The Influence of Quinolines on Coumarin 7-Hydroxylation in Bovine Liver Microsomes and Human CYP2A6.

Hirano, Yoshie; Uehara, Mayumi; Saeki, Ken-ichi; Kato, Takaaki; Takahashi, Kazuhiko; Mizutani, Takaharu

doi:10.1248/jhs.48.118

Cited by 29 publications

(18 citation statements)

References 21 publications

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“…S1). Because 7-ethoxycoumarin and coumarin are well recognized substrates of human P450 enzymes, whole-cell oxidation activity of the mutants on the conversion of 7-ethoxycoumarin and coumarin to 7-OH coumarin, a fluorescent product, was measured (Hirano et al, 2002). Colonies that showed these catalytic activities were selected, and each mutated enzyme was expressed in E. coli for purification.…”

Section: Resultsmentioning

confidence: 99%

Engineering Bacterial Cytochrome P450 (P450) BM3 into a Prototype with Human P450 Enzyme Activity Using Indigo Formation

Park¹,

Kim²,

Kim³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Errorprone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by Odeethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.

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Section: Resultsmentioning

confidence: 99%

Engineering Bacterial Cytochrome P450 (P450) BM3 into a Prototype with Human P450 Enzyme Activity Using Indigo Formation

Park¹,

Kim²,

Kim³

et al. 2010

Drug Metab Dispos

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show abstract

“…[17][18][19][20][21][22] 8,14) Our previous results showed that the activity levels of CYP2A6 and UGT in bovine liver microsomes were similar to human liver microsomes 4,28,29) but differed from rat microsomes, as rat microsomes did not involve CYP2A6 activity. Thus, it was considered that bovine microsome data were very similar to human microsome data.…”

Section: Discussionmentioning

confidence: 99%

Inhibition Mechanism of UDP-Glucuronosyltransferase 1A6 by Xanthene Food Dyes

Uesugi

Furumiya

Mizutani

2006

JOURNAL OF HEALTH SCIENCE

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We have reported that erythrosine (ET), a xanthene dye, inhibited uridine 5-diphosphate (UDP)-glucuronosyltransferase 1A6 (UGT1A6). In order to clarify the structure-inhibition relationships of these xanthene dyes, the inhibitory effect of xanthene dye on human UGT1A6 activity was investigated, such as acid red (AR), ET, phloxine (PL), and rose bengal (RB). ET, PL, and RB strongly inhibited human UGT1A6 activity, with IC 50 values = 0.05, 0.04, and 0.015 mM, respectively. Meanwhile, AR had almost no effect (IC 50 value = 1.7 mM). ET, PL and RB have four halogen atoms on their xanthene backbone, unlike AR. Meanwhile, some contrast media with high halogens on those aromatic compounds, such as ioxaglic acid, iodixanol, meglumine iotalamate, and diatriazole sodium, did not inhibit human UGT1A6 activity. These results suggest that halogens enhance the inhibitory effect of xanthene dyes. In this study, it was proved that xanthene dyes had an inhibitory effect on human UGT1A6 activity by the combination of xanthene structure and halogens on its. Part of this inhibition by xanthene dyes depends the reaction of 1 O 2 originated on xanthene dyes by light irradiation, because the inhibition was prevented by 1 O 2 quenchers, such as NaN 3 and histidine, and in the dark.

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“…The activity of CYP2A6 was measured by the previous report. 19,20) HLM (4 µg) was preincubated with MEPM in 15 µl at 0 • C for 30 min. The reaction was started by addition of the reaction mixture (15 µl) composed of 3 mM MgCl 2 , 1.3mM NADP + , 3.3 mM glucose-6-phosphate, 0.4 U glucose-6-phosphate dehydrogenase, 10 mM coumarin and 0.1 M Tris-HCl (pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

In Vitro Activation of Valproate Glucuronidation by Carbapenem Antibiotics

Mori

Mizutani

2007

JOURNAL OF HEALTH SCIENCE

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The serum concentration of valproic acid (VPA) in epilepsy patients decreased by the administration of carbapenem antibiotics, such as meropenem, panipenem or imipenem, to a sub-therapeutic level. Studies to explain the decrease were carried out using almost rats by the following steps: absorption of VPA in the intestine, glucuronidation in the liver, disposition in blood and renal excretion. It is difficult to consider the inhibition of intestinal absorption, because carbapenem antibiotics are intravenously administered and do not reach the intestine at an effective concentration. The liver is the key organ for the decrease of VPA concentration by carbapenem antibiotics, because it has been reported that no decrease of the VPA level by carbapenem was found in hepatectomized rats. The most likely mechanism in liver is the activation of UDP-glucuronosyltransferase by carbapenem antibiotics. We found a 35% increase of VPA-glucuronidation activity by the pre-incubation of human liver microsomes with meropenem. We estimated that this increase fully compensates for the decrease of serum VPA level by carbapenem antibiotics in patients. Meanwhile, we could not find the in vitro activation of CYP2A6, CYP3A4, P-glycoprotein (P-gp, MDR1) and Multidrug resistance-associated protein 2 by pre-incubation with carbapenem antibiotics. Thus, we considered that the activation of VPA glucuronidation by carbapenem antibiotics is a key point in the decrease of plasma VPA level.

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The Influence of Quinolines on Coumarin 7-Hydroxylation in Bovine Liver Microsomes and Human CYP2A6.

Cited by 29 publications

References 21 publications

Engineering Bacterial Cytochrome P450 (P450) BM3 into a Prototype with Human P450 Enzyme Activity Using Indigo Formation

Engineering Bacterial Cytochrome P450 (P450) BM3 into a Prototype with Human P450 Enzyme Activity Using Indigo Formation

Inhibition Mechanism of UDP-Glucuronosyltransferase 1A6 by Xanthene Food Dyes

In Vitro Activation of Valproate Glucuronidation by Carbapenem Antibiotics

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