Robert A. Metcalf

A comparative analysis of the tissue esterases of the white crappie (Pomoxis annularis rafinesque) and black crappie (Pomoxis nigromamaculatus lesueur) by electrophoresis and selective inhibitors

Abstract 1. 1. The multiple forms of soluble esterases were compared for the brain, liver and skeletal muscle of the white and black crappie. 2. 2. The two species exhibited a high tissue specific gene function. 3. 3. No soluble cholinesterase activity was found; however, carboxylesterase, arylesterase and acetylesterase activity was present. 4. 4. The esterases of the brain and liver were similar between species in terms of multiplicity, substrate utilization and inhibitor sensitivity. However, the skeletal muscle of the white crappie contained esterase bands inhibited by p-HMB (arylesterase) whereas the black crappie did not. 5. 5. The inhibitory properties of fourteen organophosphate and carbamate compounds were compared for the esterases of the two species.

Acute and delayed neurotoxicity of leptophos analogs

Abstract Nineteen O -halogenated-phenyl O -methyl phenylphosphonothionates were evaluated for acute toxicity (LD 50 ) to the female house fly Musca domestica L. and to the male Swiss white mouse, and for delayed neurotoxicity to the White Leghorn hen. The electron-withdrawing power of the phenyl substituents (Σσ − values) correlate with the LD 50 values to house fly and mouse, with departures from linearity attributable to the steric hindrance of di- ortho -Cl substitution and by variations in the accessibility of the anionic site of acetylcholinesterase in the two species. The relationship with delayed neurotoxicity is less predictable although it clearly depends on suitable electron-withdrawing capacity. Delayed neurotoxicity also relates to a high degree of lipophilicity and prolonged residence time of the inhibitor in the nerve axon.

Comparison of the delayed neurotoxicity of O-methyl and O-ethyl analogs of EPN, leptophos, and cyanofenphos.

Eight pairs of O-methyl and O-ethyl O-(substituted-phenyl) phenylphosphonothionates were evaluated with respect to their delayed neurotoxic activity in hens. O-methyl compounds were in all cases more active than their O-ethyl analogs. The neurotoxic potential of the O-methyl phenylphosphonothionates was 2,5-diCl greater than 4-NO2 greater than 2,4,5-triCl and 2,4,6-triCl greater than 2,4-diCl greater than 2,5-diCl-4-Br greater than 4-CN, when single oral doses were given. Both EPN-ethyl and leptophos-methyl were more neurotoxic in multiple dermal than multiple oral dosing regimens. LD50s for mice and flies were established.

Selective toxicity of analogs of methyl parathion

Abstract Analogs of methyl parathion with various substituents in the aryl ring were examined for quantitative toxicity to the female white mouse and the female house fly. Substitution in the 3-position with the halogens produced Selectivity Ratios for mouse LD 50 /fly LD 50 in the order Cl > Br > IF. The compounds with the highest Selectivity Ratios were 2Cl > 3CF 3 > 3CH 3 . The biochemical basis for the selectivity of these compounds was explored by evaluation of the kinetics for the inhibition of purified bovine and house fly acetyl cholinesterase by the PO analogs. Differences in the configuration and reactivity of the active sites of the acetyl cholinesterase of the two species explain in part the selectivity of the toxicants. However, selective metabolism also plays a substantial role.

In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs

Abstract Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O -halogenated-phenyl- O -alkyl phenylphosphonates was studied in vitro . The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The pI 50 of O -(2,6-dichlorophenyl) O -methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy compounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than desbromoleptophosoxon. Within a like group of chlorinated phenylphosphonates, a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in vivo delayed neurotoxic potential by the corresponding phosphonothionate exists. In vivo inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in vivo inhibitor. Ethoxyleptophos and leptophos have about equal in vivo esterase inhibitory properties. For desbromoleptophos and leptophos there is good agreement between the minimum dose causing delayed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.

Structure-activity relationships in the acute and delayed neurotoxicity of methyl- and ethylphosphonothionates

Abstract Data on acute toxicity to house flies and mice, and delayed neurotoxicity to hens, are presented for 24 O-alkyl O-halogenated phenyl alkyl and aryl phosphonothionates, providing comparisons of the three sets of major structural parameters involved in the toxic action of these compounds. In the house fly, topical LD50 values showed consistent similarities between phenyl- and methylphosphonothionates with comparable O-alkyl and O-phenyl substitution. The phenyl compounds were consistently less toxic than the methyl compounds and the ratio of LD50 values averaged 3.8. In both series of compounds the LD50 values decreased with increasing halogenation as predicted by summed σ values for the substituents. The degree of steric hinderance on reactivity of the central phosphorus atom was particularly significant in comparisons of phenyl- and methylphosphonothionates. Thus in the methyl series there was only a 4-fold decrease in the LD50 with increase in the size of the O-alkyl group from Me to Et to Pr but with the more bulky phenyl series, this range of increase in the size of the O-alkyl group resulted in a greater than 10-fold decrease in LD50. Similarly, in comparing the effects of 2,6-dichloro with 2,5-dichloro substitution of the O-phenyl group, the two ortho-substituents decreased the LD50 value 3-fold with the methylphosphonothionates and 12-fold with the phenylphosphonothionates. The acute mammalian toxicity of organophosphorus esters is less closely linked to structural parameters, and several compounds were less toxic (respectively more toxic) to mice than structural considerations predict. In hens, organophosphorus ester-induced delayed neurotoxicity (OPIDN) was induced by all methylphosphonothionates tested; the 2,5-Cl2 and 2,6-Cl2 compounds were the most effective delayed neurotoxicants. For p-halogenated analogs, the identity of the halogen was less important for methyl- than for phenylphosphonothionates. And, in marked contrast to the phenylphosphonothionates, there was no appreciable difference between the OPIDN potential of O-methyl and O-ethyl methylphosphonothionates. For leptophos and its methylphosphonothionate analog, inhibition of neurotoxic target enzyme (NTE) corresponded with observed symtomatology. When hens were treated with equimolar doses of the two compounds, NTE inhibition reached 80% at 24 hr, but recovery was more rapid in the leptophos-treated hens, which did not become paralyzed, than in the O-2,5-dichloro-4-bromophenyl O-methyl methylphosphonothionate, which progressed to paraplegia.

Steric, electronic, and polar parameters that affect the toxic actions of O-alkyl, O-phenyl phosphonothionate insecticides☆☆☆

Abstract The toxic action of a series of O -alkyl, O -substituted-phenyl alkyl- and aryl-phosphonates and phosphonothionates have been evaluated by correlating the linear free energy parameters for steric ( E s ), electronic (σ), and polar ( σ ∗ ) effects with topical LD 50 to the house fly and oral LD 50 to the white mouse. In molecules free from major steric interactions with the reactive P atom, variations in these linear free energy parameters account for >90% of the variations in the LD 50 values, and the degree of correlation with LD 50 is at least as precise as that with the biomolecular rate constants for inhibition of the target-site enzyme acetylcholinesterase. The value of correlations of linear free energy parameters with LD 50 in understanding quantitative structure-activity relationships is illustrated.