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Chem. Res. Toxicol. 1997, 10, 64-69
Acephate Insecticide Toxicity: Safety Conferred by Inhibition of the Bioactivating Carboxyamidase by the Metabolite Methamidophos Mahmoud Mahajna, Gary B. Quistad, and John E. Casida* Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720-3112 Received August 12, 1996X
Acephate is an important systemic organophosphorus insecticide with toxicity attributed to bioactivation on metabolic conversion to methamidophos (or an oxidized metabolite thereof) which acts as an acetylcholinesterase (AChE) inhibitor. The selective toxicity of acephate is considered to be due to facile conversion to methamidophos in insects but not mammals. We show in the present investigation that a carboxyamidase activates acephate in mice and in turn undergoes inhibition by the hydrolysis product, i.e., methamidophos; thus, the bioactivation is started but immediately turned off. These relationships are established by finding that 4 h pretreatment of mice with methamidophos ip at 5 mg/kg has the following effects on acephate action: reduces methamidophos and acephate levels in liver by 30-60% in the first 2 h after ip acephate dosage; inhibits the liver carboxyamidase cleaving [14CH3S]acephate to [14CH3S]methamidiphos with 50% block at ∼1 mg/kg; strongly inhibits 14CO2 liberation from [CH314C(O)]acephate in vivo; markedly alters the pattern of urinary metabolites of acephate by increasing O- and S-demethylation products retaining the carboxyamide moiety; greatly reduces the brain AChE inhibition following acephate treatment; doubles the LD50 of ip-administered acephate from 540 to 1140 mg/kg. Methamidophos pretreatment in rats also markedly alters the metabolism of dimethoate (another systemic insecticide) from principally carboxyamide hydrolysis to mainly other pathways. In contrast, methamidophos pretreatment of houseflies does not alter the acephate-induced toxicity and brain AChE inhibition. The safety of acephate in mammals therefore appears to be due to conversion in small part to methamidophos which, acting directly or as a metabolite, is a potent carboxyamidase inhibitor, thereby blocking further activation.
Introduction Acephate is one of the ten most important organophosphorus (OP)1 insecticides in sales volume (1) and is considered to be the safest of the plant systemics with an acute oral LD50 of about 360 and 900 mg/kg for mice and rats, respectively (2). The action of acephate on insects and its selective toxicity are attributed to more rapid bioactivation on conversion to methamidophos in insects than in mammals, offering a measure of safety (3, 4). Consistent with this proposal, biological monitoring of human exposure to acephate shows the unchanged compound but not methamidophos in the urine (5). The high acute toxicity of methamidophos (mouse and rat oral LD50 of 14-30 mg/kg) is associated with typical signs of cholinergic poisoning (1-4). Although a weak inhibitor in vitro, the poisoning by methamidophos is attributed to in vivo inhibition of acetylcholinesterase (AChE) activity (6-9). One proposal for this apparent anomaly is that, due to persistence, it slowly produces symptoms of poisoning in both rodents (7) and humans (10). An alternative proposal invokes bioactivation involving Soxidation (11, 12), but this has controversial aspects (13, 14). * To whom correspondence should be addressed at the Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 114 Wellman Hall, University of California, Berkeley, CA 94720-3112. Phone: (510) 6425424; FAX: (510) 642-6497; e-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, December 15, 1996. 1 Abbreviations: AChE, acetylcholinesterase; CI, chemical ionization; I50, concentration for 50% inhibition; LSC, liquid scintillation counting; OP, organophosphorus; SIM, selected ion monitoring.
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The present study introduces a new hypothesis for the selective toxicity of acephate (Figure 1). The first step is the widely-accepted proposal that acephate is bioactivated by the action of a carboxyamidase, yielding methamidophos as its product (3, 4); this carboxyamidase has never been directly demonstrated. The second step is the novel hypothesis that methamidophos in turn acts not only as an AChE inhibitor but also as a carboxyamidase inhibitor, blocking the enzyme involved in its formation and thereby quickly turning off the bioactivation process by shifting to alternative detoxication mechanisms that protect against poisoning.
Experimental Procedures Spectroscopy and Chromatography. NMR spectra were recorded in D2O with a Bruker WM-300 spectrometer at 300 MHz using internal tetramethylsilane for 1H and at 121.5 MHz using external trimethyl phosphate for 31P. GC/MS/chemical ionization (CI) with selected ion monitoring (SIM) involved: a Hewlett Packard 5890 gas chromatograph coupled to a 5971A mass spectrometer; a DB-5 fused-silica capillary column, 30 m × 0.25 mm i.d. (J and W Scientific, Folsom, CA); injection port temperature 250 °C; temperature program 70-250 °C over 18 min. HPLC utilized a Merck LiChrospher 100 RP-18 (5-µm) reverse-phase column with a Waters Model 600E solvent delivery system coupled to a Waters Model 994 photodiode array detector: 0.1% trifluoroacetic acid for 5 min, then a linear gradient of 0-60% methanol in water with constant 0.1% trifluoroacetic acid over 20 min, and finally a linear gradient to
© 1997 American Chemical Society
Inhibition of Acephate Bioactivation
Figure 1. Hypothesis for the metabolic activation and detoxication of acephate. Methamidophos S-oxide is proposed but not established as an intermediate. 100% methanol over an additional 10 min, each at 1.5 mL/min (monitoring at 220 nm). Organophosphorus Compounds. Caution: Some of the OP compounds considered are known to be toxic and must be used under containment conditions. The purity of the OPs was determined by 31P NMR and GC/MS/CI. Acephate and methamidophos of >97% purity were obtained from Chevron Chemical (Richmond, CA). High-purity acephate (>99%) was obtained by repeated recrystallization from ethyl acetate-ether (1:1) (15). [14CH3S]Acephate (52.1 mCi/mmol) and [CH314C(O)]acephate (37.7 mCi/mmol) were supplied by Allen Rose of Valent USA (Walnut Creek, CA) with >97% purity as used. Three candidate acephate metabolites were prepared as standards. NaO(CH3S)P(O)NHC(O)CH3 was obtained almost quantitatively as a precipitate by treating acephate with NaI (1.5 molar equiv) in refluxing dry acetone for 4 h. NMR (ppm): 1H δ 2.18 (3H, d, J ) 14.4 Hz, SCH3), 2.06 (3H, s, C(O)CH3); 31P δ 12.3. To confirm the identity, acidification of the salt followed by esterification with diazomethane gave acephate characterized by GC/MS/CI. To prepare CH3O(HO)P(O)NHC(O)CH3, acephate in D2O was stirred with a large excess of dimethyldioxirane in acetone for 24 h followed by evaporating the acetone. NMR (ppm): 1H δ 3.72 (3H, d, J ) 11.8 Hz, OCH3), 2.08 (3H, s, C(O)CH3); 31P δ -5.9. For identification by GC/MS/CI, the demethylthio product was treated with diazomethane to give (CH3O)2P(O)NHC(O)CH3 (MH+ ) 168). CH3O(CH3S)P(O)ONa was prepared by demethylation of CH3O(CH3S)P(O)OCH3 with NaI as above (16). Dimethoate (>99% pure) was obtained from Chem Service (West Chester, PA). Mice and Treatment Protocols. Male albino SwissWebster mice (18-23 g) from Simonsen Laboratories (Gilroy, CA) were treated ip with the test compounds using water (50 µL) as the carrier vehicle. Acephate was administered alone or 4 h after methamidophos to determine the effect of methamidophos on acephate distribution, metabolism, or toxicity. The mice were sacrificed by cervical dislocation and dissected to obtain the liver or brain for analysis. Extraction and Analysis of Liver. Fresh samples of liver (1 g portions) from individual mice were extracted with 25 mL of ethyl acetate for 5 min in a polytron homogenizer in the presence of 2 mL of water and 15 g of anhydrous Na2SO4. This process was repeated two more times, and the combined extracts were filtered through anhydrous Na2SO4 and evaporated to dryness under vacuum. The residue was taken up in 0.5 mL of acetone and analyzed immediately by GC/MS/CI/SIM. The ions selected for monitoring acephate were 184 and 143 and that for methamidophos was 142. The analysis involved a 1-µL aliquot injected with the Hewlett-Packard 7673A automatic sample injector. tR values for acephate and methamidophos were 13.2 and 10.3 min, respectively. Quantitation involved comparison to standard curves prepared with the authentic compounds using trimethyl phosphate as the internal standard.
Chem. Res. Toxicol., Vol. 10, No. 1, 1997 65 Acephate-Hydrolyzing Activity of Mouse Liver Preparations. Liver from control mice, or mice treated 4 h earlier with methamidophos, was homogenized at 30% (w/v) in 100 mM sodium phosphate buffer (pH 7.4) at 4 °C. The microsome-pluscytosol fraction was obtained as the supernatant by centrifuging at 10000g for 10 min at 4 °C. Further centrifugation of the supernatant at 105000g for 60 min yielded the cytosol and the microsomes. The microsomes were washed once with cold buffer and resuspended to the original volume before use. Protein was determined by the Bradford method (17). Incubation mixtures in 100 mM sodium phosphate buffer (pH 7.4) contained 0.3 mL of the liver fraction (15-90 mg of fresh liver weight equivalent) and [14CH3S]acephate (1.2 nmol added in 0.1 mL of buffer as above). Controls consisted of the heatdenatured supernatant fraction (85 °C, 15 min) or addition of tetraethyl pyrophosphate at 20 µM final concentration as an inhibitor. After incubation for 60 min, the solutions were passed through micro-spin filters (0.45 µm nylon, Lida Manufacturing, Kenosha, WI), and 100-µL aliquots of the filtrates were analyzed by adding unlabeled acephate and methamidophos (20 µg each) and HPLC as above, collecting the acephate and methamidophos fractions (tR 4.4 and 9.8 min, respectively) evident by monitoring at 220 nm, followed by liquid scintillation counting (LSC). [14CH3S]Methamidophos was the only labeled product formed in these incubations. No [14CH3S]methamidophos was present in the [14CH3S]acephate used as the substrate, but correction was made for the spontaneous hydrolysis of 3.9 pmol/h under the assay conditions. Urinary and Expired Radiocarbon from [CH314C(O)]Acephate in Mice. Mice were treated with methamidophos (0 or 5 mg/kg) and 4 h later with [CH314C(O)]acephate by the ip route at 25 µg/kg. They were immediately placed in all-glass metabolism chambers for collection of urine and expired gasses for 24 h. The air from the chamber was passed through 2-methoxyethanol and then into a 2-methoxyethanol/ethanolamine (2:1) trap for 14CO2 collection. Analysis by LSC involved 50-µL aliquots of urine and 14CO2 trapping solution. Urinary Metabolites of Acephate and Dimethoate. Mice were treated ip with acephate at 100-400 mg/kg alone or 4 h following a methamidophos dose of 5 mg/kg. On an analogous basis, Sprague-Dawley rats (100-120 g; Simonsen Laboratories) were treated ip with dimethoate at 100 mg/kg alone or following methamidophos (7 mg/kg, 4 h pretreatment) using Me2SO (50 µL) as the carrier vehicle. The 0-24 h urine in each case was filtered and lyophilized, and the viscous liquid obtained was dissolved in D2O (0.5 mL) (16). Phosphorus-containing urinary products were analyzed by 31P NMR with spectral acquisition for 24 h, and their ratio was determined by integration of the signals. Each metabolite was identified by addition of the authentic standard, resulting in an enhanced single signal in the appropriate position. Phosphoric acid is the only signal evident by 31P NMR examination of urine from control mice and rats. AChE Inhibition in Vivo. Brains from mice treated ip with acephate at 450-900 mg/kg, alone or 4 h after methamidophos at 5 mg/kg, were homogenized at 1% (w/v) in 100 mM sodium phosphate buffer (pH 7.4) for assay of AChE activity (18). Brain homogenates were incubated with acetylthiocholine and 5,5′dithiobis(2-nitrobenzoic acid) for 20 min; then eserine sulfate (500 µM) was added to stop further enzymatic hydrolysis and the yellow 5-thio-2-nitrobenzoate was measured at 412 nm with the Hewlett-Packard 8452A diode array spectrophotometer. For houseflies (see below), heads were homogenized at 3% (w/v) in 100 mM sodium phosphate buffer (pH 7.4) and assayed for AChE activity in the same way. Toxicity in Mice and Houseflies. Mice as above were treated ip for LD50 determinations 24 h later with 5 mice per dose. Adult female houseflies (Musca domestica L., SCR strain cultured in this laboratory, ∼20 mg each) were treated with acephate (alone or with 4-h methamidophos pretreatment) using acetone (0.2 µL) applied as a measured drop to the ventrum of the abdomen. LD50s or percentage mortality was determined after 24 h at 25 °C.
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Figure 3. Acephate-hydrolyzing activity of mouse liver carboxyamidase(s) in microsome-plus-cytosol fraction and its inhibition by methamidophos in vivo. (A) Effect of protein level from untreated mice assayed alone (normal) or following heat denaturation or treatment with 10 µM tetraethyl pyrophosphate (controls) and from mice pretreated with methamidophos (4 h, 5 mg/kg). (B) Effect of methamidophos dose with 4 h pretreatment assaying at 90 mg fresh liver weight equivalent. Table 1. Acephate-Hydrolyzing Activity of Mouse Liver Carboxyamidase(s) in Microsome and Cytosol Fractions and Its Inhibition by Methamidophos in Vivo hydrolysisa
Figure 2. Acephate and methamidophos levels in liver of mice as a function of time and dose after ip treatment with acephate alone or following methamidophos (5 mg/kg, 4 h pretreatment). (A) Effect of time with acephate at 450 mg/kg. (B) Effect of dose with time at 120 min.
Results Acephate and Methamidophos Levels in Liver of Mice as a Function of Time and Dose after Treatment with Acephate Alone or Acephate following Methamidophos. Acephate and methamidophos are the only acephate-derived products detected by GC/MS/ CI/SIM in the ethyl acetate extracts of the liver of mice dosed only with acephate. On treatment with acephate at 450 mg/kg, the levels of both acephate and methamidophos drop progressively with time, and at 60 and 120 min the acephate:methamidophos ratio is about 18:1 (Figure 2A). Methamidophos treatment alone (5 mg/kg, ip) does not yield detectable levels in liver (
