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.

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.

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.

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.

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.

Mechanisms of joint neurotoxicity of n-hexane, methyl isobutyl ketone and O-ethyl O-4-nitrophenyl phenylphosphonothioate in hens.

The joint neurotoxic action of simultaneous exposure to vapors of n-hexane and methyl iso-butyl ketone (MiBK) and dermally applied O-ethyl O-nitrophenyl phenylphosphonothioate (EPN) was studied in groups of five adult hens. Four groups of hens were concurrently exposed to a dermal 2.5 mg/kg of EPN, 1000 ppm of n-hexane and 100, 250, 500 or 1000 ppm of MiBK. Two groups were each exposed to binary mixtures of a dermal dose of 2.5 mg/kg of EPN and 250 ppm of MiBK or 1000 ppm of n-hexane. Another three groups of hens were exposed to either 250 ppm of MiBK, 1000 ppm of n-hexane or a dermal dose of 2.5 mg/kg of EPN. A Group of hens was kept untreated. All hens were terminated after 30 days of treatment. Hens exposed to MiBK or n-hexane vapor did not exhibit any toxicity signs. In contrast, hens treated with EPN alone or in combination with n-hexane and/or MiBK developed acute cholinergic and delayed neurotoxicity signs. Hen brain acetylcholinesterase and neurotoxic esterase activities were inhibited in hens treated concurrently with EPN, n-hexane and MiBK. MiBK alone or in combination with EPN and n-hexane induced liver microsomal cytochrome P-450 content and phenobarbital-inducible cytochrome P-450 enzyme activities. Microsomes from hens treated with EPN, n-hexane, MiBK or mixtures of EPN, n-hexane and MiBK significantly enhanced the biotransformation of EPN to the more neurotoxic oxidation metabolite O-ethyl O-4-nitrophenyl phenylphosphonate.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The role of pharmacokinetics and metabolism in species sensitivity to neurotoxic agents.

Attempts have been made to review the role of pharmacokinetics and metabolism in species and age sensitivity as well as the development of various toxic conditions of some neurotoxic chemicals. The route of administration may play a prominent role in the development of various toxic effects of some organophosphorus compounds such as DEF. Such variation was attributed to the differential metabolism which was found to be highly dependent on the route of administration. It is obvious from the data presented here that animals that are sensitive to OPIDN are less active in the metabolism and elimination of the neurotoxic chemical and/or its metabolite(s). So, a compound may stay for a longer period in the body of the sensitive animals resulting in greater accessibility of target tissues to the deleterious effects of the neurotoxic compounds. However, many of these neurotoxic chemicals require metabolic activation to exert their effect. While the insensitive species may convert the compound to its active metabolite faster than that of the insensitive species, this is circumvented by the far greater capability of the insensitive animals to metabolize the active metabolite and/or the parent compound to less toxic, more polar, excretable metabolites. However, it must be stressed that these studies are far from complete, and caution should be exercised in interpreting and correlating many of these results. It is difficult, and sometimes misleading to compare data from various studies due to differences in dosage, the number of animals used, route of administration, experimental protocols, etc. With respect to hexacarbons, species sensitivity is obvious, but not as extensively investigated as OPIDN. To our knowledge, no studies are available addressing species difference in pharmacokinetics and metabolism of these chemicals. The data presented in this review suggest that metabolism and pharmacokinetics may play an important role in the development of OPIDN. However, this does not rule out the influence of other factors such as target sensitivity. This necessitates further qualitative and quantitative metabolic studies which are carefully planned to address these issues.

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.

The joint neurotoxic action of inhaled methyl butyl ketone vapor and dermally applied O-ethyl O-4-nitrophenyl phenylphosphonothioate in hens: potentiating effect.

The neurotoxic action of inhaled technical grade methyl butyl ketone and dermally applied (O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was studied. Three groups of five hens each were treated 5 days/week for 90 days with a dermal dose of 1.0 mg/kg of EPN (85%) on the unprotected back of the neck. These groups were exposed simultaneously to 10, 50, or 100 ppm of technical methyl butyl ketone (MBK; methyl n-butyl ketone:methyl isobutyl ketone, 7:3) in inhalation chambers. A fourth group was treated only with the dose of EPN and a fifth group with only 100 ppm MBK. The control consisted of a group of five hens treated with a dose of 0.1 ml acetone. Treatment was followed by a 30-day observation period. Simultaneous exposure to EPN and MBK greatly enhanced the neurotoxicity produced when compared to the neurotoxicity produced by either chemical when applied alone. Continued exposure to EPN and MBK resulted in earlier onset and more severe signs of neurotoxicity than exposure to either individual compound. The severity and characteristics of histopathologic lesions in hens given the same daily dermal dose of EPN in combination with inhaled MBK depended on the MBK concentration. Histopathologic changes were more severe and prevalent in the 100 ppm MBK:1 mg/kg EPN group than in the others. In this group, Wallerian-type degeneration was seen along with paranodal axonal swellings. The morphology and distribution of these lesions were characteristic of those induced by MBK. In the 50 ppm MBK:1 mg/kg EPN group axonal swelling was evident but not clearly identifiable as paranodal. Hens treated with 10 ppm MBK:1 mg/kg EPN had minimal lesions with low incidence of axonal swellings. These were not as large as those seen in MBK neurotoxicity, but instead resembled the histopathologic lesions caused by EPN. The results indicate that the combined treatment gave a value for neurotoxicity coefficient which was two times the additive neurotoxic effect of each treatment alone. Pretreatment with three daily ip doses of 5 mmol/kg technical grade MBK or methyl n-butyl ketone (MnBK), equally increased chicken hepatic microsomal cytochrome P-450 content. Also, hepatic microsomes from MBK-treated hens metabolized [14C]EPN in vitro to [14C]EPN oxon to a much greater extent than those from control hens. These results suggest that MBK potentiates the neurotoxic effect of EPN, at least in part, by increasing the metabolic activation of EPN to the more neurotoxic metabolite EPN oxon.(ABSTRACT TRUNCATED AT 400 WORDS)

Pattern of neurotoxicity of n-hexane, methyl n-butyl ketone, 2,5-hexanediol, and 2,5-hexanedione alone and in combination with O-ethyl O-4-nitrophenyl phenylphosphonothioate in hens.

This investigation was designed to study the neurotoxicity produced in hens by the aliphatic hexacarbons n-hexane, methyl n-butyl ketone (MnBK), 2,5-hexanediol (2,5-HDOH), and 2,5-hexanedione (2,5-HD) following daily dermal application of each chemical alone and in combination with O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). Dermal application was carried out on the unprotected back of the neck. To assess whether the joint neurotoxic action of various chemicals is caused by the enhancement of absorption through the skin or by interaction at the molecular level, two additional experiments were performed. In the first experiment, EPN was dissolved in each of the aliphatic hydrocarbons prior to their topical application. In the second experiment, EPN was dissolved in acetone and applied at a different location from that of the aliphatic hexacarbons. Dermal application was carried out for 90 d followed by a 30-d observation period. The results show that hens treated with EPN developed severe ataxia followed by improvement during the observation period; n-hexane produced leg weakness with subsequent recovery, whereas the same dose of MnBK, 2,5-HDOH, or 2,5-HD produced clinical signs of neurotoxicity characterized by gross ataxia; concurrent dermal application of EPN with n-hexane or 2,5-HDOH at the same site or at different sites produced an additive neurotoxic action; simultaneous dermal application of EPN and MnBK at different sites resulted in an additive effect, whereas it caused potentiation when applied at the same site; and concurrent topical application of EPN and 2,5-HD produced a potentiating neurotoxic effect. While no histopathologic lesion was produced at the end of the observation period when any test chemical was applied alone, binary treatments of EPN and aliphatic hexacarbons resulted in histopathologic changes in some hens, with morphology and distribution characteristic of EPN neurotoxicity. The joint potentiating or additive action of aliphatic hexacarbons on EPN neurotoxicity was: 2,5-HD greater than MnBK greater than 2,5-HDOH greater than n-hexane. The mechanism of this joint action seems to be related both to enhancing skin absorption of EPN and/or its metabolic activation by n-hexane and its related chemicals.

Subchronic organophosphorus ester-induced delayed neurotoxicity in mallards.

Eighteen-week-old mallard hens received 0, 10, 30, 90, or 270 ppm technical grade EPN (phenylphosphonothioic acid O-ethyl-O-4-nitrophenyl ester) in the diet for 90 days. Ataxia was first observed in the 270-ppm group after 16 days, in the 90-ppm group after 20 days, in the 30-ppm group after 38 days; 10 ppm failed to produce ataxia. By the end of 90 days all 6 birds in the 270-ppm group exhibited ataxia or paralysis whereas 5 of 6 birds in the 90-ppm group and 2 of 6 birds in the 30-ppm group were visibly affected. Treatment with 30 ppm or more resulted in a significant reduction in body weight. Brain neurotoxic esterase activity was inhibited by averages of 16, 69, 73, and 74% in the 10-, 30-, 90-, and 270-ppm groups, respectively. Brain acetylcholinesterase, plasma cholinesterase, and plasma alkaline phosphatase were significantly inhibited as well. Distinct histopathological effects were seen in the 30-, 90-, and 270-ppm groups which included demyelination and degeneration of axons of the spinal cord. Additional ducks were exposed in a similar manner to 60-, 270-, or 540-ppm leptophos (phosphonothioic acid O-4-bromo-2,5-dichlorophenyl-O-methylphenyl ester) which resulted in similar behavioral, biochemical, and histopathological alterations. These findings indicate that adult mallards are probably somewhat less sensitive than chickens to subchronic dietary exposure to organophosphorus insecticides that induce delayed neurotoxicity.

Mechanisms of toxicological interactions involving organophosphate insecticides.

Administration of apparently nontoxic doses of organophosphorus compounds can greatly alter the toxicity of other compounds. These might include other organophosphates, as well as other drug or nondrug xenobiotics. Mechanisms of toxicologic interactions are discussed. Emphasis is placed upon organophosphate inhibition of noncritical tissue esterases and alterations in the metabolism and toxicity of selected ester and amide containing xenobiotics.

Neurotoxic and teratogenic effects of an organophosphorus insecticide (phenyl phosphonothioic acid-O-ethyl-O-[4-nitrophenyl] ester) on mallard development.

Phenyl phosphonothioic acid-O-ethyl-O-[4-nitrophenyl] ester (EPN) is one of the 10 most frequently used organophosphorus insecticides and causes delayed neurotoxicity in adult chickens and mallards. Small amounts of organophosphorus insecticides placed on birds' eggs are embryotoxic and teratogenic. For this reason, the effects of topical egg application on EPN were examined on mallard (Anas platyrhynchos) embryo development. Mallard eggs were treated topically at 72 hr of incubation with 25 microliter of a nontoxic oil vehicle or with EPN in the vehicle at concentrations of approximately 12, 36, or 108 micrograms/g egg, equivalent to one, three, and nine times the agricultural level of application used to spray crops. Treatment with EPN resulted in 22 to 44% mortality over this dose range by 18 days of development compared with 4 and 5% for untreated and vehicle-treated controls. EPN impaired embryonic growth and was highly teratogenic: 37-42% of the surviving embryos at 18 days were abnormal with cervical and axial scoliosis as well as severe edema. Brain weights were significantly lower in EPN-treated groups at different stages of development including hatchlings. Brain neurotoxic esterase (NTE) activity was inhibited by as much as 91% at 11 days, 81% at 18 days, and 79% in hatchlings. Examination of brain NTE activity during the course of normal development revealed an increase of nearly sixfold from Day 11 through hatching. The most rapid increase occurred between Day 20 and hatching. Brain acetylcholinesterase (AChE) activity was inhibited by as much as 41% at 11 days, 47% at 18 days, and 20% in hatchlings. Plasma cholinesterase and alkaline phosphatase activities were inhibited and plasma aspartate aminotransferase activity was increased at one or more stages of development. Hatchlings from EPN-treated eggs were weaker and slower to right themselves. Histopathological examination did not reveal demyelination and axonopathy of the spinal cord that was characteristic of delayed neurotoxicity in adult birds.

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.

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.

Sensitivity of the cat to delayed neurotoxicity induced by O-ethyl O-4-nitrophenyl phenylphosphonothioate.

Delayed neurotoxicity was produced in cats following the administration of either a single dermal dose of 22.5 to 225 mg/kg (0.2 to 5.0 times the LD50) or subchronic (90 days) administration of 0.5 to 2.0 mg/kg of technical grade O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). The study showed three differences from the condition produced in the chicken: difficulty to protect from acute poisoning, slower progression of delayed neurotoxicity, and propensity for improvement. These animals received atropine sulfate and pyridine-2-aldoxime methyl chloride (PAM) to protect them against acute poisoning, but most developed signs of acute cholinergic neurotoxicity, the degree of severity being dose dependent. Also cats given small single doses of EPN showed only leg weakness, while those treated with large doses progressed to severe ataxia and death. In cats treated with subchronic dermal daily doses of EPN, the extent and permanence of injury and progression or improvement of neurologic deficit also depended on the dose size and duration of exposure. Histopathologic changes were present in the most distal portion of the longest tracts in both the central and peripheral nervous system. Ascending tracts were most affected in the cervical spinal cord, while change in the descending tracts was concentrated in the lumbosacral spinal cord. Recovery to a varying degree from delayed neurotoxicity was seen in all surviving cats. The recovery was demonstrated as improvement in clinical signs, increase in body weight, and regeneration of peripheral nerves.

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.