Approximately 70% (by volume) of the insecticides currently used in the United States are organophosphate compounds.1 Organophosphates exert their toxicity by inhibiting cholinesterase (ChE) enzymes, resulting in the accumulation of acetylcholine in cholinergic synapses. All methods currently described for measurement of ChE activity in amphibians require terminal tissue sampling. This precludes serial sampling and discourages sampling in unstable populations. Our objectives were to: 1) determine if amphibian ChE could be measured in whole blood and/or serum samples, and if so, 2) demonstrate that ChE activity in the samples would be consistently inhibited in vitro by the addition of the organophosphate, malathion.
Serum and whole blood from free-ranging eastern hellbenders (Cryptobranchus alleganiensis) was collected in Ohio and Pennsylvania, shipped on ice, and frozen at -80°C until use. Serum and blood was collected in non-heparinized syringes and stored in additive free or lithium heparinized plastic microtainer tubes, respectively.
A colorimetric (Ellman) assay2 was used to measure ChE activity in serum (n=7) and whole blood (n=4).2 Acetylthiocholine served as the substrate, which was hydrolyzed to acetate and thiocholine by esterases in the sample. Thiocholine was reacted with a chromatophore, dithiodinitrobenzoic acid (DTNB), to produce the yellow 5-thio-2-nitrobenzoic acid. The rate of change of absorbance at 412nm was measured at 25°C over 5 minutes with a spectrophotometer. All samples were run in triplicate. Standard dilutions of electric eel acetylcholinesterase (AChE) were used to generate standard curves, providing a means to quantify esterase activity in the C. alleganiensis samples. Serum from all animals had measurable ChE activity (2-20μl volumes) and measures of serum ChE from the same animal were not significantly different. ChE activity could not be reliably measured in whole blood samples. Serum ChE activities for the 7 animals ranged between 7.65-12.54U AChE/min/mL.
Inhibition of ChE was achieved by the direct addition of malathion to serum samples, after first evaluating and refining the technique using electric eel AChE. The concentration of malathion required to inhibit electric eel AChE activity by 50% (IC50) was 5.2 ×10-4M. Malathion was diluted in methanol to 5 different molar concentrations. One μl of the malathion dilution or methanol (control) was added to 5μl of C. alleganiensis serum and incubated for 15 minutes at room temperature before measurement of activity, as described previously. For each serum sample (n=5), ChE activity was measured in 6 subsamples, one each of the 5 malathion dilutions, and 1 methanol control. The addition of malathion in vitro (final concentrations from 9.6×10-4 to 9.6×10-6M) produced a dose-response inhibition of ChE activity in serum of all animals and the IC50 value of individual animals ranged from 2.2×10-4 to 2.9×10-4M.
Our results indicate that the Ellman assay2 allows accurate measurement of serum cholinesterase activity in amphibians, and is suitable for very small amphibians, requiring as little as 2 μl of serum. Furthermore, the plasma esterase activity of C. alleganiensis showed dose-responsive inhibition by malathion that was reasonably consistent between animals.
The authors thank Drs. Barbara Wolfe and Tiffany Wolf of The Wilds (Cumberland, Ohio, USA) for providing the serum and whole blood samples used in this study.
1. CDC. 2005. Third National Report on Human Exposure to Environmental Chemicals. Centers for Disease Control, National Center for Environmental Health. Atlanta, GA
2. Ellman G, Courtney KD, Andres V, RM Featherstone. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7:88-95.