Chlorpyrifos cholinesterase inhibition

The purpose of this chapter is not to discuss the merits, or lack thereof, of using plasma cholinesterase inhibition as an adverse effect in quantitative risk assessments for chlorpyrifos or other organophosphate pesticides. A number of regulatory agencies consider the inhibition of plasma cholinesterase to be an indicator of exposure, not of toxicity. The U.S. Environmental Protection Agency, at this point, continues to use this effect as the basis for calculating the reference doses for chlorpyrifos, and it is thus used here for assessing risks. 

Based on the data from controlled human studies, the NOEL for plasma cholinesterase inhibition for a single dose of chlorpyrifos is between 0.1 and 0.5 mg/kg bw/day, and the more conservative 0.1 mg/kg bw/day (100 pg/kg bw/day) is used in this assessment as the acute NOEL for chlorpyrifos. The repeated dose NOEL in humans is 0.03 mg/kg bw/day (30 pg/kg bw/day), based on plasma cholinesterase activity, and this is the basis for the establishment of the reference dose of 0.003 mg/kg bw/day (3 pg/kg bw/day) used by the EPA in assessing dietary risk to chlorpyrifos. For the work described here, both NOELs are used as bases for assessing risks to persons who have the potential for non-dietary exposure to chlorpyrifos. For exposures that are infrequent or of short duration, the 100 pg/kg bw/day NOEL is assumed to be the more appropriate value, and the lower 30 pg/kg bw/day will be used in those situations in which exposure may be considered to be more frequent. ... 

A recent publication on PBTK/TD modeling to determine dosimetry and cholinesterase inhibition for a chemical mixture of 2 organophosphorus insecticides, chlo-rpyrifos and diazinon (Timchalk and Poet 2008), deserves some special discussion here. Based on the individual PBTK/TD models developed earlier by the same laboratory, Timchalk and Poet (2008) reported their development of a binary interaction PBTK/TD model for chlorpyrifos and diazinon. In their development of the model, Timchalk and Poet (2008) took into consideration a number of important metabolic steps (CYP450 mediated activation and detoxification of a number of esterases—B-... 

Timchalk C, Poet TS. 2008. Development of a physiologically based pharmacokinetic and pharmacodynamic model to determine dosimetry and cholinesterase inhibition for a binary mixture of chlorpyrifos and diazinon in the rat. Neurotoxicology 29 428 443. 

Cholinesterase inhibition can sometimes persist for weeks thus, repeated exposures to small amounts of this material may result in accumulation of acetylcholinesterase inhibition with possible sudden-onset acute toxicity. Chlorpyrifos may be capable of causing organophosphate-induced delayed neurotoxicity in humans a massive overdose resulted in signs characteristic of delayed neurotoxicity. Animal studies generally indicate, however, that doses several times higher than the LD50 would be required to initiate delayed neurotoxicity. 

Model refinement and validation for both the chltnpyrifos and the diazinon PBPK/PD models wa.s accomplished by conducting a scries of in vivo pharmacokinetic and pharmacodynamic studies in the rat and by evaluating the capability of the model to accurately simulate in vivo data published in the literature. The experimental details are fully described in Timchalk et ai (2002b) and Poet et at. (2004). In brief, these studies involved an acute oral exposure to chlorpyrifos or diazinon and the blood time course of the parent compounds and metabolites was determined, as well as the time course for the cholinesterase inhibition in several tissues. Representative results and model simulations are presented in Fig. 12 and 13 for the pharmacokinetic and pharmacodynamic response in rats following comparable oral doses (50 and 100 mg/kg) of chlorpyrifos and diazinon, respectively, The overall response was fairly comparable for these two insecticides, and the models reasonably simulated both dosimetry and the dose-dependent cholinesterase inhibition. These results arc very consistent with a fairly rapid oral absorption for both insecticides and subsequent metabolism and distribution of the active oxon metabolites. Figure 14 illustrates the capability of the diazinon PBPK/PD model to simulate rodent dosimetry data from the open literature and the capability of the model to accommodate alternative exposure routes (Poet et ai, 2004). In these examples, the time course of diazinon in plasma and cholinesterase inhibition in tissues (i.e.. blood,... 

Age-related differences in kinetic parameters of detoxification can partially explain the increased sensitivity of the young to acute exposure to chlorpyrifos and other OPs (Mortensen et ai, 1996 Moser et ai, 1998 Padilla et ai, 2000, 2004). Thus, in vitro assays show that differences in vivo are not due to intrinsic differences in sensitivity of the target enzyme (Mortensen el ai, 1998). Furthermore, differences in liver microsomal metabolism, which mediates activation and/or inactivation of some pesticides, do not adequately explain the increased sensitivity (Benke and Murphy, 1975 Brodeur and DuBoks, 1%7). Differences in detoxification pathways correlate better with age sensitivity. B-esterases (e.g., carboxylesterases) and A-esterase.s bind to and/or hydrolyze, and thus detoxify, some cholinesterase-inhibiting pesticides (Jokanovic et al., 1996 Maxwell, 1992). These pathways are much less well developed in the young, and maturation of these systems tracks the decreasing sensitivity to acute exposure to chlorpyrifos and other OPs (Attcrberry el ai, 1997 Benke and Murphy, 1975 Brodeur and DuBois, 1967 Chanda et ai, 1997, 2002 Mendoza, 1976 Mortensen et ai, 1996, 1998 ... 

No.strandt, A, C., Padilla, S and Moser, V, C, (1997), The relation-.ship of oral chlorpyrifos effects on behavior, cholinesterase inhibition, and muscarinic receptor density in rat. Pharmacol. Biochem. Behav. 58, 15-23. 

The effects of chlorpyrifos were also compared with other cholinesterase inhibitors using equivalent concentrations (30 p,M) of each compound demonstrated that both diazinon (an OP) and physostigmine (a CM) caused significant inhibition of DNA synthesis in C6 cells. In PC-12 cell.s, diazinon caused a significant decrement, although it was smaller than the effect of chlorpyrifos while physostigmine was Ineffective. These re,sults are consistent with those of previou.s studies in which it was demonstrated that chlorpyrifos exerts antimitotic actions on developing neural ceils independently of cholinesterase inhibition (Das and Barone, 1999 Pope, 1999 Slotkin, 1999). It was also observed that chlorpyrifos was more effective than chlorpyrifos-oxon, despite the fact that the latter is a far more potent cholinesterase inhibitor (Slotkin, 1999). 

A recent study of Moser eoworkers (2005) Mo,ser et uL (2005), using a do.se-additive design with mixtures of five commonly used OP pesticides (chlorpyrifos, diazinon, dimeihoate, acephaic, and malathion), showed a more-than-additive interaction on multiple end points blood and brain cholinesterase inhibition, motor activity, and gait score (tail-pinch response did not. show a more-than-additive interaction). This study is noteworthy because (i) relatively sensitive end points were used to test the toxic interaction of the OP pesticides, such as cholinesterase inhibition or depression of motor activity (ii) more than two OP compounds were used in the mixture and (iii) comprehensive statistical analyses of the data were performed. The pharmacokinetic interaction of two of the aimpounds in the mixture, chlorpyrifos and diazinon, has been studied in rats (Timchalk et a ., 2004). The authors found that one compound did not affect the pharmacokinetics of the other unless high doses were given, concluding that a more-than-additive interaction is unlikely at environmentally relevant concentrations. 

The UK Pesticide Safety Directorate (PSD) has decided to use the TEF approach for assessment of combined risk from exposure to mixtures of acetyl cholinesterase inhibitors (organophosphate (OP) compounds and carbamates) (PSD 1999). Despite clear differences in the action of carbamates and OP compounds, the index compounds selected for all acetyl cholinesterase inhibitors were either aldicarb (carbamate) or chlorpyrifos (OP). The POD for determining relative potency was predetermined as the dose level that produced 20% inhibition of red blood cell cholinesterase in a 90-day dietary study in rats. 

Richardson, J.R., Chambers, H.W. and Chambers, J.E. (2001) Analysis of the additivity of in vitro inhibition of cholinesterase by mixtures of chlorpyrifos-oxon and azinphos-methyl-oxon. Toxicology and Applied Pharmacology, 172, 128-139. 

Diazinon, malathion, parathion, chlorpyrifos, dichlorvos irreversible inhibition of red blood cell cholinesterase, acetylcholinestera.se, plasma cholinesterase Mild fatigue, headache, blurred vision, diz/.ines.s, numbness of extremities, nausea, vomiting, excessive. sweating and salivation, tightness in chest Moderate weaknc.ss, difficulty talking, muscular fasciculations, miosis Severe unconsciousness, flaccid paralysis, moist rales, respiratory difficulty, cardiac arrhythmias, cyanosis... 

The biomolecular inhibition rate constant (ifj) describes both the affinity and the rate of cholinesterase phosphorylation and is an indicator of inhibitory potency (Kousba et al 2004 Kardos and Sultatos, 2000 Amitai et al., 1998 Carr and Chambers, 1996). A typical determination is illustrated in Fig. 10 for the in vitro inhibition of rat BuChE with chlorpyrifos-oxon. In this example, the Kj was determined by incubating BuChE with varying concentrations of ehloropyrifos-oxon (0.25-5 nAf) the maximum inhibition ranged from 10 to 90% during a 7- to 30-min incubation period (Kousba et al., 2003). The slopes obtained from this analy.sis were then analyzed by linear regression to calculate a K (Fig. lOB). Similar in vitro approaches have been used to calculate the spontaneous first-order reactivation... 

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