Insecticides such as the organophosphorus inhibitors of acetylcholinesterase (AChE) can induce the development of tolerance to their toxicity as seen in a marked decrease and disappearance of signs of toxicity during chronic exposure. Their toxic actions can be modified through interaction with enzymes such as the carboxylesterase EC 3.1.1.1. (CarbE) which act as scavenger, reducing the toxic concentrations of inhibitors. Recent observations in rats treated with diisopropylphosphorofluoridate (DFP) suggested for rodents a resistant mechanism based on altered AChE and increased CarbE. It is postulated that the alterations of AChE associated with resistance (1) cause changes in the kinetics of inhibition with insecticides, (2) have no significant effect on the hydrolysis of the substrate acetylcholine (ACh) , (3) that the contribution of CarbE to the DFP resistance can be quantified by use of a specific inhibitor of CarbE, and (4) that the resistant enzymes are due to de novo synthesis. To test these inter-related hypotheses in rats tolerant to DFP, we will determine (1) the kinetic parameters such as Km and V-max for acetylcholine hydrolysis, (2) the kinetic constants for enzyme inhibition such as bimolecular rate constants (k-i) , as well as affinity (K-a) and phosphorylation (k-p) constants for inhibition of AChE, (3) we will pretreat rats with a CarbE inhibitor before receiving DFP in order to delineate the role of AChE from CarbE, and (4) we will study whether changes in AChE synthesis are due to changes in the rate of synthesis and/or stability of AChE mRNA. Rats will be injected daily for up to 4 weeks with DFP (0.5-0.25 mg/kg) causing tolerance with loss of signs of toxicity. Other rats will be pretreated with CBDP, an inhibitor of CarbE (2 mg/kg) 30 min before the organophosphate. Kinetic parameters of AChE and CarbE will be tested 24 hours after the first, fifth, fourteenth, and twenty-eighth injections. A better understanding of mechanisms causing resistance leads to possible management and is most likely to reduce the risk due to increased insecticide use.
| Dettbarn, W D; Yang, Z P; Milatovic, D (1999) Different role of carboxylesterases in toxicity and tolerance to paraoxon and DFP. Chem Biol Interact 119-120:445-54 |
| Milatovic, D; Dettbarn, W D (1996) Modification of acetylcholinesterase during adaptation to chronic, subacute paraoxon application in rat. Toxicol Appl Pharmacol 136:20-8 |
