethylase) of human hepatic microsomal fraction was inhibited >90% by whole antiserum or purified immunoglobulin. This decrease in activity represents complete inhibition of CYP1A2 activity, residual phenacetin O-deethylase activity being due to low-affinity enzymes. In contrast, the antibody, which does not bind to rat CYP1A2, had no effect on CYPlA2-dependent activity (high-affinity phenacetin O-deethylase) of rat hepatic microsomal fraction. The antiserum also had no effect on human hepatic microsomal debrisoquine 4-hydroxylase (CYP2D6) or coumarin 7-hydroxylase (CYP2A6) activities, indicating that inhibition was specific to human CYP1A2. These results demonstrate the importance of the region comprising residues 291–302 of human CYP1A2 in the catalytic activity of this enzyme.

Inducibility of cytochromes P-450 by dioxin in liver and extrahepatic tissues of the marmoset monkey ( Callithrix jacchus)

Cytochrome P-450 induction was investigated in the marmoset monkey, a non-human primate, using dioxins as inducing agents. Animals received a single subcutaneous dose of 1.6 nmol tetrachlorodibenzo- p-dioxin or tetrabromodibenzo- p-dioxin/kg body weight. Microsomal fractions were prepared from liver, lung and kidney, and homogenates were prepared from gut and adrenal glands. Anti-peptide antibodies which bind to CYPIA1, CYPIA2, CYP2B6 and CYP3A4 in human were used to identify related forms in the marmoset. The results indicate that CYPIA2 is constitutively expressed in liver, but not in lung, kidney, gut or adrenal gland and that CYPIAI is not expressed in any of these tissues in untreated animals. Treatment with dioxin induced both CYPIAI and CYPIA2 in liver, but only CYPIA1 in lung. No induction of CYPIA1 or CYPIA2 was found in kidney, small intestine or adrenal glands. Methoxy-, ethoxy-, pentoxy- and benzoyloxyresorufin O-dealkylases and high affinity phenacetin O-deethylase activities were induced in the liver, whereas ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities were not affected by dioxin treatment. High-affinity phenacetin O-deethylase and CYPIA2 apoprotein were detected only in liver, consistent with this activity being specifically catalysed by CYPIA2. Furafylline was found to be a competitive inhibitor of methoxyresorufin O-demethylase activity with a K i of 10 μM. In the lung the induction of CYPIAI was accompanied by 15- and 23-fold increases in ethoxyresorufin O-deethylase and methoxyresorufm O-demethylase activities, respectively, suggesting that both activities are catalysed by CYP1A1. In contrast, there was no induction of aryl hydrocarbon hydroxylase activity in lung or liver showing that, unlike in many other species, marmoset CYPIAI does not catalyse this reaction efficiently. The expression, distribution, induction and substrate specificities of marmoset monkey P-450 enzymes differ from the situation found in rodents and other species, demonstrating that caution has to be exercised when making cross-species extrapolations.

Caffeine metabolism by human hepatic cytochromes p450: Contributions of 1A2, 2E1 and 3A isoforms

Caffeine (CA) N1-, N3- and N7-demethylase, CA 8-hydroxylase and phenacetin O-deethylase activities were measured in microsomes from 18 separate human livers which had been characterized previously for a range of cytochrome P450 (CYP) isoform-specific activities and immunoreactive CYP protein contents. Correlations between the high affinity components of the three separate CA N-demethylations were highly significant ( r = 0.77–0.91, P < 0.001) and each of the three high affinity CA N-demethylations correlated significantly ( r = 0.64–0.93, P < 0.05-0.001) with the high affinity phenacetin O-deethylase, 2-acetylaminofluorene N-hydroxylation and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) mutagenicity (all predominantly CYP1A2-mediated reactions). Consistent with these observations, cDNA-expressed human CYP1A2 catalyzed the N1-, N3- and N7-demethylation of CA and apparent K m values were similar (0.24–0.28 mM) for all three reactions and comparable to those observed previously with human liver microsomes. The low affinity components of CA N1- and N7-demethylation correlated significantly ( r = 0.55–0.85, P < 0.05-0.001) with immunoreactive CYP2E1 content and the CYP2E1-specific activities 4-nitrophenol and chlorzoxazone hydroxylation. Diethyldithiocarbamate, a selective inhibitor of CYP2E1, inhibited the low affinity CA N1- and N7-demethylation, with IC 50 values of 23 μM and 11 μM, respectively. The apparent K m values for CA N1- and N7-demethylation by cDNA-expressed CYP2E1 (namely 28 and 43 mM, respectively) were of a similar order to those calculated for the low affinity microsomal activities. Significant correlations ( r = 0.87–0.97, P < 0.001) were observed between CA 8-hydroxylation and immunoreactive CYP3A content and the CYP3A-mediated reactions benzo( a)pyrene hydroxylation, omeprazole sulfoxidation and aflatoxin B1 mutagenesis. Effects of α-naphthoflavone, erythromycin, troleandomycin and nifedipine on microsomal CA 8-hydroxylation were generally consistent with CYP3A involvement. Taken together with previous data, the results indicate a major involvement of CYP1A2 in the high affinity component of all three human hepatic CA N-demethylations. In contrast, CYP2E1 appears to be the main enzyme involved in the low affinity components of CA N1- and N7-demethylation while CA 8-hydroxylation is catalysed predominantly by a CYP3A isoform(s).

Metabolism of the food derived mutagen and carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) by human liver microsomes.

Animal studies have shown that 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) undergoes both activation to a genotoxic metabolite and detoxication, catalysed by CYP enzymes. In this study, using direct chemical analysis, we have examined PhIP metabolism by the microsomal fraction of human liver for comparison to that occurring in animals. PhIP was incubated with human liver microsomes in the presence of an NADPH regenerating system and the reaction mixture then analyzed by HPLC. Only one metabolite, identified as N-hydroxy PhIP, was produced. The N-hydroxylation of PhIP by human liver microsomal fraction obeyed Michaelis-Menten kinetics, with a Km of 55 microM and a Vmax of 666 pmol/min/mg protein. Furafylline, a potent and specific inhibitor of CYP1A2 in man, inhibited this reaction by > 95%, with an IC50 of 0.6 microM. PhIP inhibited high affinity phenacetin O-deethylase activity of human liver microsomes, an activity catalysed specifically by CYP1A2, with an IC50 of about 80 microM. These data indicate that, in human liver microsomes, N-hydroxylation is the only route of oxidative metabolism of PhIP, yielding a genotoxic species, and that this reaction is catalysed almost exclusively by CYP1A2. Furthermore, the exclusive oxidative activation of PhIP by human liver is in direct contrast to PhIP metabolism in rodents and non-human primates where oxidative detoxication products predominate.

Identification of the epitope of an anti-peptide antibody which binds to CYP1A2 in many species including man

An anti-peptide antibody was raised against the sequence Thr-Gly-Ala-Leu-Phe-Lys-His-Ser-Glu-Asn-Tyr-Lys which occurs at positions 283–294 in the rat cytochrome P450 enzyme CYP1A2. Compared with its binding to the peptide used for immunization, the antibody bound with only slightly reduced affinity to the truncated peptides Thr-Gly-Ala-Leu-Phe-Lys-His-Ser and Leu-Phe-Lys-His-Ser. However, binding to the peptide Ser-Glu-Asn-Tyr-Lys-Asp-Asn, which overlaps with the C-terminal region of the immunizing peptide, was very low. Thus, a major epitope for the anti-peptide antibody is Leu-Phe-Lys-His-Ser, which corresponds to a region of CYP1A2 that is conserved in many species. The antibody was tested by immunoblotting for its ability to bind to hepatic microsomal fractions from a number of species. Where possible animals were treated with compounds which induce CYP1A2 and the results compared with those with untreated animals. It was found that the antibody bound to rat, mouse, rabbit, hamster, guinea pig, pig, marmoset monkey and human CYP1A2. No evidence was found for binding to dog CYP1A2. The region corresponding to the major epitope at residues 286–290 of rat CYP1A2 was identical in mouse, hamster, rabbit and human CYP1A2. The sequence of marmoset and guinea pig CYP1A2 are not known but are predicted to be very similar to the sequence in the rat. The lack of binding of the antibody to dog CYP1A2 may be explained by two differences in this region compared with rat CYP1A2. Maximum inhibition of CYP1A2 activity by this antibody, as measured by high-affinity phenacetin O-deethylase activity, was 20%. This is in contrast to a previously described anti-peptide antibody directed to an adjacent region which caused 65% inhibition of this activity. Thus, the edge of an inhibitory region on the surface of cytochrome P450 has been identified. The ability of the antibody to bind to CYP1A2 from a number of animals should make this antibody of use for studying the levels of CYP1A2 apoprotein in many species.

Furafylline is a potent and selective inhibitor of cytochrome P450IA2 in man.

1. Furafylline (1,8-dimethyl-3-(2'-furfuryl)methylxanthine) is a methylxanthine derivative that was introduced as a long-acting replacement for theophylline in the treatment of asthma. Administration of furafylline was associated with an elevation in plasma levels of caffeine, due to inhibition of caffeine oxidation, a reaction catalysed by one or more hydrocarbon-inducible isoenzymes of P450. We have now investigated the selectivity of inhibition of human monooxygenase activities by furafylline. 2. Furafylline was a potent, non-competitive inhibitor of high affinity phenacetin O-deethylase activity of microsomal fractions of human liver, a reaction catalysed by P450IA2, with an IC50 value of 0.07 microM. 3. Furafylline had either very little or no effect on human monooxygenase activities catalysed by other isoenzymes of P450, including P450IID1, P450IIC, P450IIA. Of particular interest, furafylline did not inhibit P450IA1, assessed from aryl hydrocarbon hydroxylase activity of placental samples from women who smoked cigarettes. 4. It is concluded that furafylline is a highly selective inhibitor of P450IA2 in man. 5. Furafylline was a potent inhibitor of the N3-demethylation of caffeine and of a component of the N1- and N7-demethylation. This confirms earlier suggestions that caffeine is a selective substrate of a hydrocarbon-inducible isoenzyme of P450 in man, and identifies this as P450IA2. Thus, caffeine N3-demethylation should provide a good measure of the activity of P450IA in vivo in man. 6. Although furafylline selectively inhibited P450IA2, relative to P450IA1, in the rat, this was at 1000-times the concentration required to inhibit the human isoenzyme, suggesting a major difference in the active site geometry between the human and the rat orthologues of P50IA2.

High affinity phenacetin O-deethylase is catalysed specifically by cytochrome P450d (P450IA2) in the liver of the rat

Phenacetin is metabolized primarily by O-deethylation to paracetamol (POD activity), a reaction catalysed by cytochrome P450. The high affinity component of POD activity is inducible in rat liver by treatment of the animals with polycyclic aromatic hydrocarbons. Following treatment with hydrocarbons such as 3-methylcholanthrene (MC) and isosafrole (ISF) both cytochromes P450c (P450IA1) and P450d (P450IA2) are also induced in rat liver. Studies with the reconstituted enzymes have shown that both forms of P450 catalyse phenacetin O-deethylation at rates that exceeded that of the high affinity component of activity of hepatic microsomal preparations from 3-methylcholanthrenetreated rats (at 4μM phenacetin: P450c, 440 ± 40 pmol/nmol/min; P450d, 1030 ± 10 pmol/nmol/min; microsomal fraction, 163 pmol/mg/min). Specific inhibitory antibodies (both monoclonal and monospecific polyclonal) were used to define the specificity of microsomal POD activity. These studies have shown that hepatic high affinity POD activity is exclusively catalysed by cytochrome P450d in both untreated rats and in rats pretreated with MC.

Differential expression and regulation of members of the cytochrome P450IA gene subfamily in human tissues

Cigarette smoking increases phenacetin O-deethylase (POD) activity in both the liver and placenta in man, but aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity is increased only in the placenta. Whilst there was no correlation between hepatic POD and AHH activities (rs = 0.42, P greater than 0.1), there was a highly significant correlation between these two activities in placenta (rs = 0.76, P less than 0.02). On Western blotting of human liver samples with an antibody specific to cytochrome P450IA2 in the rat, only the orthologue of P450IA2 could be detected. This antibody inhibited greater than 70% of hepatic high-affinity POD activity but had no effect on the placental activity. Furafylline, a methylxanthine that acts as a highly specific inhibitor of P450IA2-dependent activities in man, inhibited all of the high-affinity component of POD activity in human liver, but was at least three orders of magnitude less potent an inhibitor of placental POD and of both hepatic and placental AHH activities. As previously shown in the rat, exposure of man to polycyclic aromatic hydrocarbons, present in cigarette smoke, differentially induces P450IA2 in the liver and P450IA1 in extrahepatic tissues, at least in the placenta. Again, as in the rat, POD activity in the liver is catalysed by P450IA2, but in the placenta of women exposed to polycyclic aromatic hydrocarbons in cigarette smoke POD activity is catalysed by another isoenzyme, most likely P450IA1. Thus, tissue-dependent induction and substrate specificity of members of the P450IA family in man, at least in the placenta, appear to be the same as previously shown in the rat.