Extremely sensitive biomarker of acute organophosphorus insecticide exposure.

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.

Variable response of cholinesterase activities following human exposure to different types of organophosphates.

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.

Ecotoxicology of phenylphosphonothioates.

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.

Disposition, elimination and metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate after subchronic dermal application in male cats.

The metabolism, distribution, and excretion of the insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) were studied in the male cat. Each cat was given a daily dermal dose of 0.5 mg/kg [14C]EPN for 10 consecutive days. Fifteen days after the last dose, the cats had excreted 62% of the cumulative dose in the urine and 10% in the feces. No 14CO2 was detected in the expired air. O-Ethyl phenylphosphonic acid (EPPA) was identified as the major urinary and fecal metabolite. Phenylphosphonic acid (PPA) was the second highest metabolite. Only traces of the intact EPN were recovered in the urine and feces. The disposition studies performed 1, 5, 10 and 15 days after the administration of the last dose showed that EPN was the major compound identified in the brain, spinal cord, sciatic nerve, adipose tissue, plasma and kidney. Most of the radioactivity in the liver was identified as EPPA followed by PPA. The time course of plasma EPN, determined after the 10th daily dose was biphasic. The slower process had a half-life of 17.0 days. After tissue distribution was completed, tissue elimination was adequately represented as a single first-order process.

Consideration of dose levels for delayed neurotoxicity testing in hens: the relationship of neurotoxic dosages to acute LD50 values.

The oral LD50 of a compound in hens does not reliably predict the neurotoxic dose of that compound. The oral LD50 in rats does not predict either the oral LD50 in hens or the neurotoxic dose in hens, so there is no justification for using the rat value to predict the neurotoxic dosage in hens. A strategy is presented for a stepwise approach to neurotoxicity testing in hens.

Effect of subchronic dermal application of O-ethyl O-4-nitrophenyl phenylphosphonothioate on producing delayed neurotoxicity in hens.

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.

Toxicological significance in the cleavage of esterase-beta-glucuronidase complex in liver microsomes by organophosphorus compounds.

Egasyn is an accessory protein of beta-glucuronidase (beta-G) in the liver microsomes. Liver microsomal beta-G is stabilized within the luminal site of the microsomal vesicles by complexation with egasyn which is one of the carboxylesterase isozymes. We investigated the effects of organophosphorus compounds (OPs) such as insecticides on the dissociation of egasyn-beta-glucuronidase (EG) complex. The EG complex was easily dissociated by administration of OPs, i.e. fenitrothion, EPN, phenthionate, and bis-beta-nitrophenyl phosphate (BNPP), and resulting beta-G dissociated was released into blood, leading to the rapid and transient increase of plasma beta-G level with a concomitant decrease of liver microsomal beta-G level. In a case of phenthionate treatment, less increase in plasma beta-G level was observed, as compared with those of other OPs. This may be explained by the fact that phenthionate was easily hydrolyzed by carboxylesterase. Similarly, carbamate insecticides such as carbaryl caused rapid increase of plasma beta-G level. In contrast, no significant increase of plasma beta-G level was observed when pyrethroid insecticides were administered to rats. This is due to the fact that pyrethroids such as phenthrin and allethrin were easily hydrolyzed by A-esterase as well as carboxylesterase. On the other hand, addition of OPs to the incubation mixture containing liver microsomes caused the release of beta-G from microsomes to the medium. From these in vivo and in vitro data, it is concluded that increase of the plasma beta-G level after OP administration is much more sensitive biomarker than cholinesterase inhibition to acute intoxication of OPs and carbamates.

Acute toxicity, accumulation and excretion of organophosphorous insecticides and their oxidation products in killifish.

Acute toxicity, accumulation and excretion of four organophosphorous insecticides (diazinon, malathion, fenitrothion and EPN) and their oxidation products (diazinon oxon, malaoxon, fenitrothion oxon and EPN oxon) were studied for killifish (Oryzias latipes). The 48-hr LC50 was 4.4 mg l-1 for diazinon, 1.8 mg l-1 for malathion, 3.5 mg l-1 for fenitrothion, 0.58 mg l-1 for EPN, 0.22 mg l-1 for diazinon oxon, 0.28 mg l-1 for malaoxon, 6.8 mg l-1 for fenitrothion oxon, and 0.16 mg l-1 for EPN oxon. The bioconcentration factors (BCF) of diazinon oxon 0.5, malaoxon 1.1, fenitrothion oxon 2.3 and EPN oxon 11 in the whole body of the fish were much lower than those of diazinon 49, malathion 11, fenitrothion 122 and EPN 1124. As reference data, partition coefficients between n-octanol and water (Pow) were measured for these chemicals. The BCF values of each pesticide and its oxidation product were consistent with the Pow values. The excretion rate constants (k) from the whole body of the fish were 0.12 hr-1 for diazinon, 0.27 hr-1 for malathion, 0.11 hr-1 for fenitrothion, 0.02 hr-1 for EPN, 0.30 hr-1 for fenitrothion oxon and 0.59 hr-1 for EPN oxon. The rates of diazinon oxon and malaoxon could not be measured, but were presumed to be as rapid as or more rapid than those of fenitrothion oxon and EPN oxon. The results suggest that the contamination of fish and other aquatic organisms by the oxidation products in the environment is very low.

Separation and aquatic toxicity of enantiomers of the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN).

Enantioselectivity in separation and toxicity of chiral pesticides has become important research areas in environmental science, because these studies give a deeper insight into the environmental effect of chiral pesticides. In this study, enantiomeric separation of the organophosphorus pesticide and acaricide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was investigated by chiral high-performance liquid chromatography (HPLC) with two chiral stationary phases. The racemate and separated enantiomers of EPN were tested for aquatic toxicities assay using Daphnia magna and zebrafish (Danio rerio) embryo test. The enantiomers of EPN were completely separated on Chiralpak AD and Chiralpak AS columns coupled with a circular dichroism detector at 236 nm. Better separations were achieved with lower temperatures (e.g., 20°C) and lower levels of polar modifiers (e.g., 1%). A significant difference was found between the enantiomers in their acute aquatic toxicity; the (+)-enantiomer was about 10 times more toxic than its antipode. On the contrary, the (-)-enantiomer induced crooked body, yolk sac edema and pericardial edema significantly more than (+)-enantiomer in the zebrafish embryo test. These results suggest that biological toxicity of chiral pesticides should be assessed by using their individual enantiomers with more comprehensive methods.

Distribution and metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate after a single oral dose in one-week old chicks.

The toxicokinetics and metabolism of a single 1 mg (2.7 muCi/kg) oral dose of uniformly phenyl-labeled [14C]EPN (O-ethyl O-4-nitrophenyl [14C]phenylphosphonothioate) have been studied in 1-week old chicks. One control and three treated chicks were killed at each of the following time intervals: 0.5, 2, 4, 8, and 12 days. Radioactivity was rapidly absorbed from the gastrointestinal tract and distributed in all tissues. 14C in tissues reached a peak of 16.9% of the dose after 0.5 day and decreased to 0.6% at 4 days. The tissues of the gastrointestinal tract had the highest concentration of radioactivity, followed by bile and liver. Among nervous tissues, concentration of the 14C was highest in the peripheral nerves. The spinal cord had the next highest concentration, while the brain had the least. After 4 days 91.3% of the 14C had been eliminated in the combined urinary-fecal excreta. By the end of the 12-day experiment this percentage reached 93.1%. No 14C was detected in the expired CO2. Following the oral administration of [14C]EPN, a monophasic body level curve was observed. The half-life for the elimination of 14C from chick body was 16 h, corresponding to a rate constant of 0.04 h -1. Most of the excreted 14C materials were identified as O-ethyl phenylphosphonic acid, phenylphosphonic acid, and O-ethyl phenylphosphonothioic acid.

Lack of promoting activity of four pesticides on induction of preneoplastic liver cell foci in rats.

Four pesticides were examined for hepatopromoting activity using a medium-term bioassay based upon induction of glutathione S-transferase placental form (GST-P) positive foci in the rat liver. Male F344 rats were initially injected with diethylnitrosamine (DEN; 200 mg/kg body weight) intraperitoneally and 2 weeks later were treated with O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN; 75 and 150 ppm), diazinon (500 and 1,000 ppm), phenthoate (500 and 1,000 ppm), or iprobenfos (500 and 1,000 ppm) in the diet for 6 weeks and then killed, all rats being subjected to partial hepatectomy at week 3. All of the pesticides gave negative results, the numbers and areas of GST-P positive foci not exceeding the control values for animals given DEN alone. Indeed, a significant reduction of foci development was seen for EPN (75 ppm). These findings provide experimental evidence that the presently examined four pesticides do not have hepatocarcinogenic potential in rats.

Stereospecificity in toxicity of the optical isomers of EPN.

While the optical isomers of EPN were equally toxic to mice, (+)-EPN was 2.9 fold and 4.0 fold more toxic to houseflies and rice stem borer larvae, respectively, than the (-)-isomer. In addition, (-)-EPN produced paralysis of the legs in hens about 10 to 14 days after dosing, whereas (+)-EPN caused no paralytic effects. Thus, (+)-EPN appears to be a more appropriate insecticide than the racemic compound, since it combines high toxicity to insects and no delayed neruotoxicity in hens.

The influence of chirality on the delayed neuropathic potential of some organophosphorus esters: neuropathic and prophylactic effects of stereoisomeric esters of ethyl phenylphosphonic acid (EPN oxon and EPN) correlate with quantities of aged and unaged neuropathy target esterase in vivo.

Organophosphate-induced delayed polyneuropathy (OPIDP) is thought to result from organophosphorylation of neuropathy target esterase (NTE), followed by an "aging" of the phosphorylated NTE. Prophylactic against OPIDP should thus be achieved by production of an inhibited but "nonaging" NTE. Resolved stereoisomers of ethyl phenylphosphonic acid esters produce two forms of inhibited NTE; in vitro one form ages rapidly and the other only negligibly. The present study examined the in vivo effects of two preparations of incompletely resolved isomers of EPN oxon (ethyl 4-nitrophenyl phenylphosphonate) and its thionate on adult hen brain and spinal cord NTE and the relationship of inhibition and aging to the development of OPIDP. Single doses of the L-(-)-isomers (Preparation A, 7:3 proportion of isomers, or Preparation B, 9:1) caused severe neuropathy after doses which produced 70% aged inhibited NTE and mild effects after 50-60%. Single doses of the D-(+)-isomers produced either equal amounts of aged and unaged inhibited NTE (Preparation A) or predominantly unaged (Preparation B): the amount of aged was never more than 50% and no clinical OPIDP occurred. Doses of D-(+) which produced 50% unaged inhibited NTE were protective: challenge with the highly neuropathic phenyl saligenin cyclic phosphate did not cause OPIDP. All effects are consistent with the two-stage initiation process which requires both inhibition of NTE and subsequent modification of the protein by an "aging" process. Previously reported neuropathic effects of D-(+)-EPN probably reflect a substantial proportion of L-(-)-isomer present in the test material. Neuropathic studies with chiral OP esters should consider the possibility of production of protective unaged inhibited NTE in test animals.

Teratogenic evaluation of the pesticides baygon, carbofuran, dimethoate and EPN.

Baygon was administered IG once daily to CD rats (5 to 50 mg/kg), on the 7th-19th day of gestation or to CD-1 mice (5 to 60 mg/kg) on days 6-16 of gestation. Baygon, at dose levels which were not maternally lethal, did not produce fetotoxicity, fetal lethality or malformations in the fetuses. Baygon was not teratogenic in the CD rat or CD-1 mouse at maternally nontoxic dose levels. Carbofuran was administered IG once daily to CD rats (0.05 to 5.0 mg/kg), on the 7th-19th day of gestation or to CD-1 mice (0.1 to 20 mg/kg) on days 6-16 of gestation. At dose levels which were not maternally lethal, carbofuran did not produce fetotoxicity, fetal lethality or malformations in the fetuses. Carbofuran was not teratogenic in the CD rat or CD-1 mouse at maternally nontoxic dose levels. Dimethoate was administered IG once daily to CD-1 mice (10 to 80 mg/kg), on the 6th-16th day of gestation. At dose levels which were not maternally lethal, dimethoate did not produce fetotoxicity, fetal lethality or malformations in the fetuses. Dimethoate was not teratogenic in the CD-1 mouse at maternally nontoxic dose levels. EPN was administered IG once daily to CD-1 mice (1.0 to 12.0 mg/kg) on the 6th-16th day of gestation. EPN, at dose levels up to those which were maternally lethal, did not produce fetotoxicity, fetal lethality or an increase in malformations. EPN was not teratogenic in the CD-1 mouse at maternally nontoxic dose levels.

Six-month daily treatment of sheep with neurotoxic organophosphorus compounds.

The delayed neurotoxic effects of tri-o-cresyl-phosphate (TOCP), O-methyl-O-(4-bromo-2,5-dichlorophenyl) phenylphosphonothioate (leptophos), and O-ethyl O-(4-nitrophenyl) phenylphosphonothioate (EPN) at 5, 5, and 1 mg/kg/day, respectively, on male sheep were studied during 6 months of daily oral treatment under field conditions. A vehicle-control group of sheep given corn oil (0.1 ml/kg/day) only was used for comparison. All sheep were killed 24 h after the 180th daily treatment. Blood, brain, spinal cord, and sciatic nerve tissues were taken for histological and/or biochemical examinations. The results indicated that leptophos induced severe ataxia and paralysis in sheep following about 4 months of treatment. TOCP produced either mild ataxia or lameness in two of four sheep during the last week of experiment. On the other hand, none of the EPN-treated sheep showed clinical signs of neurotoxicity during the course of the experiment at the dosage tested. These clinical results were supported by histological findings and also by biochemical results with neurotoxic esterase (NTE) measurements. In the case of leptophos-treated sheep, numerous prominent degenerative lesions of axons were observed in spinal cords and brains. Similar but somewhat less numerous lesions were noted in sheep treated with TOCP. No histological changes were observed in similar tissues taken from EPN-treated sheep. The results also indicated that, for chronic exposure to these neurotoxic organophosphorus compounds in sheep, a threshold in excess of 60-70% prolonged inhibition of brain NTE, or 50-60% inhibition of spinal cord NTE must be exceeded to initiate clinical and/or histological neurotoxic effects.

Neurotoxicity of organophosphorus insecticides Leptophos and EPN.

Phosfolan, chlorpyrifos, and stirophos when applied to white mice at sublethal doses did not induce any delayed neurotoxic effect. On the other hand, Leptophos and EPN when administered orally at sublethal or lethal levels clearly produced a delayed neurotoxic ataxia in treated mice. The five tested organophosphorus insecticides were compared for their ability to inhibit cholinesterase, neurotoxic esterases and monoamine oxidase. I50 values were estimated for each case. The results revealed that all five compounds were inhibitors of cholinesterase, but only Leptophos and EPN were shown to be potent inhibitors for both neurotoxic esterase and monoamine oxidase in the mouse brain. Additional particular properties of both Leptophos and EPN were found in their ability to cause delayed neurotoxic ataxia in chickens and sheep fed once on sublethal doses of these compounds. It is believed that the phosphonate ester configuration of EPN and Leptophos has a specific mode of toxic action which is mainly located at the central nervous system. It is also postulated that these delayed neurotoxic agents might inhibit postganglionic sympathetic neurons, thus resulting in chronic paralytic effects.