DDE increases the toxicity of parathion to coturnix quail

Adult male Japanese quail (Coturnix coturnix) were exposed to DDE or chlordane in the diet and subsequently dosed with parathion or paraoxon. Pretreatment with 5 or 50 ppm DDE in the diet for 12 weeks resulted in increased cholinesterase (ChE) activity in plasma, but not in the brain. Dietary concentrations of 5 and 50 ppm DDE caused increased susceptibility of quail that were challenged with parathion or paraoxon. The increased mortality resulting from DDE pretreatment was reflected in brain ChE inhibition. The synergistic action of DDE was apparent after 3 days of exposure to 50 ppm DDE and 1 week of exposure to 5 ppm DDE. Birds exposed for 3 weeks to 5 or 50 ppm DDE retained their DDE-potentiated sensitivity to parathion after 2 weeks on clean diet. Chlordane pretreatment resulted in decreased susceptibility (antagonism) to parathion, but not to paraoxon dosage. Implications of differing responses in ChE and mortality among controls, DDE-, and chlordane-pretreated birds after parathion or paraoxon dosage are discussed.     

Insensitive acetylcholinesterase,high glutathione-S-transferase,and hydrolytic activity as resistance factors in a tetrachlorvinphos-resistant strain of house fly

The gene producing insensitive acetylcholinesterase in a tetrachlorvinphos-resistant strain of house fly was introduced into the genome of a susceptible strain. The resistance to a number of organophosphorus insecticides, which was high in the original strain (234- and more than 800-fold for paraoxon and tetrachlorvinphos, respectively), was much lower in the resulting strain, ranging from 53-fold for paraoxon to 3.9-fold for tetrachlorvinphos. Synergists further decreased these factors. The reduction in sensitivity of the acetylcholinesterase in vitro was about 20-fold for both inhibitors. The Michaelis parameters for hydrolysis of acetylcholine and acetylthiocholine of the mutant and normal acetylcholinesterase showed only minor differences. An increased rate of metabolism of the insecticides was found in the original strain. In vitro, glutathione-dependent detoxication was from 9- to 120-fold that of a susceptible strain, depending on the insecticide and the conditions used. The enzyme de-alkylated parathion and methylparaoxon, but there was no de-arylation of parathion, although it is important in other strains. In vitro, paraoxon and methylparaoxon were also hydrolysed, and this is not so in the susceptible strain. The high resistance present in the original strain seems to be due to joint action of at least three mechanisms.     

The relationship of esterases to organophosphorus insecticide tolerance in mosquitofish

The levels of peripheral acetylcholinesterase and carboxylesterases and their organophosphate sensitivities were studied in two populations of mosquitofish (Gambusia affinis). One population, highly resistant to organochlorine insecticides, demonstrated a low tolerance to parathion and methyl parathion. The organochlorine resistant fish possessed higher levels of both peripheral acetylcholinesterase and carboxylesterases. The sensitivities of these esterases to organophosphate inhibition were the same in both populations. The esterases were more sensitive to paraoxon than to methyl paraoxon. Carboxylesterases were far more sensitive to organophosphate inhibition than was acetylcholinesterase. Carboxylesterases, by their higher affinity for the organophosphates, may serve to protect acetylcholinesterase from inhibition.     

Organophosphate degradation by insecticide-resistant and susceptible populations of mosquitofish (Gambusia affinis)

Phosphorothionate and phosphate degradation was investigated as a factor which could influence the tolerance of organochlorine compound-resistant and susceptible mosquitofish (Gambusia affinis) to parathion and methyl parathion. The greater toxicity of methyl parathion than parathion can be attributed in part to a higher rate of degradation of methyl paraoxon than paraoxon (7-fold), but not to any difference in phosphorothionate dearylation. Resistant fish possess higher levels of microsomal mixed-function oxidases which can degrade methyl parathion (1.3-fold); these higher levels could contribute to the increased methyl parathion tolerance by this population over the susceptible population. Environmentally induced tolerance to parathion in the resistant population may be the result of increased levels of parathion degradation by induced mixed-function oxidases which can dearylate parathion. The increased tolerance of either insecticide by the resistant population is not caused by degradation of the phosphates by phosphotriesterases.     

Degradation of parathion applied to peach leaves.

Parathion was applied to peach trees in three different formulations 70 days before harvest. Leaf samples were taken periodically through the 70-day period and gas-liquid chromatographic analyses were conducted for dislodgable and penetrated residues. Analyses were also conducted for paraoxon and the s-ethyl isomer of parathion. Punched samples were compared to whole-leaf samples; generally residue levels for both types corresponded closely. A new experimental formulation, encapsulated parathion, produced highest levels of total parathion throughout the 70-day study, but even this formulation resulted in low total residue levels around 1 ppm at time of harvest. Degradation of the s-ethyl isomer of parathion was generally very rapid in all formulations studied. Dislodgable residues of paraoxon may be significant in some formulations and should be included in parathion degradation studies. Much of the parathion found on peach leaves throughout the growing season was dislodgable residue, but this depended considerably on the formulation used.     

Action of pesticides on earthworms. Part I: The toxicity of cholinesterase-inhibiting insecticides to earthworms as evaluated by laboratory tests

The action of seven cholinesterase-inhibiting pesticides [aldicarb, carbaryl, carbofuran, oxamyl, paraoxon (diethyl 4-nitrophenyl phosphate) parathion and trichloronate], the organochlorine insecticide, gamma-HCH, and the nematicide potassium N-hydroxymethyl-N-methyl(dithiocarbamate) (PHMD) on four earthworm species was investigated by laboratory toxicity tests. Eisenia foetida was the most tolerant species to the pesticides tested. Aldicarb was the most toxic pesticide to this species, causing severe dehydration prior to death or at sublethal concentrations. Aldicarb was also toxic to the other species (Allolobophora caliginosa, A. chlorotica and Lumbricus rubellus), while oxamyl, the other oxime carbamate, was not toxic to any of them. Carbaryl and carbofuran at low concentrations were lethal to A. caliginosa, A. chlorotica and L. rubellus, but E. foetida could tolerate high concentrations without dying, although low concentrations severely affected its ability to work the soil or to disappear from the soil surface. Paraoxon, parathion, trichloronate and gamma-HCH were moderately toxic with low lethal effect to all species. The ability to work the soil was moderately affected by parathion, trichloronate and gamma-HCH. PHMD was toxic to all the species. The lethal and non-lethal effects of the pesticides are discussed in relation to their possible biochemical mode of action in earthworms, and the data are compared with published information from field trials.     

Action of pesticides on earthworms. Part II: Elimination of parathion by the earthworm Eisenia foetida (Savigny)

The earthworm, Eisenia foetida, eliminated parathion and carbofuran at first order rates when continually rinsed in water after treatment with the pesticides. This experiment was also carried out on Lumbricus rubellus for comparison. Carbofuran which is more soluble in water, was eliminated quicker than parathion. The later rate of elimination was very similar for the two species, but immediately after injection the rate was much higher in E. foetida. The metabolism of 1-ethyl¹⁴C labelled parathion and paraoxon (diethyl 4-nitrophenyl phosphate) was studied in E. foetida. The worm was able to convert parathion to paraoxon by a rather slow process although this metabolite could not be detected in the worms due to its rapid transformation to diethyl hydrogen phosphate. Indirectly, paraoxon can be postulated as a parathion metabolite because of a progressive depression of cholinesterase level observed after treatment with parathion. Small amounts of diethyl hydrogen phosphate were detected as a metabolite of parathion; this is also an indication of paraoxon formation. During the 30 h following injection of parathion, only 4.4% of the applied dose was recovered as water-soluble metabolites (2.8% in the worms and 1.6% in the sand surrounding them), while 52% was recovered as unmetabolised parathion. Because of inefficient injection, only 70-59% of the dose thought to be injected was recovered. Therefore the part of the actual applied dose that remained unmetabolised was probably even greater (88%). Five days after injection of parathion, 15 and 9.3 % of the recovered radioactivity in the surrounding sand and in the worm extracts, respectively, was identified as O,O-diethyl O-hydrogen phosphorothioate, 3.7 and 7.0% as diethyl hydrogen phosphate, 8.8 and 3.3% as O-ethyl O-4-nitrophenyl O-hydrogen phosphorothioate (desethylparathion) and/or O-4-aminophenyl O,O-diethyl phosphorothioate, while 70.3 and 80.4% was unmetabolised parathion. Paraoxon was very quickly hydrolysed to diethyl hydrogen phosphate in vivo and in vitro. The in-vitro hydrolysis was associated with a microsomal fraction and was not inhibited by ethylenediaminetetra-acetic acid or 4-(chloromercuri)benzoic acid, and incompletely by aldicarb. Cholinesterase and arylesterase were therefore excluded as enzymes responsible for the activity.     

The toxicological significance of paraoxon deethylation

The in vivo formation of deethylation and hydrolytic products of paraoxon degradation after parathion or paraoxon administration was nearly equal in control male rats, and the relative abundance of metabolites was not appreciably altered by pretreatment of rats with enzymeinducing agents. However, pretreatment with inducers dramatically increased the oxidative paraoxon O-deethylase of male rat liver while having little effect on hydrolytic enzymes. Prior to induction, the hepatic O-deethylase activity was greatly inferior to the various hydrolytic enzymes, but nearly equal levels of both enzyme systems were found after induction. These results indicate that a large portion of the hepatic hydrolases detected in vitro is not active in vivo. It also appears that the majority of the induced hepatic deethylase was not involved in vivo at the dosage levels employed. The in vivo metabolism of monoethyl paraoxon was also demonstrated. The predominant metabolite of ethyl-[1-¹⁴C]monoethyl paraoxon is ¹⁴CO₂, while phenyl-[1-¹⁴C]monoethyl paraoxon yielded 4-nitro[1-¹⁴C]phenol. Paraoxon deethylation was also shown to be an important detoxication mechanism in female rats and male mice and must be considered in interpreting the toxicological properties of parathion and paraoxon.     

Factors affecting the sequential acquisition by Danish houseflies (musca domestica L.) of resistance to organophosphorus insecticides

The prolonged use of dimethoate, introduced into Denmark to control houseflies (Musca domestica L.) that had become resistant to parathion and diazinon, resulted ultimately in dimethoate resistance. Selection with dimethoate led to the disappearance of the hydrolytic phosphatase, a major mechanism of resistance to parathion and diazinon, and its replacement by the acetylcholinesterase AChER with somewhat decreased sensitivity to inhibition by organophosphorus (OP) insecticides. The hydrolytic phosphatase probably disappeared because low substrate turn-over made it ineffective against dimethoxon (O, O-dimethyl S-methylcarbamoylmethyl phosphorothioate, also known as omethoate). which accumulates at higher concentrations than paraoxon (diethyl4-nitrophenyl phosphate) in the haemolymph. Dimethoate selected AChER preferentially because it improved the chances of houseflies surviving against the relatively poor AChE inhibitor dimethoxon, whereas its relatively small insensitivity to OP insecticides, unimportant against good inhibitors such as paraoxon, prevented its selection by parathion.     

Metabolism of [14C]parathion and [14C]paraoxon in isolated,perfused rat livers

Perfusion of ¹⁴C-(ring)-parathion or ¹⁴C-(ring)-paraoxon with blood through isolated, intact rat livers resulted in the rapid degradation of these insecticides. Degradation was negligible in the absence of rat liver (controls), thus demonstrating the capacity of the liver per se to effectively degrade these compounds. Of the total radiocarbon recovered after liver perfusion with [¹⁴C]parathion, 33 % could be attributed to unchanged [¹⁴C]parathion (similarly distributed between the liver and the blood) while 67.9 % was degraded to water soluble compounds and 2.5% was converted to organic soluble paraoxon and traces of p-nitrophenol. Nearly all of the [¹⁴C]paraoxon, however, was degraded by the intact rat liver, resulting in water soluble products that amounted to 98.5% of the total radiocarbon recovered. Unexplained losses of radiocarbon with the perfusion apparatus used were lower in the presence of rat liver which degraded the insecticides to more water soluble compounds. The water soluble degradation products produced from [¹⁴C]parathion and [¹⁴C]paraoxon were non-toxic to mosquito larvae (Aedes aegypti L.). These ring-labelled products were found to be conjugated p-nito-phenol. Nearly all of the water soluble radiocarbon was located in the perfused blood, while only small amounts (1.8 to 3.0% of recovered) were excreted via the bile or were associated with the liver tissue (1.3 to 1.8 % of recovered).     

Action of pesticides on earthworms. Part III: Inhibition and reactivation of cholinesterases in Eisenia foetida (Savigny) after treatment with cholinesterase-inhibiting insecticides

The degree of inhibition and the rate of recovery of total cholinesterase level were investigated after in-vivo treatment of the earthworm Eisenia foetida (Savigny) with aldicarb, carbaryl, carbofuran, oxamyl, and the O-analogues of bromophos (bromo-phosoxon), parathion (paraoxon), parathion-methyl (paraoxon-methyl), and trichloronate (trichloronatoxon). The results can be explained by the presence of two cholinesterases (E1 and E2), which were demonstrated by in-vitro inhibition studies. E1 was the most sensitive to all the inhibitors tested. The in-vivo reactivation rate after inhibition with paraoxon was very high for E1 and very low for E2. Therefore, pretreatment with paraoxon probably increased the toxicity of carbaryl. It was concluded that the toxicity towards E. foetida can be explained in terms of cholinesterase inhibition, provided that the action on the two cholinesterases and the worms' tolerance for a transient but almost total cholinesterase depression are taken into account. Data are given showing that two other species of earthworms also contain different types of cholinesterases with respect to reactions with inhibitors.     

Paraoxon formation and hydrolysis by mammalian liver

In vitro studies of the desulfuration of parathion at 37°C by hepatic tissue from males and females of nine mammalian species revealed sex and species variation in initial rates of parathion desulfuration and arylesterase-catalyzed hydrolysis of the oxygen analogue, paraoxon. Double reciprocal plots of initial rates of parathion activation for representative males and females of each species gave K_m values ranging from 0.2 × 10^?4?1.0 × 10^?4M parathion. Guinea pigs and rats were the only animals showing sex differences in activation, males possessing higher desulfurating abilities than the corresponding females. Based upon the sex possessing the higher desulfurating ability, the species pattern of decreasing activity was hamster > guinea pig > mouse > rat > rabbit > bovine > dog > porcine > cat. Studies of paraoxon hydrolysis indicated that only rats showed sex differences in hydrolysis, males possessing higher arylesterase activity than females. The species pattern of decreasing hydrolytic activity was in the order mouse > bovine > rat > guinea pig > rabbit > hamster > cat > dog > porcine.     

Toxicity in the brain of organophosphate insecticides: Comparison of the toxicities of metabolites with parent compounds using an intracerebral injection method

The toxicity in the brain of several parathion, fenthion, and fensulfothion insecticides and their toxic metabolites was determined by a technique of directly injecting the compounds into the region of the third ventricle of conscious mice, an area rich in cholinesterase activity. The results were compared on a body weight basis to the toxicity of these compounds when given by ip and oral routes. The results show that there is a direct relationship between the relative inhibition of cholinesterase activity in the brain by the organophosphates (e.g., methyl paraoxon, Sumioxon, and some members of the fenthion series) and the toxicity of these compounds in the brain. Methyl paraoxon and Sumioxon were found to be very toxic in the brain, Sumioxon being three to four times less toxic than methyl paraoxon. This is of the same order of effect of these compounds in inhibiting cholinesterases. It is concluded that any selective effects of Sumithion compared with methyl parathion must be due to the greater rate of metabolism of Sumithion to less toxic metabolites as well as to the lower toxicity of the oxon metabolite and not due to the relative rates of penetration of the toxic oxygen metabolites as previously suggested [J. Miyamoto, Agr. Biol. Chem.28, 422 (1964)]. A gas-liquid chromatographic method was employed to assess the distribution in the brain following intracerebral injection of the parathion-type compounds. The results suggest that there may be intracerebral metabolism of thionophosphates in vivo.     

The photodegradation of parathion and chlormephos

Irradiation (～350 nm) of oxygenated acetonitrile solutions of parathion containing sensitisers such as benzoin and α-dicarbonyl compounds leads to oxidative desulphurisation at phosphorus, producing paraoxon in reasonable yield. Other degradation reactions also occur, probably as a result of the presence of the 4-nitrophenyl group in parathion. Under similar irradiation conditions, chlormephos is desulphurised at both the thionate and thiolate moiety. The reactions are rationalised as occurring via peroxy radicals which are generated from the sensitisers. Reaction was not observed when dyes, e.g. rose bengal, were used as sensitisers, which accords with the view that thionates and thiolates are not reactive towards singlet oxygen.     

Photochemistry of parathion in the plant cuticle environment: Model reactions in the presence of 2-propanol and methyl 12-hydroxystearate

On UV-irradiation (λ > 280 nm), photodegradation of parathion dissolved in 2-propanol took place mainly by reduction of the phenylnitro group. The photoreduction intermediates so formed then combined, yielding primarily azoxyparathion which, upon further irradiation, rearranged into 2-hydroxyazoparathion. When parathion was irradiated in the presence of the cutin acid 12-hydroxystearic acid (as the methyl ester), azoparathion, azoxyparathion and 2-hydroxyazoparathion were the dominant photoproducts if the reaction was performed in thin-layer films of the 12-hydroxystearate. Irradiation of parathion dissolved in a solution of the 12-hydroxystearate in cyclohexane yielded mainly paraoxon. Furthermore, in all experiments, photolysis of the P—O ester bonds of the parent compound as well as of the photoproducts was observed at low levels.     

A-esterase activities in relation to the differential toxicity of pirimiphos-methyl to birds and mammals

Pirimiphos-methyloxon (2-diethylamino-6-methylpyrimidin-4-yl dimethyl phosphate) the phosphate analogue of pirimiphos-methyl, and paraoxon (diethyl 4-nitrophenyl phosphate) the phosphate analogue of parathion were used as substrates to determine the esterase activity in plasma. Aryl groups released were measured by high-performance liquid chromatography or with a recording spectrophotometer. In a survey of 14 species of birds representing six different avian orders, the plasma esterase activities (expressed as nmol min^?1 ml^?1 of plasma) were always low, ranging from 0-71 for pirimiphos-methyloxon and from 0-0.63 for paraoxon. By contrast, mammalian activities were very much higher than these, and in no case was a sample of mammalian plasma less than 13 times more active than any sample of avian plasma using the same assay procedure. It is concluded that birds are deficient in A-esterase activity towards pirimiphos-methyloxon and paraoxon. The importance of this deficiency in determining the relatively high susceptibility of birds to these and other organophosphorus insecticides is discussed.     

Effect of parathion on water intake in the rat

The effect of several subtoxic doses of parathion on water intake was studied in conscious rats with centrally induced thirst. A potent and long-lasting antidipsogenic effect was obtained when single doses ranging from 100 to 600 μg of parathion/100 g body wt were ip given to rats in which thirst was induced either by 24 hr water deprivation or sc injection of hypertonic NaCl solution. A significant thirst inhibition was also obtained when paraoxon or dichlorvos (DDVP) was given. The antidipsogenic effect was prevented by the previous administration of diacetylmonoxime (DAM), an acetylcholinesterase reactivator, but not prevented by pralidoxime (2-PAM), another reactivator that is unable to cross the blood-brain barrier (BBB). Thirst inhibition induced by parathion administration was also prevented when muscarinic cholinergic blockers were previously given, such as atropine sulfate. Acetylcholinesterase activity (AChE) was not inhibited on either blood or hypothalamic tissue with the different doses of parathion given. Locomotor activity and emotionality also remained unaltered. The results obtained indicate that the antidipsogenic effect of the organophosphorus pesticide is centrally produced by stimulation of muscarinic cholinergic pathways.     

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

The metabolism in vivo and in vitro of [¹⁴C]parathion and [¹⁴C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina. Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.     

Glutathione-dependent degradation of parathion and its significance for resistance in the housefly

Glutathione-dependent degradation of parathion was studied in six strains of houseflies to find out whether it might be important as a cause of resistance. When supernatant fractions of high-speed centrifuged homogenates were fortified with glutathione and incubated with parathion, water-soluble products were formed. The rate of parathion detoxication was highest in a malathion-resistant strain (c. 4 μg parathion degraded per abdomen per hour), lowest in a susceptible strain, and intermediate in some other organophosphate-resistant strains. In one of the latter strains, E₁, the gene for glutathione-dependent degradation is located on the second chromosome, closely linked with gene cm⁺. This is the same chromosome on which gene a for low ali-esterase activity and hydrolytic detoxication of paraoxon is located. It is not likely that the gene for glutathione-dependent degradation is identical with gene a, since it is also present in strain Nie which lacks gene a, and, therefore, the presence of a separate gene which is called gene g is postulated.     

Mode of action of N-ethylmaleimide as a parathion synergist in Triatoma infestans

N-Ethylmaleimide (NEM) injected in sublethal doses enhanced the triatomicide effect of parathion. The study of the possible causes responsible for this synergistic effect in Triatoma infestans showed (1) a depletion of SH groups in the hemolymph and the central nervous system (CNS) caused by NEM; (2) protein and lipid covalent binding of [¹⁴C]NEM in the CNS; (3) partial inhibition by NEM of the glutathione S-transferase detoxifying pathway; (4) enhanced penetration of parathion and paraoxon into the insect CNS pretreated with NEM; (5) significant change in the electrophoretic and histochemical pattern of acetylcholinesterase inhibition in NEM-pretreated insects. The results obtained suggest that NEM synergizes parathion in adult T. infestans by inhibiting glutathione-dependent detoxification and enhancing penetration into the CNS.