Reptile cholinesterase characterization and use in monitoring anti-cholinesterases

Date

2003-12

Journal Title

Journal ISSN

Volume Title

Publisher

Texas Tech University

Abstract

Cholinesterase (ChE) activity measurement in blood, brain, or liver of an animal is a useful tool for obtaining evidence of exposure to ChE-inhibiting compounds such as organophosphate (OP) or carbamate insecticides. Baseline ChE values and exposure to anti-ChEs in humans, other mammals, and birds have been assessed, but very little has been done to study reptiles and amphibians. Tissues from 10 species of Reptilia were characterized with regard to substrate affinity and inhibitor concentrations in the Ellman ChE assay. Optimized substrate (acetylthiocholine) and iso-OMPA (a differential BChE inhibitor) concentrations ranged from 1.0 mM to 3.16 mM and 100 uM to 5,620 uM, respectively. Total plasma CbiE (umoles AThCh hydrolyzed/min/ml) and percent AChE (and number of individuals assayed) were determined for representatives of the following species: Spotted Turtle {Clemmys guttata) - 0.409, 7.53% (184); River Cooter {Pseudemys concinna) - 0.166, 7.3% (8); Loggerhead Sea Turtle {Caretta caretta) - 0.089, 55.4% (51); Eastern Cottonmouth (Agkistrodonpiscivorous) - 0.232, 27.2% (7); Western Diamondback Rattlesnake (Crotalus atrox) - 1.53, 6% (4); and American Alligator (Alligator mississippiensis) - 0.65, 5% (18). Cottonmouth and rattlesnake brain contained 25% BChE, a finding dissimilar to the other reptiles studied with < 4% BChE. Kinetic constants determined for AChE and BChE with the substrates, acetylthiocholine and butyrylthiocholine showed that blood and brain AChE Kms, for a single species, were very similar.

Inhibitory concentrations of a selection of OP oxygen analogues were tested in tissue samples of each of these species to determine species-specific sensitivity patterns. In vitro brain and plasma IC50s for chlorpyrifos-oxon (CPF-0), paraoxon (PAR-0), and diazoxon (DIA-0) were determined. Overall, brain AChE IC50 values ranged from 12.9 nM to 98.6 nM, 70.3 nM to 617 nM, and 206 nM to 3,150 nM for CPF-0, PAR-0, and DIA-0, respectively. Generally, brain AChEs were more sensitive than plasma AChEs, with sensitivity to CPF-0 > PAR-0 > DIA-0. Snake brain BChE had >100 times increased sensitivity to CPF-0 than plasma BChE, while brain AChE was 10 times more sensitive than plasma AChE. Within their respective families, turtles, homed lizards, and snakes had similar AChE IC50s regardless of tissue or compound. Between families, terrestiial turtle brain AChE was the least sensitive, followed by lizards and then snakes.

Three field-oriented studies applied the methods developed in this study to investigate the relationship between age and ChE activity in Morelet's Crocodile (Crocodylus moreletii), seasonal ChE variability in Loggerhead Sea Turtles, and Spotted Turtle ChE in Massachusetts cranberry bogs. Decreasing ChE with age was foimd in the crocodiles with the juvenile ChE significantly different from sub-aduH and adult ChE. Loggerhead ChEs did not show a relationship with month or water temperature. Spotted turtle ChE tended to be lower in the fall than in the summer though evidence did not support direct effects of pesticides on study animals.

These studies show that reptilian ChEs share characteristics similar to birds and mammals, are sensitive to OPs and are likely affected by antiChE compounds under field conditions, and are suitable as biomarkers for monitoring exposure to cholinesterase inhibiting compounds.

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