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
N1 - Funding Information:
This work was supported in part by Grants-in-Aid for Scientific Research (17390035 and 18590116) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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 - CYP2E1
KW - Chlorzoxazone 6-hydroxylation
KW - Cynomolgus monkey
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
C2 - 17935737
AN - SCOPUS:35548975119
SN - 0024-3205
VL - 81
SP - 1436
EP - 1445
JO - Life Sciences
JF - Life Sciences
IS - 19-20
ER -