Structural requirements of organophosphorus insecticides (OPI) to inhibit chicken yolk sac membrane kynurenine formamidase related to OPI teratogenesis.

This paper provides new information related to the mechanism of OPI (organophosphorus insecticides) teratogenesis. The COMFA (comparative molecular field analysis) and COMSIA (comparative molecular similarity indices analysis) suggest that the electrostatic and steric fields are the best predictors of OPI structural requirements to inhibit in ovo chicken embryo yolk sac membrane kynurenine formamidase, the proposed target for OPI teratogens. The dominant electrostatic interactions are localized at nitrogen-1, nitrogen-3, nitrogen of 2-amino substituent of the pyrimidinyl of pyrimidinyl phosphorothioates, and the oxygen of crotonamide carbonyl in crotonamide phosphates. Bulkiness of the substituents at carbon-2 and carbon-6 of the pyrimidinyls and/or N-substituents and carbon-3 substituents of crotonamides are the steric structural components that contribute to superiority of those OPI as in ovo inhibitors of kynurenine formamidase.

Separation and toxicity of enantiomers of organophosphorus insecticide leptophos.

Enantiomers of leptophos were separated by high-performance liquid chromatography with a Whelk-O1 column using 3% dichloromethane in n-hexane as mobile phase. Toxicity tests of leptophos enantiomers and racemate were performed with daphnia. Enzyme inhibition of leptohpos was carried out by using butyryl cholinesterase from horse serum and acetylcholinesterase from housefly heads. From the inhibition test of butyrylcholinesterase, the half-inhibitory concentrations, IC(50), of (+)-leptophos, (-)-leptophos, and (+/-)-leptophos were 0.241, 1.17, and 1.05 gmL(-1), respectively. No significant difference in IC(50) in (-)-leptophos and (+/-)- leptophos was found. However, the IC(50) of (+)-leptophos was significantly different from those of the others. In the inhibition test of acetylcholinesterase, the IC(50) values of (+)-leptophos, (-)-leptophos, and (+/-)-leptophos were 14.01, 24.32, and 13.22 gmL(-1), respectively. There was no significant difference in IC(50) in (+)-leptophos and (+/-)-leptophos, although the IC(50) of (-)-leptophos was significantly different from those of the others. From these results, leptophos-both enantiomers and racemate-seems to have higher neurotoxicity for mammals than for the target insects. In the toxicity test of daphnia, the half-lethal concentrations, LC(50), of (+)-leptophos, (-)-leptophos, and (+/-)-leptophos were 0.0387, 0.802, and 0.0409 gL(-1), respectively. There is no significant difference in LC(50) in (+)-leptophos and (+/-)-leptophos. The LC(50) of (-)-leptophos is significantly higher than those of the others. From these results, (-)-leptophos has lower toxicity to daphnia.

The effect of Calcicol as calcium tonic on delayed neurotoxicity induced by organophosphorus compounds.

To examine whether delayed neuropathy is prevented or alleviated when Ca is administered to experimental animals before or after organophosphorus compounds (OPs) dosing, we observed the effects of Calcicol administration as a calcium tonic on delayed neurotoxicity by OPs in hens. The hens (n=28) were randomly divided into seven groups (four in each group). One group received glycerol formal as vehicle group, two groups received 30 mg/kg leptophos or 40 mg/kg triortho-cresyl phosphate (TOCP) (L group and T group), two groups received 2.4 mg/kg Ca(2+) (0.3 ml/kg Calcicol) 24 h before leptophos or TOCP administration, and the last two groups received 2.4 mg/kg Ca after leptophos or TOCP administration, respectively. Although delayed polyneuropathy induced by OPs could not be prevented completely by Calcicol, the clinical signs of organophosphorus-induced delayed neuropathy (OPIDN) in hens that received Calcicol soon before or after OPs administration were less severe than those in hens that received only OPs and there were significant differences in OPIDN score between groups (P<0.05). This shows that polyneuropathy and the recovery function of nerves and muscles suffering from polyneuropathy can be alleviated, as long as calcium tonic is administered before the clinical signs develop. This study offers hope of recovery to humans who are exposed to these OPs because of work, attempted suicide, accidental ingestion or other accidents, etc. Meanwhile, our results indicate further that there is a relationship between a decrease in Ca(2+) concentration in tissues and induction of delayed neuropathy.

Neurotoxic potential of six organophosphorus compounds in adult hens.

The neurotoxic potential of trichlorfon, diazinon, phosmet, dichlorvos, phosphamidon and coumaphos was evaluated for their ability to inhibit brain neurotoxic esterase (NTE) activity in adult hens. Leptophos was used as a reference neurotoxic agent. All compounds were administered at high single oral doses and the NTE and acetylcholinesterase (AchE) activities were measured at 24 h and 6 w later. With the exception of leptophos, all compounds produced severe cholinergic signs associated with > 80% inhibition of brain AchE at 24 h. On the other hand, brain NTE activity was 86% inhibited by leptophos and to lesser extents by trichlorfon (76%), phosphamidon (74%), coumaphos (70%) and dichlorvos (70%). However, none of the latter compounds produced clinical delayed neurotoxicity as was observed with leptophos. It was concluded that trichlorfon, phosphamidon, coumaphos and dichlorvos are potentially neurotoxic because of their ability to inhibit brain NTE activity, but the extent of inhibition required for development of clinical delayed neurotoxicity (> 80%) is not likely to occur with any of these compounds due to their severe cholinergic activity.

Transfer of leptophos in hen eggs and tissues of embryonic rats.

To estimate the delayed neurotoxic effect of OPs on the next generation, we tried two examinations; one was on the distribution of leptophos in tissues and eggs of hens which are highly susceptible to the delayed neurotoxic effect of OPs but have no placenta, and the other was on the concentration of OPs in tissues of both pregnant and embryonic rats which are not susceptible to the delayed neurotoxic effect but have placenta, after leptophos was administered to the mother in both experiments. First, organophosphorus compound-induced delayed neurotoxicity (OPIDN) was checked in 4 hens and the concentration of leptophos was determined in the other 16 hens after 20 adult laying hens were given 30 mg/kg leptophos (iv), a neurotoxic organophosphate. Three out of 4 hens treated with leptophos showed OPIDN. The concentration of leptophos decreased sharply in the blood, liver, brain and spinal cord from 24 to 48 hr after leptophos administration, but clearance of leptophos was relatively slow in the ovary. Leptophos in laid egg yolk was detected every day for 10 days, and the highest concentration of leptophos in egg yolk was observed on the 6th day after administration to hens. Secondly, in order to investigate the transfer of leptophos to the embryo through the placenta, we divided the thirty-two pregnant rats into 2 groups. The first group received 10 mg/kg leptophos intraperitoneally on the 17th day of pregnancy and the second received 20 mg/kg leptophos on the same day. The time-course of leptophos concentration in the tissues of pregnant and embryonic rats was checked, and the correlation between findings in the pregnant rats and the embryos was determined. The time-course of leptophos concentration in the blood, liver, brain and placenta of the rats was similar to that in hens. Leptophos concentration in the liver and brain of the embryos was equal to approximately 60% of leptophos concentration in each tissue of the pregnant rats, and the concentration of leptophos in the liver and brain of embryonic rats correlated with that in the blood and placenta of pregnant rats (p < 0.01). In both groups treated with 10 and 20 mg/kg leptophos, the concentrations of leptophos in the liver and brain of embryos were lower than that of pregnant rats in the early period after dosing, but the concentrations in embryos were inversely higher than those in pregnant rats in the latter period (48 hr). Compared with the biological half-lives of leptophos in the liver and brain of pregnant rats, these parameters in embryonic rats were 1.58 and 1.87 times, respectively. These results indicate that some of the fat-soluble organophosphorus compounds readily pass through the blood-placenta barrier into the embryos and accumulate there. Therefore, the neurobehavioral development of F1 rats exposed to some organophosphorus compounds through the placenta of pregnant rats should be further examined.

Effects of various post-treatment by phenylmethylsulfonyl fluoride on delayed neurotoxicity induced by leptophos.

Delayed neurotoxicity induced by leptophos, an organophosphorus insecticide, was intensified in hens when phenylmethylsulfonyl fluoride (PMSF) at dose of 30, 60, and 120 mg/kg body weight was administered at different time intervals (24 hr, 3 days, and 5 days) for each dose of PMSF after the hens were exposed to 30 mg/kg (i.v.) of leptophos. The scores for organophosphorus-induced delayed neuropathy (OPIDN) in all groups treated with 120 mg/kg PMSF were significantly higher than those in the group treated with leptophos only (P<0.05 or P<0.01) and the initial signs of OPIDN appeared 2 or 3 days earlier in the former groups than in the latter group. Further, the greater the PMSF post-treatment dose, the more severe were the signs of OPIDN. These findings indicate that post-treatment with PMSF promotes leptophos-induced OPIDN and reduces the period to OPIDN onset. We also examined the effects of various time intervals between PMSF administration and exposure to leptophos on the development of OPIDN. The OPIDN scores in the two groups of hen treated with PMSF on days 3 and 5 after leptophos exposure were high, especially the score of the 5 days treated group became significantly higher on the 18th and 19th day after leptophos administration than even that of the 24 hr treated group with PMSF (P<0.05). These findings suggest that variations in both the dose of PMSF and the time intervals of PMSF post-treatment may affect the delayed neurotoxicity induced by leptophos. Moreover, these results also indicate that PMSF should not be used for either the treatment or the prevention of OPIDN.

Acetylcholinesterase activity and degenerative changes in various segments of the spinal cord of hens induced by ingestion of subchronic levels of leptophos.

Changes in acetylcholinesterase (AChE) levels in the brain and various segments of the spinal cord of birds fed subchronic repeated oral doses of 0-(4-bromo-2,5 dichlorophenyl) 0-methyl phenylphosphonothioate, (leptophos, 15 mg/kg/day) is reported. The effect of leptophos on the histological structure of the spinal cord has also been described. Three birds each of four groups tested were sacrificed 7, 14, 21 and 35 days after treatment. AChE levels in the cervical, thoracic and lumbar cord were depressed from 29%-33% after 7 days to 34%-56% after 14 days of leptophos administration. This was followed by a gradual recovery at 21 days post treatment with a further decrease (23%-48%) at 35 days post treatment. Similar decreases in brain AChE levels were also observed. Spinal cord lesions in the cervical and thoracic segments were restricted to the anterior and lateral columns, while lumbar cord lesions were restricted to the anterior column. It is concluded that routine histopathology correlated with AChE levels in the cervical, thoracic and lumbar sections of the spinal cord may be useful in monitoring the onset of clinical neuropathy in laboratory animals fed prolonged subacute doses of neurotoxicants.

Developmental toxicity of desbromoleptophos in chicks: enzyme inhibition, malformations and functional deficits.

The relationship among inhibition of acetylcholinesterase (AChE), inhibition of neuropathy target enzyme (NTE), and developmental toxicity of the organophosphorus ester desbromoleptophos (DBL) was evaluated in chicks exposed on day 3 or day 15 of incubation or 10 days posthatching. DBL induced prolonged inhibition of AChE and NTE when administered either early or late in incubation, structural malformations if administered before organogenesis, posthatching paresis if administered after organogenesis, and delayed deficits of gait if administered after hatching. The posthatching paresis and abnormal gait are not determined solely by either AChE inhibition of NTE inhibition, since they occur in the absence of the latter and are not invariably seen in the presence of the former (Toxicology 49: 253-261; 1988).

Delayed neurotoxicity and toxicokinetics of leptophos in hens given repeatedly by low-dose intravenous injections.

The repeated intravenous injections (RIVInj) of 5 mg/kg/day leptophos [O-(4-bromo-2, 5-dichlorophenyl) O-methyl phenylphosphonothioate] for 3 consecutive days caused delayed ataxia in 4 out of 9 hens (44.4%). And one out of 9 hens (11.1%) given RIVInj of 3 mg/kg leptophos for 5 days was affected with ataxia. Twenty hens, however, which received a single intravenous injection (SIVInj) of 15 mg/kg leptophos did not exhibit any delayed neuropathic signs at all. Thus, delayed neurotoxicity was increased by the subdividing RIVInj of the critical dose which was shown in the SIVInj of leptophos. The leptophos concentration in plasma and liver decreased very rapidly after finish of either SIVInj or RIVInj. Although no significant differences were observed in the biological half life of leptophos in plasma by different dosages, the mean level of leptophos decreased significantly with frequency of injections. On the contrary, the evident accumulation of leptophos was observed in only sciatic nerve with RIVInj. Leg muscle maintained relatively high level of leptophos after the last injection. These results suggest that leptophos seems to transfer from blood to affinitive tissues such as sciatic nerve or leg muscles and to accumulate there easily in initial stage after repeated iv injections, and that this causes the enhancement of neuropathy with repeated administrations of divided critical dose of leptophos in both iv and oral administration.

Effects of fenthion, fenitrothion and desbromoleptophos on gait, acetylcholinesterase, and neurotoxic esterase in young chicks after in ovo exposure.

Previous studies have demonstrated that gait is affected in chicks exposed to organophosphorus esters (OPs) that induce delayed neurotoxicity (OPIDN) in adult hens. To investigate the developmental relationship between such functional deficits and OPIDN, chicks were exposed to 3 OPs with different OPIDN potential. Desbromoleptophos (DBL) induces OPIDN in adult hens; fenthion (FEN) has uncertain OPIDN potential; fenitrothion (FTR) does not induce OPIDN. Chicks were treated by injection into the egg on day 15 of incubation, after the presumed period of OP-induced structural teratogenesis. AChE and neurotoxic esterase (NTE) were assayed during incubation and in parallel with post-hatching evaluations of gait. DBL, 125 mg/kg in ovo, caused paralysis in 70% of chicks after hatching. The gait of surviving chicks was affected for at least 6 weeks and marked by toes curling under. NTE was inhibited until 10 days post-hatching and AChE until hatching. FEN did not inhibit NTE significantly, but AChE was significantly inhibited until hatching. Chicks exposed as embryos to FEN were hyperactive and aggressive. Gait was still affected 6 weeks after treatment with 3 mg/kg FEN. FTR at 125 mg/kg inhibited AChE until day 10 post-hatching, but neither inhibited NTE nor affected gait. The growth of OP-exposed chicks was not significantly decreased, so the decreased length and increased width of the stride could not be ascribed to stunted growth. We conclude that OPs cause irreversible effects on gait that are not related to their defined neurotoxic effects, since altered gait (1) occurs below the age of sensitivity to OPIDN, (2) is seen in the absence of NTE inhibition and (3) does not invariably accompany AChE inhibition.

Effects of multiple dosing of fenthion, fenitrothion, and desbromoleptophos in young chicks.

The effects of multiple doses of desbromoleptophos, fenitrothion, and pure fenthion on brain acetylcholinesterase (AChE), brain neurotoxic esterase (NTE), and walking were investigated in immature chicks, below the age of sensitivity to organophosphorus ester-induced delayed neurotoxicity (OPIDN). Ten milligrams per kilogram per day of delayed neurotoxicant desbromoleptophos (DBL), 15 mg/kg.d of the non-neurotoxicant fenitrothion (FTR), and 3 mg/kg.d of the suspected neurotoxicant fenthion (FEN) were given orally for 7 d to 3-d-old chicks. Behavioral testing was performed for treated and control chicks on various days after treatment. Brain NTE and AChE assays were carried out for treated and control chicks on each day of behavioral testing. DBL altered gait and inhibited both NTE and AChE; FEN altered gait and inhibited AChE but not NTE; and FTR did not affect gait, while inhibiting AChE but not NTE. NTE and AChE inhibition were 70% and 55%, respectively, 24 h after the last treatment, for the chicks treated with DBL. NTE returned to normal levels by around d 25 and AChE by 20 d after the last treatment. FTR caused more than 50% AChE inhibition but no NTE inhibition, 24 h after last treatment. NTE inhibition for the FEN-treated chicks never exceeded 11% during the whole period of the experiment, whereas 54% inhibition of AChE was seen 1 d after last treatment. DBL and FEN significantly altered the gait of treated chicks, but the non-OPIDN-inducing FTR did not. This study confirms that alterations in the gait of young chicks are not direct consequences of either NTE or AChE inhibition, and that fenthion-induced functional deficits can be distinguished from classical OPIDN.

Toxicological screening for organophosphorus-induced delayed neurotoxicity: complications in toxicity testing.

The acute biocidal effects of organophosphorus pesticides are a central feature of modern agricultural chemistry, and also define the concerns of regulatory toxicology. Less well known, but more complex and idiosyncratic, is the potential for some agents to produce a delayed and progressive polyneuropathy--Organophosphorus Induced Delayed Neurotox-icity (OPIDN). On three occasions during the past ten years, the National Institute for Occupational Safety and Health (NIOSH) had been asked to evaluate human delayed neurotoxicity from three commercially available pesticides. These were leptophos, fenthion, and isofenphos. In each case, human disease was either observed or suggested by specialized toxicity testing. The reasons that federally recommended screening measures failed to identify a potential for human neurotoxicity were not accidental, but stem from a systematic approach that focuses on a traditional definition of acute lethal toxicity. The oral single dose study on one species appears to be insufficient for recognizing the delayed neurotoxic hazard of many representatives of this chemical class. The recent addition of a recommended biochemical assay--neurotoxic esterase (NTE)--to federal guidelines potentially improves sensitivity, but it is purely adjunctive and does not amend underlying ambiguities in selecting the dose and route of administration. It is also quite probable that human neurotoxicity may be a potential hazard from exposure to more than the handful of organophosphorus pesticides that have been described in the literature.

Delayed neurotoxicity in the wild mallard duckling caused by the organophosphorus insecticides cyanofenphos and leptophos.

The susceptibility of wild mallard ducklings to the delayed neurotoxic effect of the neurotoxic organophosphorus insecticides cyanofenphos and leptophos was evaluated following a daily dosing regimen. Ducklings were treated daily with either cyanofenphos or with leptophos at different dose levels for 90 days, or until they died, or became paralyzed. A control group of ducklings given corn oil at 1 ml/kg daily for 90 days was used for comparison. All treated birds were observed daily for any clinical signs of neurotoxicity during the course of this study. All of the surviving ducklings that were treated with cyanofenphos at 4 mg/kg/day or leptophos at 10 mg/kg/day developed clinical signs of delayed neurotoxicity after 7 to 11 weeks of intoxication. Symptoms included leg weakness, ataxia, severe ataxia and paralysis. The observed clinical signs were confirmed by histological changes found in the spinal cords of the treated birds. These changes were of the type associated with organophosphorus-induced delayed neuropathy (OPIDN). These results demonstrate that wild mallard ducklings are susceptible to OPIDN and this avian species can be used in screening organophosphorus compounds for such effect.

In vivo inhibition of chicken brain acetylcholinesterase and neurotoxic esterase in relation to the delayed neurotoxicity of leptophos and cyanofenphos.

An equimolal single dose (1 mmole/kg) of leptophos or cyanofenphos was given orally to chickens to assay the clinical and biochemical neurotoxic effects of these two organophosphorus insecticides. Parathion and TOCP at 2 and 1000 mg/kg of chicken body weight were tested in the same manner as negative and positive neurotoxicants, respectively. Three birds of each of five groups tested were sacrificed 1,2,3,7,14,21 and 28 days after treatment and the brains were taken for the biochemical tests. Acetylcholinesterase (AChE) and neurotoxic esterase (NTE) activities were determined in the brain microsomal fractions. In addition, the AChE activity in the brain soluble fractions was measured. Clinical observations indicated that leptophos-, cyanofenphos- and parathion-treated chickens became acutely poisoned but recovered from the typical cholinergic signs in a day or two. However, about 10 to 15 days later leptophos- and cyanofenphos-treated chickens developed the characteristic leg weakness and unrecoverable ataxia seen in birds given TOCP. The biochemical results indicated that cyanofenphos followed by leptophos and parathion produced more in vivo AChE inhibition than that produced by TOCP in both chicken brain soluble and microsomal fractions. Results suggested that there are no correlations between the in vivo effect of TOCP, leptophos and cyanofenphos on AChE and phenyl valerate-total hydrolyzing activities and the ability of these chemicals to produce neuropathy in hens. The results obtained from this study of the in vivo effect of the tested compounds on chicken brain NTE activity present an acceptable correlation between the inhibition of this enzyme and the ability of these chemicals to induce neuropathy. The mechanism and explanation for this correlation are presented. The in vivo effect of the tested compounds on the chicken brain NTE activity was determined using the indirect and a new direct method. The data presented in this report suggested that the new direct technique of assaying NTE activity using 4-nitrophenyl valerate (4-NPV) as substrate, can be useful in the in vivo screening studies of organophosphates for their ability to induce neuropathy in hens.

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.

Peripheral nerve damage in chicks following treatment with organophosphorus compounds in ovo.

Chick embryos were treated with tri-o-cresyl phosphate (TOCP) or leptophos, organophosphorus compounds that cause delayed neurotoxicity. Embryos received either TOCP (62 or 250 microliter/kg egg) or leptophos (125 to 750 mg/kg egg) Day 14 of incubation and were examined after hatching for nerve damage. The high doses caused high embryo mortality. Chicks which survived the high doses were grossly ataxic from hatching until the study was ended at 3 weeks posthatching. On Posthatching Day 2, many degenerating nerve fibers were observed in the profundus/superficialis peroneus nerve in chicks surviving the high doses. TOCP-treated chicks were followed in detail for neuromuscular changes. Twenty days after hatching there were fewer large nerve fibers in the distal ischiadic nerve compared with controls and the largest nerve fibers were absent in the peroneus profundus nerve. Consistent with the evidence of denervation there was increased terminal branching of motor axons in femoral (sartorius) and tibial (external gastrocnemius and peroneus longus) leg muscles. The leg nerves of chicks treated with the low dose of TOCP did not show either an excessive number of degenerating nerve fibers or a detectable loss of large nerve fibers. However, terminal branching of motor axons was increased in the external gastrocnemius and peroneus longus muscles of 5- and 15-day-old chicks, followed by recovery by Day 25. The evidence is interpreted as a distal axonopathy in chicks treated with TOCP during late embryonic development.