Biotransformation of Metamitron by Human P450 Expressed in Transgenic Tobacco Cell Cultures

In the present investigation, the oxidative metabolism of ¹⁴C-labeled metamitron was examined in plant cell cultures of tobacco overexpressing human P450 enzymes CYP1A1 or CYP1A2; special interest was in the aromatic hydroxylation of the herbicide. The oxidative metabolites deaminometamitron (DAM) and 4-hydroxydeaminometamitron (4-HDAM) were found in the untransformed control culture as well as in the transgenic culture. The transgenic cultures, however, exhibited higher turnover rates after 48 h of incubation with 20 μg ¹⁴C-metamitron per assay (untransformed: 40%, CYP1A1: 80%, CYP1A2: 100%). Primary metabolite 4-HDAM was partially found in glucosylated form in the transgenic cultures. As minor oxidative metabolites, 6-hydroxyphenyl-3-methoxymethyl-1,2,4-triazine-5(4H)-one and 3-hydroxymethyl-6-phenyl-1,2,4-triazine-5(4H)-one were identified in the transgenic cultures by GC-MS, LC-MS. Additionally, it could be demonstrated that both foreign enzymes (CYP1A1, CYP1A2) also catalyzed the deamination of metamitron. In a large-scale study (up to 400 μ g per assay) with the transgenic culture expressing CYP1A2, the high efficiency of this P450 system toward metamitron was demonstrated: turnover of the xenobiotic was almost complete with 400 μ g. Since large portions of unglucosylated 4-H-DAM were found, the activity of foreign CYP1A2 apparently exceeded that of endogenous O-glucosyltransferases of the tobacco cell culture. We concluded that in comparison to the nontransformed cell culture, the extent of metabolism was considerably higher in the transgenic cultures. The transgenic cell cultures expressing human CYP1A1 or CYP1A2 are thus suitable tools for the production of large quantities of primary oxidized metabolites of metamitron.

Key Words:

• Transgenic plant cell cultures
• Tobacco
• Human CYP1A1 and CYP1A2
• Metamitron
• Metabolism
• Oxidative metabolites

ACKNOWLEDGMENTS

The authors wish to thank Bayer CropScience AG, Monheim (formerly Bayer AG GB Pflanzenschutz) for the support with the radiolabeled substances, Dr. J. Hollender (Institute of Hygiene and Environmental Medicine, University Hospital Aachen, Medical Faculty of RWTH Aachen University) for support concerning LC-MS, Professor Dr. F. P. Guengerich (Vanderbilt University School of Medicine, Nashville, TN, USA) for providing cDNA of human CYP1A1 and CYP1A2, and H. Patti for technical assistance.

Notes

^a Fresh weight of cell culture at end of experiment.

^b Nonextractable residues.

^c Percentages of recovered metamitron were calculated from TLC data of media and cell extracts.

^d Two replicates.

^a Five replicates.

^a n.d. = not detectable.