Inhibitor Report for: EPN

Hydrolytic "A"-esterase activities of various tissues of rat (plasma, liver, kidney, brain and intestinal mucosa) against selected OP esters of diverse structure as potential substrates (paraoxon, di-n-propyl paraoxon, di-n-butyl paraoxon, chlorpyrifos oxon, di-(4-phenyl butyl) phosphorofluoridate and the chiral isomers of ethyl 4-nitrophenyl phenylphosphonate) were studied. We have developed a sensitive and widely applicable assay depending on measuring decline in residual inhibitory power of any chosen OP against horse serum cholinesterase: for seven compounds examined so far I50s against BCHE ranged from 0.07 to 70 nM, and it is easy to monitor loss of OP starting from an initial 25 microM concentration. Progressive destruction rates were always highest in liver and plasma with activity sometimes detectable in kidney, brain but not in intestinal mucosa, but the ratios of activity between tissues differed for different substrates. At 25 microM/37 degrees/pH 7.2 hydrolysis rates ranged from 8500 nmol/min/g liver for di-(4-phenylbutyl) phosphorofluoridate down to 0.8 nmol/min for the butyl analogue of paraoxon; the rate for L(-) isomer of EPN oxon (23 nmol/min/g liver) was greater than 2x that for the D(+) isomer and for paraoxon. From our data we conclude that several OP hydrolases exist whose identity may be further characterised by use of selective substrates

The phenylphosphonothioate insecticides EPN and leptophos, and several analogs, were evaluated with respect to their delayed neurotoxic effects in hens and their environmental behavior in a terrestrial-aquatic model ecosystem. Acute toxicity to insects was highly correlated with sigma sigma of the substituted phenyl group (regression coefficient r = -0.91) while acute toxicity to mammals was slightly less well correlated (regression coefficient r = -0.71), and neurotoxicity was poorly correlated with sigma sigma (regression coefficient r = -0.35). Both EPN and leptophos were markedly more persistent and bioaccumulative in the model ecosystem than parathion. Desbromoleptophos, a contaminant and metabolite of leptophos, was seen to be a highly stable and persistent terminal residue of leptophos.

Ten day old chick sympathetic ganglia cultured in a microslide assembly were treated with a selected group of organophosphate pesticides to evaluate their cytotoxicity ranges, and the usefulness of such a model for screening pesticides. Examination by phase contrast and light microscopy for chemically-induced morphological alteration of nerve fibers, glial cells and neurons provided the criteria for quantitation and assessment of the toxic effects. Concentrations that produced half-maximal effects ranged from 1 x 10(-6)M (severely toxic) for methylparathian, diazinon, paraoxon, mevinphos, diisopropylfluorophosphate, tri-o-tolyl phosphate and its mixed isomers to a 1 x 10(-3)M (intermediate) for malathion, leptophos, coumaphos, mono- and dicrotophos. Some or no effects were evident at 1 x 10(2-)M for O'ethyl-O-p-nitrophenyl phenyl phosphonothioate, tri-m-tolylphosphate, chlorpyriphos and triphenyl phosphate. In all instances, nerve fibers were more sensitive than neurons or glial cells to insecticides. All cellular growth was inhibited at 1 x 10(-2)M (except triphenyl phosphate). Below 1 x 10(-7)M, no inhibitory effects were evident. The secondary abnormalities included decreased cellular migration, diffuse cellular growth pattern, increased vacuolization, nerve fiber swelling and cellular degeneration. The cytotoxic effects of these chemicals do not appear to be related to in vivo toxicity or cholinesterase inhibition potential.

We investigated the red blood cell (RBC) acetylcholinesterase (AChE) activities and butyrylcholinesterase (BChE) activities at presentation to the emergency department (ED) and at 24 h after presentation following poisoning by dichlorvos, fenitrothion, or ethyl p-nitrophenol thio-benzene phosphonate (EPN). Although the patients from different groups had similar characteristics at presentation such as time interval from ingestion to presentation to the ED and the amount of organophosphate ingested, the dichlorvos group had significantly lower BChE levels than the fenitrothion group and lower RBC cholinesterase activity than the EPN group. Patients poisoned with EPN or dichlorvos had significantly higher inhibition of BChE activities from baseline than RBC AChE activities at presentation. Twenty four hours after administration of pralidoxime, RBC AChE activities had increased in patients in the dichlorvos and EPN groups, while RBC AChE activities had slightly decreased in the fenitrothion group. BChE activities increased significantly in the dichlorvos group but decreased in the EPN group. The recovery patterns of RBC AChE and BChE activities did not match in any particular individual. This study showed that the patterns of inhibition and recovery of the activities of two cholinesterases after treatment are highly variable according to the organophosphate and in different individuals.

Egasyn-beta-glucuronidase complex is located at the luminal site of liver microsomal endoplasmic reticulum. When organophosphorus insecticides (OP) are incorporated into the liver microsomes, they become tightly bound to egasyn, a carboxylesterase isozyme, and subsequently, beta-glucuronidase (BG) is dissociated and released into blood. Consequently, the increase in plasma BG activity becomes a good biomarker of OP exposure. Thus, the single administration of EPN (O-ethyl O-p-nitrophenylphenylphosphonothioate), acephate and chlorpyrifos increased plasma BG activity in approximately 100-fold the control level in rats. The increase in plasma BG activity after OP exposure is a much more sensitive biomarker of acute OP exposure than acetylcholinesterase (AChE) inhibition.

Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.

Hydrolytic "A"-esterase activities of various tissues of rat (plasma, liver, kidney, brain and intestinal mucosa) against selected OP esters of diverse structure as potential substrates (paraoxon, di-n-propyl paraoxon, di-n-butyl paraoxon, chlorpyrifos oxon, di-(4-phenyl butyl) phosphorofluoridate and the chiral isomers of ethyl 4-nitrophenyl phenylphosphonate) were studied. We have developed a sensitive and widely applicable assay depending on measuring decline in residual inhibitory power of any chosen OP against horse serum cholinesterase: for seven compounds examined so far I50s against BCHE ranged from 0.07 to 70 nM, and it is easy to monitor loss of OP starting from an initial 25 microM concentration. Progressive destruction rates were always highest in liver and plasma with activity sometimes detectable in kidney, brain but not in intestinal mucosa, but the ratios of activity between tissues differed for different substrates. At 25 microM/37 degrees/pH 7.2 hydrolysis rates ranged from 8500 nmol/min/g liver for di-(4-phenylbutyl) phosphorofluoridate down to 0.8 nmol/min for the butyl analogue of paraoxon; the rate for L(-) isomer of EPN oxon (23 nmol/min/g liver) was greater than 2x that for the D(+) isomer and for paraoxon. From our data we conclude that several OP hydrolases exist whose identity may be further characterised by use of selective substrates

Inhibition of rat liver microsomal carboxylesterase (CEase) by O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN) and binding of EPN oxygen analog to microsomal CEase were enhanced by addition of NAD or NADP. This was more prominent in addition of NAD than NADP. No potentiation of anti-CEase action of EPN by NAD was seen when pure esterase (E.C. 3.1.1.1) instead of liver microsomes was used as an enzyme source. This effect of NAD in microsomal CEase was significantly decreased when N-ethylmaleimide or p-chloromercuribenzoic acid was added. From these findings, it is strongly suggested that NAD-mediated potentiation of the anti-CEase action of EPN might be attributed to the increase in formation of NADH from NAD by microsomal dehydrogenase(s) containing a sulfhydryl group, leading to a subsequent increase in formation of the EPN oxygen analog from EPN, and in turn, CEase inhibition was enhanced.

Daily dermal administration for 90 days of 0.01 to 10 mg/kg of O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) technical grade (85%) in acetone (0.1 ml) on the unprotected back of the neck produced delayed neurotoxicity. Hens given 2.5 to 10 mg/kg daily doses also received daily doses of atropine sulfate for 5 or 6 days to protect against cholinergic acute toxicity. Severity of the clinical condition depended on the concentration of the daily dermal dose of EPN; i.e., while hens given small doses showed only ataxia, those treated with large doses progressed to paralysis and died. The most consistent histopathologic alteration was the degeneration of axons and myelin in the spinal cord which was identical to that found in positive control hens that received daily dermal doses of 5 or 10 mg/kg tri-o-cresyl phosphate (TOCP). Some of the hens treated daily with the smallest tested dose of EPN (0.001 mg/kg) which did not show clinical signs of delayed neurotoxicity showed equivocal histological changes in the spinal cord. EPN and TOCP treatments had a more profound effect on the activity of plasma butyrylcholinesterase than that of brain acetylcholinesterase (AchE). by contrast O,O,-diethyl O-4-nitrophenyl phosphorothioate (parathion) was more inhibitory to brain AChE. Negative control hens that were treated with 90 daily dermal doses of 1 mg/kg of parathion initially showed leg weakness followed by recovery. A group of hens that received the same volume of acetone (0.1 ml) daily remained normal.

Five organophosphorous insecticides: Leptophos, EPN, Cyanofenphos, trichloronate and salithion proved to cause irreversible ataxia not only to chicken but also to mice and sheep. TOCP was included as a reference. Cyanofenphos blocked the catecholamine B-receptor binding activity with 3H-norepinephrine at a level similar to that of the specific inhibitor propranolol in the mouse heart preparation. In the lamb heart preparation, the B-receptor was more sensitive to Leptophos, salithion and TOCP than to propranolol. The six compounds and their oxons were screened for their in-vitro inhibition to monamine oxidase (MAO), acetyl cholinesterase (AChE) and neurotoxic esterase (NTE) in the brain of either mouse, lamb or chicken. It is believed that their AChE inhibition stands for their acute toxicity, while NTE inhibition is responsible for their paralytic ataxia.

The phenylphosphonothioate insecticides EPN and leptophos, and several analogs, were evaluated with respect to their delayed neurotoxic effects in hens and their environmental behavior in a terrestrial-aquatic model ecosystem. Acute toxicity to insects was highly correlated with sigma sigma of the substituted phenyl group (regression coefficient r = -0.91) while acute toxicity to mammals was slightly less well correlated (regression coefficient r = -0.71), and neurotoxicity was poorly correlated with sigma sigma (regression coefficient r = -0.35). Both EPN and leptophos were markedly more persistent and bioaccumulative in the model ecosystem than parathion. Desbromoleptophos, a contaminant and metabolite of leptophos, was seen to be a highly stable and persistent terminal residue of leptophos.

Ten day old chick sympathetic ganglia cultured in a microslide assembly were treated with a selected group of organophosphate pesticides to evaluate their cytotoxicity ranges, and the usefulness of such a model for screening pesticides. Examination by phase contrast and light microscopy for chemically-induced morphological alteration of nerve fibers, glial cells and neurons provided the criteria for quantitation and assessment of the toxic effects. Concentrations that produced half-maximal effects ranged from 1 x 10(-6)M (severely toxic) for methylparathian, diazinon, paraoxon, mevinphos, diisopropylfluorophosphate, tri-o-tolyl phosphate and its mixed isomers to a 1 x 10(-3)M (intermediate) for malathion, leptophos, coumaphos, mono- and dicrotophos. Some or no effects were evident at 1 x 10(2-)M for O'ethyl-O-p-nitrophenyl phenyl phosphonothioate, tri-m-tolylphosphate, chlorpyriphos and triphenyl phosphate. In all instances, nerve fibers were more sensitive than neurons or glial cells to insecticides. All cellular growth was inhibited at 1 x 10(-2)M (except triphenyl phosphate). Below 1 x 10(-7)M, no inhibitory effects were evident. The secondary abnormalities included decreased cellular migration, diffuse cellular growth pattern, increased vacuolization, nerve fiber swelling and cellular degeneration. The cytotoxic effects of these chemicals do not appear to be related to in vivo toxicity or cholinesterase inhibition potential.