Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes

Nobumitsu Hanioka, Maki Yamamoto, Hiroyuki Iwabu, Hideto Jinno, Toshiko Tanaka-Kagawa, Shinsaku Naito, Takefumi Shimizu, Kazufumi Masuda, Takashi Katsu, Shizuo Narimatsu

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Cytochrome P450 2E1 (CYP2E1) is an enzyme of major toxicological interest because it metabolizes various drugs, precarcinogens and solvents to reactive metabolites. In this study, human and cynomolgus monkey CYP2E1 cDNAs (humCYP2E1 and monCYP2E1, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate CYP2E1s. The enzymatic properties of CYP2E1 proteins were characterized by kinetic analysis of chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation. humCYP2E1 and monCYP2E1 enzymes showed 94.3% identity in their amino acid sequences. The functional CYP content in yeast cell microsomes expressing humCYP2E1 was 38.4 pmol/mg protein. The level of monCYP2E1 was 42.7% of that of humCYP2E1, although no significant differences were statistically observed. The Km values of microsomes from human livers and yeast cells expressing humCYP2E1 for CYP2E1-dependent oxidation were 822 and 627 μM for chlorzoxazone 6-hydroxylation, and 422 and 514 μM for 4-nitrophenol 2-hydroxylation, respectively. The Km values of microsomes from cynomolgus monkey livers and yeast cells expressing monCYP2E1 were not significantly different from those of humans in any enzyme source. Vmax and Vmax / Km values of human liver microsomes for CYP2E1-dependent oxidation were 909 pmol/min/mg protein and 1250 nl/min/mg protein for chlorzoxazone 6-hydroxylation, and 1250 pmol/min/mg protein and 2990 nl/min/mg protein for 4-nitrophenol 2-hydroxylation, respectively. The kinetic parameter values of cynomolgus monkey livers were comparable to or lower than those of human liver microsomes (49.5-102%). In yeast cell microsomes expressing humCYP2E1, Vmax and Vmax / Km values for CYP2E1-dependent oxidation on the basis of CYP holoprotein level were 170 pmol/min/pmol CYP and 272 nl/min/pmol CYP for chlorzoxazone 6-hydroxylation, and 139 pmol/min/pmol CYP and 277 nl/min/pmol CYP for 4-nitrophenol 2-hydroxylation, respectively, and the kinetic parameters of monCYP2E1 exhibited similar values. These findings suggest that human and cynomolgus monkey CYP2E1 enzymes have high homology in their amino acid sequences, and that their enzymatic properties are considerably similar. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.

Original languageEnglish
Pages (from-to)1436-1445
Number of pages10
JournalLife Sciences
Volume81
Issue number19-20
DOIs
Publication statusPublished - Oct 27 2007

Fingerprint

Cytochrome P-450 CYP2E1
Hydroxylation
Macaca fascicularis
Cytochrome P-450 Enzyme System
Chlorzoxazone
Liver
Yeast
Yeasts
Cells
Liver Microsomes
Microsomes
Proteins
Kinetic parameters
Oxidation
Amino Acid Sequence
Enzymes
Amino Acids
Xenobiotics
Metabolites
Extrapolation

Keywords

  • 4-Nitrophenol 2-hydroxylation
  • Chlorzoxazone 6-hydroxylation
  • Cynomolgus monkey
  • CYP2E1
  • Cytochrome P450 (CYP)
  • Human
  • Primates

ASJC Scopus subject areas

  • Pharmacology

Cite this

Hanioka, N., Yamamoto, M., Iwabu, H., Jinno, H., Tanaka-Kagawa, T., Naito, S., ... Narimatsu, S. (2007). Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes. Life Sciences, 81(19-20), 1436-1445. https://doi.org/10.1016/j.lfs.2007.09.002

Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes. / Hanioka, Nobumitsu; Yamamoto, Maki; Iwabu, Hiroyuki; Jinno, Hideto; Tanaka-Kagawa, Toshiko; Naito, Shinsaku; Shimizu, Takefumi; Masuda, Kazufumi; Katsu, Takashi; Narimatsu, Shizuo.

In: Life Sciences, Vol. 81, No. 19-20, 27.10.2007, p. 1436-1445.

Research output: Contribution to journalArticle

Hanioka, N, Yamamoto, M, Iwabu, H, Jinno, H, Tanaka-Kagawa, T, Naito, S, Shimizu, T, Masuda, K, Katsu, T & Narimatsu, S 2007, 'Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes', Life Sciences, vol. 81, no. 19-20, pp. 1436-1445. https://doi.org/10.1016/j.lfs.2007.09.002
Hanioka N, Yamamoto M, Iwabu H, Jinno H, Tanaka-Kagawa T, Naito S et al. Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes. Life Sciences. 2007 Oct 27;81(19-20):1436-1445. https://doi.org/10.1016/j.lfs.2007.09.002
Hanioka, Nobumitsu ; Yamamoto, Maki ; Iwabu, Hiroyuki ; Jinno, Hideto ; Tanaka-Kagawa, Toshiko ; Naito, Shinsaku ; Shimizu, Takefumi ; Masuda, Kazufumi ; Katsu, Takashi ; Narimatsu, Shizuo. / Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes. In: Life Sciences. 2007 ; Vol. 81, No. 19-20. pp. 1436-1445.
@article{0360505184654c5189c7cba7ae36b525,
title = "Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes",
abstract = "Cytochrome P450 2E1 (CYP2E1) is an enzyme of major toxicological interest because it metabolizes various drugs, precarcinogens and solvents to reactive metabolites. In this study, human and cynomolgus monkey CYP2E1 cDNAs (humCYP2E1 and monCYP2E1, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate CYP2E1s. The enzymatic properties of CYP2E1 proteins were characterized by kinetic analysis of chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation. humCYP2E1 and monCYP2E1 enzymes showed 94.3{\%} identity in their amino acid sequences. The functional CYP content in yeast cell microsomes expressing humCYP2E1 was 38.4 pmol/mg protein. The level of monCYP2E1 was 42.7{\%} of that of humCYP2E1, although no significant differences were statistically observed. The Km values of microsomes from human livers and yeast cells expressing humCYP2E1 for CYP2E1-dependent oxidation were 822 and 627 μM for chlorzoxazone 6-hydroxylation, and 422 and 514 μM for 4-nitrophenol 2-hydroxylation, respectively. The Km values of microsomes from cynomolgus monkey livers and yeast cells expressing monCYP2E1 were not significantly different from those of humans in any enzyme source. Vmax and Vmax / Km values of human liver microsomes for CYP2E1-dependent oxidation were 909 pmol/min/mg protein and 1250 nl/min/mg protein for chlorzoxazone 6-hydroxylation, and 1250 pmol/min/mg protein and 2990 nl/min/mg protein for 4-nitrophenol 2-hydroxylation, respectively. The kinetic parameter values of cynomolgus monkey livers were comparable to or lower than those of human liver microsomes (49.5-102{\%}). In yeast cell microsomes expressing humCYP2E1, Vmax and Vmax / Km values for CYP2E1-dependent oxidation on the basis of CYP holoprotein level were 170 pmol/min/pmol CYP and 272 nl/min/pmol CYP for chlorzoxazone 6-hydroxylation, and 139 pmol/min/pmol CYP and 277 nl/min/pmol CYP for 4-nitrophenol 2-hydroxylation, respectively, and the kinetic parameters of monCYP2E1 exhibited similar values. These findings suggest that human and cynomolgus monkey CYP2E1 enzymes have high homology in their amino acid sequences, and that their enzymatic properties are considerably similar. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.",
keywords = "4-Nitrophenol 2-hydroxylation, Chlorzoxazone 6-hydroxylation, Cynomolgus monkey, CYP2E1, Cytochrome P450 (CYP), Human, Primates",
author = "Nobumitsu Hanioka and Maki Yamamoto and Hiroyuki Iwabu and Hideto Jinno and Toshiko Tanaka-Kagawa and Shinsaku Naito and Takefumi Shimizu and Kazufumi Masuda and Takashi Katsu and Shizuo Narimatsu",
year = "2007",
month = "10",
day = "27",
doi = "10.1016/j.lfs.2007.09.002",
language = "English",
volume = "81",
pages = "1436--1445",
journal = "Life Sciences",
issn = "0024-3205",
publisher = "Elsevier Inc.",
number = "19-20",

}

TY - JOUR

T1 - Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes

AU - Hanioka, Nobumitsu

AU - Yamamoto, Maki

AU - Iwabu, Hiroyuki

AU - Jinno, Hideto

AU - Tanaka-Kagawa, Toshiko

AU - Naito, Shinsaku

AU - Shimizu, Takefumi

AU - Masuda, Kazufumi

AU - Katsu, Takashi

AU - Narimatsu, Shizuo

PY - 2007/10/27

Y1 - 2007/10/27

N2 - Cytochrome P450 2E1 (CYP2E1) is an enzyme of major toxicological interest because it metabolizes various drugs, precarcinogens and solvents to reactive metabolites. In this study, human and cynomolgus monkey CYP2E1 cDNAs (humCYP2E1 and monCYP2E1, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate CYP2E1s. The enzymatic properties of CYP2E1 proteins were characterized by kinetic analysis of chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation. humCYP2E1 and monCYP2E1 enzymes showed 94.3% identity in their amino acid sequences. The functional CYP content in yeast cell microsomes expressing humCYP2E1 was 38.4 pmol/mg protein. The level of monCYP2E1 was 42.7% of that of humCYP2E1, although no significant differences were statistically observed. The Km values of microsomes from human livers and yeast cells expressing humCYP2E1 for CYP2E1-dependent oxidation were 822 and 627 μM for chlorzoxazone 6-hydroxylation, and 422 and 514 μM for 4-nitrophenol 2-hydroxylation, respectively. The Km values of microsomes from cynomolgus monkey livers and yeast cells expressing monCYP2E1 were not significantly different from those of humans in any enzyme source. Vmax and Vmax / Km values of human liver microsomes for CYP2E1-dependent oxidation were 909 pmol/min/mg protein and 1250 nl/min/mg protein for chlorzoxazone 6-hydroxylation, and 1250 pmol/min/mg protein and 2990 nl/min/mg protein for 4-nitrophenol 2-hydroxylation, respectively. The kinetic parameter values of cynomolgus monkey livers were comparable to or lower than those of human liver microsomes (49.5-102%). In yeast cell microsomes expressing humCYP2E1, Vmax and Vmax / Km values for CYP2E1-dependent oxidation on the basis of CYP holoprotein level were 170 pmol/min/pmol CYP and 272 nl/min/pmol CYP for chlorzoxazone 6-hydroxylation, and 139 pmol/min/pmol CYP and 277 nl/min/pmol CYP for 4-nitrophenol 2-hydroxylation, respectively, and the kinetic parameters of monCYP2E1 exhibited similar values. These findings suggest that human and cynomolgus monkey CYP2E1 enzymes have high homology in their amino acid sequences, and that their enzymatic properties are considerably similar. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.

AB - Cytochrome P450 2E1 (CYP2E1) is an enzyme of major toxicological interest because it metabolizes various drugs, precarcinogens and solvents to reactive metabolites. In this study, human and cynomolgus monkey CYP2E1 cDNAs (humCYP2E1 and monCYP2E1, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate CYP2E1s. The enzymatic properties of CYP2E1 proteins were characterized by kinetic analysis of chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation. humCYP2E1 and monCYP2E1 enzymes showed 94.3% identity in their amino acid sequences. The functional CYP content in yeast cell microsomes expressing humCYP2E1 was 38.4 pmol/mg protein. The level of monCYP2E1 was 42.7% of that of humCYP2E1, although no significant differences were statistically observed. The Km values of microsomes from human livers and yeast cells expressing humCYP2E1 for CYP2E1-dependent oxidation were 822 and 627 μM for chlorzoxazone 6-hydroxylation, and 422 and 514 μM for 4-nitrophenol 2-hydroxylation, respectively. The Km values of microsomes from cynomolgus monkey livers and yeast cells expressing monCYP2E1 were not significantly different from those of humans in any enzyme source. Vmax and Vmax / Km values of human liver microsomes for CYP2E1-dependent oxidation were 909 pmol/min/mg protein and 1250 nl/min/mg protein for chlorzoxazone 6-hydroxylation, and 1250 pmol/min/mg protein and 2990 nl/min/mg protein for 4-nitrophenol 2-hydroxylation, respectively. The kinetic parameter values of cynomolgus monkey livers were comparable to or lower than those of human liver microsomes (49.5-102%). In yeast cell microsomes expressing humCYP2E1, Vmax and Vmax / Km values for CYP2E1-dependent oxidation on the basis of CYP holoprotein level were 170 pmol/min/pmol CYP and 272 nl/min/pmol CYP for chlorzoxazone 6-hydroxylation, and 139 pmol/min/pmol CYP and 277 nl/min/pmol CYP for 4-nitrophenol 2-hydroxylation, respectively, and the kinetic parameters of monCYP2E1 exhibited similar values. These findings suggest that human and cynomolgus monkey CYP2E1 enzymes have high homology in their amino acid sequences, and that their enzymatic properties are considerably similar. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.

KW - 4-Nitrophenol 2-hydroxylation

KW - Chlorzoxazone 6-hydroxylation

KW - Cynomolgus monkey

KW - CYP2E1

KW - Cytochrome P450 (CYP)

KW - Human

KW - Primates

UR - http://www.scopus.com/inward/record.url?scp=35548975119&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=35548975119&partnerID=8YFLogxK

U2 - 10.1016/j.lfs.2007.09.002

DO - 10.1016/j.lfs.2007.09.002

M3 - Article

VL - 81

SP - 1436

EP - 1445

JO - Life Sciences

JF - Life Sciences

SN - 0024-3205

IS - 19-20

ER -