5 ml/kg of dimethoate 40% emulsifiable concentrate lacking cyclohexanone (EC35; Cheminova A/S, Harboøre, Denmark), by gavage all followed by 60 ml of water. The quantity of each compound in each study represented the quantity present in a 2.5 ml/kg dose of agricultural dimethoate EC40. This allowed the results of each study to be compared with the original dimethoate EC40 study. The initial dose of dimethoate EC40 was selected as being towards the middle range of the estimated dose in human self-poisoning Z-VAD-FMK in vivo (bottle sizes 100–400 ml (Eddleston et al., 2005), mean weight of self-poisoned patients 50 kg (Eddleston et al., 2000); likely dose range 0.1 to 8 ml/kg). Dose response studies with a 50% reduction in dimethoate

EC40 dose caused mild poisoning that did not require high doses of noradrenaline (Eddleston et al., manuscript in preparation). The

severe poisoning elicited by 2.5 ml/kg dimethoate EC40 allowed the components of the toxicity to be studied. Noradrenaline was administered to maintain a MAP >55 mmHg, with a target MAP of 65 mmHg. Two hours post-dimethoate (EC or AI) or saline administration, a bolus of pralidoxime chloride (8 mg/kg) was given over 30 min followed by an infusion of 3.5 mg/kg/h until the end of the study. Atropine Screening Library supplier was administered as required to control muscarinic features. The study was ended by euthanasia using pentobarbital or anaesthetic overdose after 12 h. Cardiovascular data were collected 30 and 10 min before poisoning and 15 min intervals thereafter using LiDCO. Arterial blood samples were taken at −40, −10, and 30 min, and then every hour, and lactate analysed using an i-STAT (Abbott, NJ, USA). Analyses for red cell AChE activity were performed as previously described (Worek et al., 1999 and Eddleston et al., 2005). Dimethoate and its active metabolite omethoate were detected by LC-ESI-MS/MS and FI-ESIMS/MS (Eddleston et al., 2005 and John

et al., 2010). Cyclohexanone and cyclohexanol were quantified using a Thermo Scientific Trace gas chromato-graph fitted with an AS2000 autosampler and a flame ionisation detector. Plasma samples were prepared by thawing from −80 °C at room temperature, then 1 ml aliquots were spun in a micro-centrifuge for 5 min at 10,000 rpm to pellet any solid matter. 200 μl of supernatant was added to an autosampler vial containing 20 μl of 2 g/100 ml iso-amyl alcohol (internal standard) in water. One μl volumes Thymidylate synthase of this mixture were injected and analysed using a HP-Innowax 30 m × 0.53 mm × 1 μm film thickness capillary column and the following conditions: injector temperature 240 °C, split ratio 6:1, carrier gas (helium) flow rate 1.8 ml/min, oven temperature programmed between 80 and 200 °C (2 min at 80 °C, then 15 °C/min increase to 200 °C); detector temperature 270 °C with hydrogen and air flow rates of 35 and 350 ml/min, respectively. Cyclohexanol, cyclohexanone and ethanol were quantified using an internal standard method with calibration over the range 0–10 mM.