Environ. Sci. Technol. 2009, 43, 5507–5513
Mechanistic Approach to Understanding the Toxicity of the Azole Fungicide Triadimefon to a Nontarget Aquatic Insect and Implications for Exposure Assessment J O H N F . K E N N E K E , * ,† CHRISTOPHER S. MAZUR,† K R I S T E N A . K E L L O C K , ‡,§ A N D J A Y P . O V E R M Y E R ‡,| National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia 30605, and Department of Entomology, University of Georgia, Athens, Georgia
Received February 9, 2009. Revised manuscript received May 11, 2009. Accepted May 18, 2009.
Mechanistic and stereoselective based in vitro metabolism assays were utlilized to gain insight into the toxic mode of action of the 1,2,4-triazole fungicide, triadimefon, with black fly (Diptera: Simuliidae) larvae. Based on results from enzyme inhibitor studies, the metabolism of triadimefon in black fly larvae microsomes was found to occur predominantly via an oxidative P450-mediated pathway; triadimenol was formed via the stereoselective reduction of the prochiral carbonyl group of triadimefon. The relatively minor contribution of carbonyl reduction suggests that triadimefon may inhibit ecdysone 20-monooxygenase and disrupt insect molting hormone biosynthesis. 48-h LC50 tests for triadimefon and triadimenol with black fly larvae yielded median values (with 95% confidence intervals) of 6.1 (5.8-6.4) and 22.3 (20.3-24.1) mg/L, respectively. The exposure of black fly larvae to sublethal concentrations of triadimefon resulted in increased microsomal P450 activity and affected the microsomal rates of both triadimefon depletion and triadimenol formation. In contrast to trout, black fly larvae produced a higher fraction of the more toxic triadimenol stereoisomers, which may explain in part why triadimefon exhibited a significantly greater toxicity with black fly larvae than trout. These results illustrate that while LC50 tests conducted with commercial triadimenol would presumably expose each organism to the same relative abundance of the four triadimenol stereoisomers, LC50 tests with triadimefon ultimately expose each organism to a unique set of triadimenol stereoisomers depending upon the organism’s stereoselective metabolism.
Introduction Alterations in enzyme activity, whether due to enzyme induction or inhibition, may result in an organism being * Corresponding author fax: 706-355-8202; e-mail: kenneke.john@ epa.gov. † U.S. Environmental Protection Agency. ‡ University of Georgia. § Currently with the School of Forestry and Natural Resources, University of Georgia, Athens, GA. | Currently with Syngenta Crop Protection, Greensboro, NC. 10.1021/es900351w CCC: $40.75
Published on Web 06/09/2009
2009 American Chemical Society
more susceptible to other toxic chemical contaminants or, conversely, may provide a means of enhanced detoxification (e.g., chemical resistance) (1, 2). Changes in enzyme activity may also lead to changes in endogenous biochemical pathways. For example, specific insect cytochrome P450 monooxygenases (P450) involved in xenobiotic metabolism are also key enzymes in the biosynthesis of ecdysteroids and juvenile hormones, which are critical for insect growth, development and reproduction (3); changes in P450 levels or inhibition of these enzymes can adversely affect these essential biochemical processes. P450s are found in nearly all aerobic organisms including insects, plants, mammals, and microbes. While information on P450-mediated oxidative pathways for xenobiotic metabolism is well established, the roles of specific enzymes involved in reductive pathways are far less understood. Carbonyl reduction is a major biotransformation step in the metabolism of endogenous and exogenous compounds in both prokaryotes and eukaryotes. In invertebrates, the primary function of carbonyl reducing enzymes is to convert lipid hormones to their corresponding alcohols. In several cases, these enzymes have also been shown to participate in xenobiotic metabolism (4-6). Recently, 1,2,4-triazole and imidazole analogs of metyrapone were developed as selective larvicides whose mode of action is inhibition of the P450 enzyme, ecdysone 20monooxygenase (E-20-M). E-20-M catalyzes the 20-hydroxylation of the insect steroid ecdysone to yield the molting hormone, 20-hydroxyecdysone (4-6). Insecticidal activity of the metyrapone analogs is influenced by the presence of a carbonyl group, which was found to readily undergo reduction in microsomes from vertebrate and invertebrate species but not insects (6). Carbonyl reduction of these analogs in vertebrates is a significant pathway since it results in less cytotoxic and more easily excretable alcohol-containing metabolites and thus provides a route for the development of more selective and safer insecticides (i.e., nontarget vertebrate species such as humans can metabolize and detoxify the insecticide while insects cannot) (4). The 1,2,4-triazole fungicide, triadimefon, is structurally similar to many of the insecticidal metyrapone azole analogs (Figure 1), which begs the question as to whether or not triadimefon possesses any insecticidal activity. Here, we investigate the potential insecticidal activity of triadimefon using larval black fly (Diptera: Simuliidae). Results from sublethal exposures and in vitro metabolism assays are used to delineate the role of P450 oxidizing and non-P450 carbonyl reducing enzymes and develop a mechanistic-based understanding of triadimefon’s mode of toxicity.
Materials and Methods Reagents. Triadimefon and triadimenol were obtained from the U.S. Environmental Protection Agency National Pesticide Standard Repository (Fort Meade, MD). β-Nicotinamide adenine dinucleotide 2′-phosphate (NADP), glucose-6phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), magnesium chloride (MgCl2), phosphate buffer (pH 7.4), glycyrrhetinic acid, and 1-aminobenzotriazole (ABT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals were analytical grade or better and used as received. Black Flies. Fourth-fifth instar black fly larvae, S. vittatum (Zetterstedt) cytospecies IS-7, were obtained from the University of Georgia (Athens, GA, USA) colony and were reared following the protocols of Gray and Noblet (7). VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Reduction of the prochiral carbonyl in triadimefon and resulting four triadimenol stereoisomers. Metyrapone and its insecticidal azole analogs (4) are shown for comparison. Toxicity Tests and Sublethal Exposures. The toxicity of triadimefon and triadimenol to S. vittatum IS-7 was assessed using a 48-h orbital shaker toxicity test (8) with slight modifications (9). Fifteen fourth-fifth instar larvae were transferred with larval forceps into 250-mL flat bottom extraction flasks containing 145 mL of test water and were placed on a New Brunswick Scientific G-10 Gyratory shaker (Edison, NJ, USA). Five flasks were used per concentration and included a test-water and acetone control; each of the fungicide concentrations was replicated three times. Tests were conducted at 20 ( 0.5 °C and a 16:8 h light:dark photoperiod. Results were considered valid if control mortality was
