Neonicotinoid Insecticide Metabolism and Mechanisms of Toxicity in Mammals

Neonicotinoids are the most important class of insecticides. Seven commercial neonicotinoids currently account for approximately 25% of the total insecticide market and are increasing in use as they replace other major classes such as organophosphates and methylcarbamates. Neonicotinoids are extensively metabolized in plants and mammals to produce over 100 metabolites. The overall goal of this study was to better understand three aspects of neonicotinoid metabolism in mice: the importance of aldehyde oxidase (AOX) in vivo, the fate of a new neonicotinoid, cycloxaprid (CYC), and the production of formaldehyde-generating intermediates from the hepatotoxicant, thiamethoxam (TMX).Neonicotinoids are metabolized in vitro by cytochrome P450s (CYPs) via oxidation reactions and by AOX on reduction of the nitroimino group. AOX metabolizes many xenobiotics in vitro but its importance in vivo is unknown relative to CYPs and other detoxification systems. Here we establish the relative importance of AOX and CYPs in vivo in neonicotinoid metabolism using the mouse model. AOX activity was reduced in mice by 45% with tungsten, 61% with hydralazine and 81% in AOX-deficient mice relative to controls and CYP activity was not affected. When mice were treated intraperitoneally with the major neonicotinoid imidacloprid (IMI), metabolism by CYP-oxidation reactions was not appreciably affected whereas the AOX-generated nitrosoguanidine metabolite was decreased by 30% with tungsten, 56% with hydralazine and 86% in the AOX-deficient mice. Another IMI nitroreduction metabolite, desnitro-IMI, was decreased by 55, 65, and 81% with tungsten, hydralazine and in the AOX-deficient mice, respectively. Thus, decreasing liver AOX activity by three quite different procedures gave a corresponding decrease for in vivo reductive metabolites in the liver of IMI-treated mice. Possible AOX involvement in IMI metabolism in insects was evaluated using AOX-expressing and AOX-deficient Drosophila, but no differences were found in IMI nitroreduction or sensitivity between the two strains. This is the first study to establish the in vivo relevance of AOX in neonicotinoid metabolism in mammals and one of the first for xenobiotics in general. The candidate novel insecticide, CYC, has a unique heterocyclic ring system and cis-nitro substituent. Although it is not yet registered for use, it is proposed to control IMI-resistant pests by binding to a different site on the nicotinic acetylcholine receptor. CYC is potentially a proinsecticide, metabolized to the active nitromethylene-imidazole (NMI) analog of IMI. The metabolic pathways of CYC and NMI are unknown. Metabolites in the brain, liver and plasma of CYC- or NMI-treated mice were analyzed by liquid chromatography/ mass spectrometry at 15 and 120 min post-treatment. The major metabolites of CYC were mono- and dihydroxylation products (CYC m/z +16 and +32) and NMI. All metabolites dissipated by 24 h. NMI was metabolized only to a small extent to one hydroxylation and one nitroso product. Although CYC may be a proinsecticide, the major metabolic pathways in mice do not involve high or persistent levels of NMI as an intermediate. Not all neonicotinoid metabolites are detoxification products. TMX, one of the most commonly-used neonicotinoids, is hepatotoxic and hepatocarcinogenic in mice but not rats. Earlier studies established that TMX is a much better substrate for mouse liver microsomal CYPs than the corresponding rat or human enzymes in forming desmethyl-TMX (dm-TMX), which is also hepatotoxic, and clothianidin (CLO), which is not hepatotoxic or hepatocarcinogenic. It was proposed that TMX hepatotoxicity/ hepatocarcinogencity is due to dm-TMX and a further metabolite, desmethyl-CLO (dm-CLO) (structurally analogous to a standard inducible nitric oxide synthase inhibitor), acting synergistically. Here we considered formation of formaldehyde (HCHO) and N-methylol intermediates as an alternative mechanism of TMX hepatotoxicity/ hepatocarcinogenicity. Comparison of neonicotinoid metabolism by mouse, rat and human microsomes with NADPH showed two important points. First, TMX and dm-TMX yield more HCHO than any other commercial neonicotinoid. Second, mouse microsomes give much higher conversion than rat or human microsomes. These observations provide an alternative hypothesis of HCHO and N-methylol intermediates from CYP-mediated oxidative oxadiazinane ring cleavage as the bioactivated hepatotoxicants. However, the proposed mono-N-methylol CYP metabolites are not observed, possibly further reacting in situ. Thoroughly characterizing the metabolism and mechanisms of toxicity of neonicotinoids is important for future pesticide design especially as the demand for and use of these compounds continues to increase.