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.

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.

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.

Toxin-induced inhibition of nerve terminal growth.

An in vivo model to permit the quantitative study of the effects of neurotoxins upon nerve terminal growth has been developed and used in practice to test the ability of methyl mercury chloride, leptophos, ethanol, morphine and acrylamide to inhibit nerve terminal growth. The model utilized the sympathetic innervation of rat submaxillary salivary glands. Nerve terminal growth was induced following crush of the sympathetic axons which innervated the submaxillary gland and quantified by measuring the return towards normal values of the density of sympathetic innervation in denervated glands. The nerve crush reduced the density of sympathetic innervation to a very low value but nerve growth began within 9 days and was sufficient to restore the density of innervation to approximately 70% of normal values in three weeks. Restoration of the original density of innervation took between 40 and 60 days. The major advantage of the model is its ability to detect and quantify 30% - 100% inhibition of nerve terminal growth. The major disadvantage is the time required to measure the density of sympathetic innervation though this can be minimized in a number of ways which are discussed. Acrylamide inhibited sympathetic nerve terminal growth without causing degeneration of sympathetic neurones. This demonstrates that toxins can selectively inhibit nerve terminal growth which will result in altered neuronal circuitry and abnormal function. Methyl mercury chloride, leptophos, ethanol and morphine did not inhibit sympathetic nerve terminal growth.

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.

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.

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).

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.

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.

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.

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.

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.