title: Substrate specificity of rabbit aldehyde oxidase for nitroguanidine and nitromethylene neonicotinoid insecticides.
abstract: The nitroguanidine or nitromethylene moiety of the newest major class of insecticides, the neonicotinoids, is important for potency at insect nicotinic receptors and selectivity relative to mammalian receptors. Aldehyde oxidase (AOX) was recently identified as the imidacloprid nitroreductase of mammalian liver, producing both nitrosoguanidine and aminoguanidine metabolites. The present study considers the ability of AOX, partially purified from rabbit liver, to reduce five commercial nitroguanidine (i.e., imidacloprid, thiamethoxam, clothianidin, and dinotefuran) and nitromethylene (i.e., nitenpyram) neonicotinoid insecticides and three derivatives thereof (i.e., the N-methyl and nitromethylene analogues of imidacloprid and desmethylthiamethoxam). LC/MS/MS was used to demonstrate that AOX reduces nitroguanidines to both nitroso- and aminoguanidines, while nitromethylenes are reduced only to the corresponding nitroso metabolites. Additionally, nitrosonitenpyram was found to spontaneously dehydrate to form a 2-cyanoamidine metabolite, mimicking a predominant photoreaction. The substrate specificity of AOX was characterized as follows: Neonicotinoids with a tertiary nitrogen (N-methylimidacloprid and thiamethoxam) are poor substrates; nitroguanidines are metabolized faster than nitromethylenes; and clothianidin is the most rapidly reduced. Kinetic constants were measured for reduction of three nitroguanidines at two concentrations of AOX. At 2 mg protein/mL, only nitroso metabolites were detected, with Km values of 1.03, 2.99, and 2.41 mM and Vmax values of 5.13, 2.54, and 0.98 nmol/min/mg protein measured for clothianidin, imidacloprid, and dinotefuran, respectively. At 5 mg protein/mL, both amino and nitroso metabolites were detected. However, with each nitroguanidine, the formation of nitroso metabolites did not saturate at substrate levels up to 4 mM, whereas amino metabolite formation exhibited Km values of 0.052, 0.16, and 0.084 mM with corresponding Vmax values of 0.80, 1.24, and 0.79 nmol/min/mg protein for clothianidin, imidacloprid, and dinotefuran, respectively. These in vitro observations show large structural differences in the rates of AOX-catalyzed reduction and help to interpret the extensive studies on in vivo metabolism of neonicotinoid insecticides.
authors.affiliation: Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112, USA.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Dick
authors.forename: David B
authors.initials: DB
authors.lastname: Kanne
authors.forename: John E
authors.initials: JE
authors.lastname: Casida
mesh_terms: D042931:Aldehyde Oxidase / Q000737:chemistry / Q000378:metabolism*; D000691:Anabasine / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D006146:Guanidines / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D000073943:Neonicotinoids; D009574:Nitro Compounds / Q000737:chemistry / Q000378:metabolism*; D009603:Nitroso Compounds / Q000032:analysis / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D010084:Oxidation-Reduction; D011725:Pyridines / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity; D000077922:Thiamethoxam; D013844:Thiazoles
chemical_list: D006146:Guanidines; D007093:Imidazoles; D007306:Insecticides; D000073943:Neonicotinoids; D009574:Nitro Compounds; D009603:Nitroso Compounds; D010078:Oxazines; D011725:Pyridines; D013844:Thiazoles; C465368:dinotefuran; C464843:nitenpyram; C082359:imidacloprid; D000077922:Thiamethoxam; D042931:Aldehyde Oxidase; D000691:Anabasine; C056179:nitroguanidine; C004479:pimagedine