Identification of enzymes involved in the metabolism of atrazine, terbuthylazine, ametryne, and terbutryne in human liver microsomes.
Compounds of the s-triazine family are among the most heavily used herbicides over the last 30 years. Some of these derivatives are suspected to be carcinogens. In this study the identity of specific phase-I enzymes involved in the metabolism of s-triazine derivatives (atrazine, terbuthylazine, ametryne, and terbutryne) by human liver microsomes was determined. Kinetic studies demonstrated biphasic kinetics for all pathways examined (S-oxidation, N-dealkylation, and side-chain C-oxidation). Low K(m) values were in a range of about 1-20 microM, whereas high K(m) values were up to 2 orders of magnitude higher. For a correlation study, 30 human liver microsomal preparations were screened for seven specific P450 activities, and these were compared to activities for the metabolites derived from these s-triazines. A highly significant correlation in the high-affinity concentration range was seen with cytochrome P450 1A2 activities. Chemical inhibition was most effective with alpha-naphthoflavone and furafylline at low s-triazine concentrations and additionally with ketoconazole and gestodene at high substrate concentrations. Studies with 10 heterologously expressed P450 forms demonstrated that several P450 enzymes are capable of oxidizing these s-triazines, with different affinities and regioselectivities. P450 1A2 was confirmed to be the low-K(m) P450 enzyme involved in the metabolism of these s-triazines. A potential participation of flavin-containing monooxygenases (FMOs) in sulfoxidation reactions of the thiomethyl derivatives ametryne and terbutryne in human liver was also evaluated. Sulfoxide formation in human liver microsomes as a function of pH, heat, and chemical inhibition indicated no significant involvement of FMOs. Finally, purified recombinant FMO3, the major FMO in human liver, exhibited no significant activity (< 0.1 nmol (nmol of FMO3)-1 min-1) in the formation of the parent sulfoxides of ametryne and terbutryne. Therefore, P450 1A2 alone is likely to be responsible for the hepatic oxidative phase-I metabolism of the s-triazine derivatives in exposed humans.
Lang
D H
DH
Department of Medicinal Chemistry, University of Washington, Seattle 98195, USA. dilang@u.washington.edu
Rettie
A E
AE
Böcker
R H
RH
D000328:Adult; D000368:Aged; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism; D006540:Herbicides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008862:Microsomes, Liver / Q000737:chemistry / Q000201:enzymology*; D008875:Middle Aged; D014227:Triazines
D006540:Herbicides; D007527:Isoenzymes; D014227:Triazines; D003577:Cytochrome P-450 Enzyme System