ToxSci Advance Access originally published online on May 16, 2007
Toxicological Sciences 2007 98(2):605-606; doi:10.1093/toxsci/kfm118
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Published by Oxford University Press 2007.
Reply to Letter to the Editor
Department of Pharmaceutical Property Assessment, TargeGen, Inc., 9380 Judicial Drive, San Diego, California 92121. Fax: (858) 678-0762
E-mail: akousba{at}targegen.com
Received May 7, 2007; accepted May 8, 2007
We would like to thank Dr Padilla for her comments on our manuscript, in which we reported age-related ki (bimolecular inhibition rate) differences for brain acetylcholinesterase (AChE) inhibition with chlorpyrifos-oxon but not diazinon-oxon in the preweanling rat. As Dr Padilla indicates these results are different than what her laboratory reported based on in vitro IC50 determinations in preweanling (postnatal day 4) and adult rat brains (Mortensen et al., 1998
). Based on IC50 determinations, Mortensen et al. (1998) reported a similar brain AChE sensitivity for preweanling and adult rats to inhibition but also noted that the dose-response curve shifted to the left for both age groups when "extrinsic" factors were removed by immunoprecipitation. Likewise, removal of extrinsic factors in plasma and liver, also resulted in a leftward shift, which suggest that extrinsic factors in all these tissues can impact AChE inhibition response and appear to be at least partially responsible for age-related differences in plasma and liver enzyme inhibition.
As noted by Mortensen, "Although IC50 values may not indicate the inherent sensitivity of the enzyme to the anticholinesterase pesticide, tissue IC50 values may be useful in determining which tissues or species are more vulnerable in ‘real-life’ situations." They go on to suggest that the most relevant information may be the sensitivity of the enzyme within the tissue matrix versus the isolated enzyme. Their reported results strongly support this assessment, which we agree with, and the design of our experiments which utilized crude tissue homogenates is based on the premise that not just the enzyme but other factors within the tissue are important and key modulators of in vivo enzyme activity.
In our study, we measured the bimolecular inhibition rate constant (ki) which includes an affinity and phosphorylation constant since the inhibitory potency is a function of both binding affinity and rate of phosphorylation (Main, 1964; Rosenfeld et al., 2001). As noted by Richardson et al. (1993) and cited by Mortensen "... the bimolecular inhibition rate constant ki (versus IC50) is a better tool in determining the potency of an organophosphate for cholinesterase inhibition ...." For our study, we were interested in evaluating the age-related ki changes for both chlorpyrifos-oxon and diazinon-oxon in the whole brain homogenate. These ki values determined using the modified Ellman assay did reveal age-related differences for chlorpyrifos-oxon but not for diazinon-oxon. Since these two oxon molecules are structurally similar, the observed differences were surprising, yet the results also suggests that the homogenate system and incubation conditions were sufficient to show a chemical-specific effect. Because we were fully cognizant of the work from Dr Padilla's group, we did postulate several potential factors that are consistent with a number of the extrinsic factors suggested by Mortensen et al. (1998) which include: (1) instability of the chlorpyrifos-oxon within the incubation system (i.e., metabolism); (2) additional protein/lipid binding in brain homogenates that could modify in vitro–binding affinity to chlorpyrifos-oxon, and (3) the potential role of peripheral binding sites. Based on the finding of Mortensen et al. (1998), we would postulate that similar ki determinations utilizing a more purified enzyme system, such as the suggested immunoprecipitated AChE, would most likely result in different results particularly if extrinsic factors are modifying binding affinity of the oxon with AChE. Although determining the ki with an immunoprecipitated AChE, as suggested by Dr Padilla, has strong merit and should be pursued to better understand the role of extrinsic factors, the determination of a ki value using the whole tissue is particularly important for understanding "real-world" in vivo sensitivity.
In an attempt to better understand what experimental factors may have contributed to the observed difference in ki versus IC50 results with preweanling and adult brain AChE, we have summarized the different experimental conditions between the two studies (see Table 1). If we assume that differences in extrinsic factors present in the assays are key factors associated with the observed results, then the enzyme sources (crude homogenate versus supernant) and tissue dilutions (750 versus 10) may be of particular importance. Secondly, Mortensen et al. (1998) homogenized the brain in buffer containing 1% Triton X-100; Rosenfeld et al. (2000) reported that measurement of AChE activity after enzyme was exposed simultaneously to Triton X-100 and oxon (paraoxon) could be problematic. They reported that the presence of detergent markedly affected the interaction of paraoxon and AChE. In addition, if differences in the binding of chlorpyrifos-oxon with tissue lipids are important extrinsic factors, then the use of detergents in the assay may likewise contribute to observed differences. The age-dependent differences in ki determined in our study suggest that the in vivo-binding constant for chlorpyrifos-oxon with AChE may change as a function of age in the rat and therefore may be of importance in understanding age-related differences in pharmacodynamic response to the insecticide chlorpyrifos. Nonetheless, as suggested in our manuscript and by Dr Padilla additional investigation is warranted.
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REFERENCES
Rosenfeld C, Kousba A, Sultatos LG. Interactions of rat brain acetylcholinesterase with the detergent triton X-100 and the organophosphate paraoxon. Toxicol. Sci. (2001) 63:208–213.
Richardson RJ, Moore TB, Kayyali US, Fowke JH, Randall JC. Inhibition of hen brain acetylcholinesterase and neurotoxic esterase by chlorpyrifos in vivo and kinetics of inhibition by chlorpyrifos oxon in vitro: Application to assessment of neoropathic risk. Fundam. Appl. Toxicol. (1993) 20:273–279.[CrossRef][Web of Science][Medline]
Main AR. Affinity and phosphorylation constants for the inhibition of esterases by organophosphates. Science (1964) 144:992–993.
Mortensen SR, Brimijoin S, Hooper MJ, Padilla S. Comparison of the in vitro sensitivity of rat acetylcholinesterase to chlorpyrifos-oxon: What do tissue IC50 values represent? Toxicol. Appl. Pharmacol. (1998) 148:46–49.[CrossRef][Web of Science][Medline]
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