Recommendations of the IUBMB Nomenclature Committee: Comments concerning classification and nomenclature of esterases hydrolysing organophosphorus compounds

Organophosphate degrading enzymes are of great interest in light of their ability to detoxify chemical warfare agents. The diisopropylfluorophosphatase (DFPase) from Loligo vulgaris is characterized by its high stability and broad substrate specifity. Here we report the production of large amounts of active, recombinant DFPase using an Escherichia coli expression system. The enzyme was purified to homogeneity using a combination of immobilized metal affinity and ion exchange chromatography. CD-spectroscopy indicates a well folded protein with a high amount of β-sheet structure. Limited proteolysis was used to gain a deeper insight into the structural organization of the protein. DFPase possesses an internal protease-sensitive region located between amino acids 146 and 149. The two proteolytic fragments remain as a tight complex retaining a DFPase activity comparable to the intact enzyme. Overexpression clones for each fragment were constructed with the expression resulting in the formation of inclusion bodies. Upon isolation and refolding active protein is only formed when both fragments are present. Thus, the two proteolytic fragments are probably part of a stable single-domain protein with multiple contacts between them and only one protease accessible surface loop.

Young organisms are often more sensitive to the toxic effects of pesticides, and this finding has spurred research on further characterization of this susceptibility. The neurotoxic effects of cholinesterase (ChE)-inhibiting pesticides are of particular concern for human health risk assessment due to the widespread exposure potential in children. This study evaluated age-related differences in susceptibility for a carbamate (aldicarb) and an organophosphorus pesticide (methamidophos). Comparisons were made between preweanling (Postnatal Day 17, PND17), postweanling (PND27), and adult (∼PND70) male and female rats. All were acute studies using oral administration. Sensitivity was quantified by (1) determination of maximally—tolerated doses (MTDs); (2) measurement of brain and blood ChE inhibition; and (3) neurobehavioral evaluation using end points known to be sensitive indicators of exposure to anticholinesterases. MTD data showed that preweanling rats were twice as sensitive as adults to aldicarb, but there was no differential sensitivity to methamidophos. The dose–response data for brain ChE inhibition followed a similar pattern of age-related differences, and similar levels of inhibition were measured at the MTD regardless of age. Dose–response and time course studies of neurobehavioral end points indicated that differential effects due to age depend on the behavioral end point examined. Following aldicarb administration, the dose–response curves for a few end points overlapped; however, the young rats otherwise showed fewer signs of toxicity than did the adults despite similar levels of brain ChE inhibition. Motor activity assessment showed that aldicarb did not produce any activity depression in PND17 rats, whereas the data for the PND27 and adult rats overlapped. With methamidophos, the dose–response curves for most end points for preweanling and adult rats were quite similar. Aldicarb-induced ChE inhibition was readily reversible in all age groups, whereas with methamidophos, enzyme activity recovered more rapidly in the young. Most behavioral alterations had recovered by 24 h with either pesticide. The results of these studies indicate that (1) ChE-inhibiting pesticides are not all the same regarding relative sensitivity of the young; (2) age-related differences were reflected in both the MTDs and degree of ChE inhibition; and (3) age-related differences in neurobehavioral measures depended on the pesticide and on the end points examined.

The present study shows the existence of both Ca²⁺-dependent and EDTA-resistant hydrolysing activities against HDCP and paraoxon in the particulate and soluble fractions of hen, rat and rabbit liver. HDCP was more extensively hydrolysed than paraoxon in both subcellular fractions and each of three individuals of the three animal species under study in spite of wide interindividual variations. However the ratio of HDCP versus paraoxon hydrolysing activity (HDCPase/paraoxonase), although within the same order of magnitude, cannot be considered as constant as it ranges one- to seven-fold between individuals of the same species. Also there is no constant ratio of Ca²⁺-dependent/EDTA-resistant activities. Rabbit liver showed the highest rates of Ca²⁺-dependent hydrolysis for both organophosphorus compounds whereas the hen paraoxonase activity was not inhibited by EDTA. The stereospecific hydrolysis of HDCP was mostly a Ca²⁺-dependant one, the S-HDCP isomer being hydrolysed faster than the R-HDCP one. The suggestion is made that HDCP could be conveniently used to measure PTE activity in the liver.

O-Hexyl, O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral compound that induces delayed neuropathy in hens. This compound is hydrolyzed by a phosphotriesterase known as HDCPase in hen and rat plasma, liver and brain. We studied the stereospecificity of HDCPase in hen tissues and in human and rabbit plasma employing a chromatographic method for analysis and quantification of HDCP stereoisomers. Hen and human plasma HDCPases were not stereospecific. However, rabbit plasma showed a remarkable stereospecificity to S-(−)-HDCP. High levels of stereospecific HDCPase were found in the particulate fraction of hen liver, where S-(−)-HDCP is hydrolyzed faster than R-(+)-HDCP. However, in hen brain the stereospecificity was found in the soluble fraction, where R-(+)-HDCP is hydrolyzed faster than S-(−)-HDCP. It is concluded that liver particulate fraction must be the main tissue responsible for the HDCP stereospecific biotransformation in hens. In an oral administration, the steroisomer R-(+)-HDCP would survive after passing through the liver and would interact with acetylcholinesterase and neuropathy target esterase in the nervous system.

Young rats are more sensitive than adults to a single oral dose of chlorpyrifos, an organophosphorus pesticide. A direct comparison of chlorpyrifos effects in young (postnatal day 17; PND17), adolescent (PND27), and adult (70 days) Long–Evans rats was conducted to determine quantitative and possibly qualitative differences in sensitivity in terms of behavioral changes and cholinesterase (ChE; total cholinesterase activity) inhibition at these three ages. Male and female rats were administered chlorpyrifos orally at one of two doses (PND17, 5 or 20 mg/kg; PND27, 20 or 50 mg/kg; adult, 20 or 80 mg/kg) and tested at either 3.5 or 6.5 h after dosing. Behavioral testing included observational evaluations and measurements of motor activity and was followed immediately by tissue collection for ChE determination in brain and blood. For both behavioral changes and ChE inhibition, peak effects occurred at 3.5 h in adult male and PND27 rats (both sexes) and at 6.5 h in adult female and PND17 rats (both sexes). Comparisons of the 20 mg/kg dose across ages showed generally less ChE inhibition and fewer behavioral effects with increasing age, except that the adult females were similar to the PND27 rats. The high dose used for each age group produced similar brain ChE inhibition (80–90%) and generally similar behavioral effects. Interestingly, a few endpoints in the young rats were less affected than in adults at this level of ChE inhibition. The degree of ChE inhibition in the brain more closely paralleled the blood inhibition in the younger rats, compared to the adults. Carboxylesterase (CaE) and A-esterase are known to play an important role in the detoxification of organophosphates and may be partially responsible for these sensitivity differences. Liver and plasma CaE and A-esterase activities were measured in untreated male rats on PND1, 4, 7, 12, 17, and 21 and in adults of both sexes (82–92 days old). Preweanling rats had considerably less activity of both enzymes, and adult females had less liver CaE activity than males. These differences in detoxifying enzymes correlate with the age-related differences in behavioral and biochemical effects, as well as the gender differences seen in adult rats, and thus may be a major influence on the differential sensitivity to chlorpyrifos.