In Vitro Metabolism of the Fungicide and Environmental Contaminant trans-bromuconazole and Implications for Risk Assessment

Abstract

trans-Bromuconazole is a chiral chemical representative of a class of triazole derivatives known to inhibit specific fungal cytochrome P-450 (CYP) reactions. Kinetic measurements and delineation of metabolic pathways for triazole chemicals within in vitro hepatic microsomes are needed for accurate risk assessment and predictive in vivo physiological modeling. The studies described here were conducted with rat liver microsomes to determine Michaelis–Menten saturation kinetic parameters (V _max and K _M) for trans-bromuconazole using both substrate depletion and product formation reaction velocities. Kinetic parameters determined for trans-bromuconazole depletion at varying protein levels incubated at physiological temperature 37°C resulted in a K _M value of 1.69 μM and a V _max value of 1398 pmol/min/mg protein. The concomitant linear formation of two metabolites identified using liquid chromatography/time-of-flight mass spectrometry (LC/MS-TOF) and LC-MS/MS indicated hydroxylation of the trans-bromuconazole dichlorophenyl ring moiety. K _M values determined for the hydroxylated metabolites were 0.87 and 1.03 μM, with V _max values of 449 and 694 pmol/min/mg protein, respectively. Chemical inhibition assays and studies conducted with individual purified human recombinant enzymes indicated the CYP3A subfamily was primarily responsible for biotransformation of the parent substrate. Additionally, trans-bromuconazole was found to undergo stereoselective metabolism as evidenced by a change in the enantiomeric ratio (trans−/trans +) with respect to time.

The authors express appreciation to Tom Sack, Cather Brown, and John Evans and for their assistance in conducting laboratory analyses. In addition, the authors acknowledge Tim Collette for his consultation and expertise. This article has been reviewed in accordance with the U.S. Environmental Protection Agency's peer and administrative review policies and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation of use.

Notes

¹Calculated, (1398 pmol/min/mg∗ 45 mg microsomal protein/g liver)/1.69 nmol/ml = 37.2 nl/min/g liver.

Department for Environment, F. A. R. A., Pesticide Safety Directorate. 1996 .Food and Environment Protection Act, 1985, Part III Issue 160. Control of Pesticide Regulations 1986