Comparative metabolism of Bensulide by Soil fungus Cunninghamella elegans and Human liver microsome

Abstract

Bensulide [O-O-diisopropyl S-2-phenylsulfonylaminoethyl phosphoro -dithioate] is an organophosphorous herbicide, acting as a inhibitor of cell division in meristematic root tissues and seedling growth by conjugation of acetyl co-enzyme A. This study was performed to investigate in vitro metabolism of bensulide with soil fungi, Cunninghamella elegans(Cunn. elegans), human liver microsomes(HLMs) and characterize the specific isoforms of cytochrome P450 involved in metabolic reaction.

In the presence of Cunn. elegans, a well-known fungal species with its strong resemblance of the xenobiotic metabolism of the mammalian system, bensulide was biodegraded to its oxygen analog, bensulide oxon (N-[(2-(diisopropoxyphosphinoylthio-)-1-ethyl)]-benzenesulonamiade). In sterilized Cunn. elegans, the metabolite did not form, indicating that Cunn. elegans metabolized bensulide. Bensulide degradation pattern showed that day 1 showed 5% of degradation, day 3, 89%, day 5, 99% and by day 7 bensulide in all three replicates degraded completely. By day 3 metabolite was detected.

With presense of NADPH, bensulide was metabolized by HLMs to give identical metabolite as microbial degradation, its oxygen analog, bensulide oxon. With boiled-denatured microsomes the metabolite did not form but with heat-denatured microsomes, the metabolite did form, indicating that metabolic enzymes are cytochrome P450s. In enzyme kinetic studies, Vmax(counts/min/mg protein) of 18.5, Km of 11.7 were obtained. A screen of 9 human cDNA-expressed CYP isoforms for metabolic ability with respect to the production of bensulide oxon demonstrated that 2 (CYP 3A4, CYP 2C19) CYP isoforms which are responsible for bensulide metabolism. Enzyme kinetics of those two CYP isoforms also demonstrated that CYP 3A4 has the highest affinity to bensulide.