Identification of the main human cytochrome P450 enzymes involved in safrole 1'-hydroxylation.
Safrole is a natural plant constituent, found in sassafras oil and certain other essential oils. The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, followed by sulfonation to an unstable sulfate that reacts to form DNA adducts. To identify the main cytochrome P450 (P450) involved in human hepatic safrole 1'-hydroxylation (SOH), we determined the SOH activities of human liver microsomes and Escherichia coli membranes expressing bicistronic human P450s. Human liver (n = 18) microsomal SOH activities were in the range of 3.5-16.9 nmol/min/mg protein with a mean value of 8.7 +/- 0.7 nmol/min/mg protein. In human liver (n = 3) microsomes, the mean K(m) and V(max) values of SOH were 5.7 +/- 1.2 mM and 0.14 +/- 0.03 micromol/min/nmol P450, respectively. The mean intrinsic clearance (V(max)/K(m)) was 25.3 +/- 2.3 microL/min/nmol P450. SOH was sensitive to the inhibition by a CYP2C9 inhibitor, sulfaphenazole, and CYP2E1 inhibitors, 4-methylpyrazole and diethyldithiocarbamate. The liver microsomal SOH activity showed significant correlations with tolbutamide hydroxylation (r = 0.569) and chlorzoxazone hydroxylation (r = 0.770) activities, which were the model reactions catalyzed by CYP2C9 and CYP2E1, respectively. Human CYP2C9 and CYP2E1 showed SOH activities at least 2-fold higher than the other P450s. CYP2E1 showed an intrinsic clearance 3-fold greater than CYP2C9. These results demonstrated that CYP2C9 and CYP2E1 were the main P450s involved in human hepatic SOH.
National Research Institute of Chinese Medicine, 155-1, Department of Pharmacology, National Yang-Ming University, 155, Li-Nong Street, Section 2, Taipei, Republic of China, ueng@cma23.nricm.edu.tw
Yune-Fang
YF
Ueng
Chih-Hang
CH
Hsieh
Ming-Jaw
MJ
Don
Chin-Wen
CW
Chi
Li-Kang
LK
Ho
D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000201:enzymology*; D012451:Safrole / Q000378:metabolism*
D065607:Cytochrome P-450 Enzyme Inhibitors; D018736:DNA Adducts; D004791:Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D012451:Safrole