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.     

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.     

Methoxyresorufin as a substrate for the fluorometric assay of insect microsomal O-dealkylases

Methoxy-, ethoxy-, propoxy-, and butoxyresorufin were prepared and tested as substrates for the fluorometric assay of O-dealkylation by the mixed-function oxidase system in house flies, Musca domestica L. Methoxyresorufin proved to be the most suitable substrate because of the favorable reaction rates. This sensitive assay can be performed with a minimum of microsomal protein (<0.3 mg/ml) in 5 min or less. The apparent K_m and maximum velocity were calculated as 2.88 μM and 0.27 nmol of resorufin produced/min/mg of microsomal protein, respectively. The O-dealkylation reaction required O₂ and NADPH and was inhibited by CO.     

Metabolism of cisanilide (Cis-2,5-dimethyl-1-pyrrolidinecarboxanilide) by rat liver microsomes

Metabolism of [phenyl-¹⁴C] and [(2,5) pyrrolidine-¹⁴C] cisanilide was investigated in vitro with microsomal preparations from rat liver. Microsomal activity was associated with a mixed-function oxidase system that required O₂ and NADPH and was inhibited by CO. Two major ether-soluble metabolites were isolated. They were identified as primary oxidation products: 2-hydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide (A) and 4′-hydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide (B). Minor ether-soluble metabolites were also isolated. Precursor product studies and qualitative thin layer chromatography analysis of [pyrrolidine-¹⁴C] and methylated [phenyl-¹⁴C] hydrolysis products suggested that these metabolites were secondary oxidation products formed from metabolites A or B. One of these metabolites appeared to be the dihydroxy product 2,4′-dihydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide. Crude microsomal preparations (postmitochondrial supernatant fractions) also formed small quantities (<10%) of polar metabolites. Enzyme hydrolysis with β-glucuronidase (Escherichia coli) indicated that approximately 50% of these metabolites were glucuronides. Similarities and differences in cisanilide oxidation in vivo in plants and in vitro with rat liver microsomal preparations were discussed.     

Monooxygenases from soybean root nodules: Aldrin epoxidase and cinnamic acid 4-hydroxylase

The metabolism of aldrin and trans-cinnamic acid within the root nodules of soybean (Glycine max. cv. Forrest) was investigated. In vitro studies with microsomal preparations revealed the presence of two distinct monooxygenase enzymes requiring NADPH and molecular oxygen. This was shown by different distributions in membrane fractions from sucrose density gradients. In addition, cinnamic acid hydroxylase was sensitive to carbon monoxide (CO), similar to cytochrome P-450-dependent monooxygenases, whereas aldrin epoxidation was not. Cyanide ion, strongly inhibited the epoxidase, without affecting hydroxylase activity. The superoxide radical (O₂⁻) scavengers, superoxide dismutase and norepinephrine, inhibited aldrin epoxidation but allowed greater cinnamic acid 4-hydroxylase activity. These results indicate aldrin epoxidase, in contrast to cinnamic acid hydroxylase, does not involve cytochrome P-450, and may instead utilize a peroxidase-like hemoprotein.     

Role of a microsomal carboxylesterase in reducing the action of malathion in eggs of Triatoma infestans

A microsomal malathion carboxylesterase present in Triatoma infestans eggs was active from the first day of embryonic development. This microsomal egg malathionase (MEM) showed a unique band in polyacrilamide gel electrophoresis (PAGE) when malathion was used as substrate. In vivo metabolism of [¹⁴C]malathion during all embryonic development showed a 10% degradation due to carboxylesterases. The in vitro evaluation of the same metabolic pathway catalyzed by the microsomal fraction of T. infestans eggs showed partial inhibition by paraoxon. α- and β-malathion monoacids were identified as the main metabolites of the in vivo and in vitro metabolic pathways. The carboxylesterase band that appeared in PAGE (MEM) from the first day of embryonic development could be the main cause of malathion tolerance in T. infestans eggs.     

The in vitro metabolism of azinphosmethyl by mouse liver

The in vitro metabolism of methoxy-¹⁴C- or ³²P-azinphosmethyl by subcellular fractions from mouse liver was studied. The major degradative activity was associated with the microsomal and soluble fractions. Since the microsomal activity required NADPH and was inhibited by carbon monoxide, it is reasonable to assume that the mixed function oxidases were involved. The microsomal system catalyzed the dearylation reaction resulting in the formation of dimethyl phosphorothioic acid and dimethyl phosphoric acid. The system was also responsible for the oxidative desulfuration of azinphosmethyl resulting in the formation of azinphosmethyl oxygen analog.     

An evaluation of the role of oxidative enzymes in Colorado potato beetle resistance to carbamate insecticides

Carbofuran and carbaryl LD₅₀ values were determined with and without piperonyl butoxide pretreatment for a resistant (New Jersey) and two susceptible (Utah and Netherland) populations of Colorado potato beetle larvae. Similar bioassays were conducted with carbofuran for resistant (Rutgers) and susceptible (NAIDM) adult house flies. The degree of resistance development by New Jersey Colorado potato beetles (RR = 848) was greater than that of the laboratory-selected colony of Rutgers house flies (RR = 583). Comparisons of synergist difference calculations including “percentage synergism” (%S), “log percentage synergism” (L%S), and “relative percentage synergism (R%S) for the resistant (R) and the susceptible (S) populations indicated the possibility that monooxygenases and other resistance mechanisms may be involved in Colorado potato beetle resistance to these carbamates. Monooxygenase involvement in resistance of Rutgers house flies was demonstrated in vitro by a 4-fold enhancement of p-nitroanisole O-demethylation over that of NAIDM house flies. O-demethylation of p-nitroanisole could not be demonstrated for potato beetle larvae. Colorado potato beetle resistance was associated with increases in microsomal levels of NADPH-cytochrome c reductase (ca. 2-fold) and NADPH oxidation (1.2-fold). The inability to measure O-demethylation in Colorado potato beetles may have been due to the solubilization of NADPH-cytochrome c reductase during microsomal preparation. Significant differences between resistant and susceptible Colorado potato beetle larvae were not observed in the penetration of [¹⁴C]carbaryl. Excretion of the radiocarbon may have been significantly greater in the resistant New Jersey population, but some of the insecticide may have also rubbed off the cuticle. This increased capacity for excretion, combined with increased levels of monooxygenase enzymes, could account for the high resistance level of this population.     

The biochemical basis of malathion resistance in the sheep blowfly,Lucilia cuprina

The in vivo and in vitro metabolism of [¹⁴C]malathion was studied in susceptible (LS) and malathion resistant (RM) strains of the sheep blowfly, Lucilia cuprina (Wiedemann). No difference was found between strains in the penetration, excretion, storage, or inhibitory potency of the insecticide. However, RM degraded malathion to its α- and β-monocarboxylic acid metabolites more rapidly than LS, both in vivo and in vitro. This enhanced degradation of [¹⁴C]malathion occurred in vitro in both mitochondrial and microsomal fractions of resistant flies. Kinetic analysis revealed that these fractions degraded malathion by discrete mechanisms. The enzymes from the mitochondria of both strains had the same K_m, whereas the microsomal enzyme from the RM strain had a fivefold higher K_m than that from the LS strain. Studies of esterase activities and the effect of enzyme inhibitors showed that both the mitochondrial and microsomal resistance mechanisms were the result of enhanced carboxylesterase activity. It was concluded that increased carboxylesterase detoxification of malathion adequately explained the high level of malathion resistance in RM if rate-limiting factors such as cuticular penetration were taken into account.     

Effect of hepatic enzyme inducers on the in vivo and in vitro metabolism of dicrotophos,dimethoate, and phosphamidon in mice

Daily 75 mg/kg phenobarbital ip injections for 3 days or 25 ppm dieldrin in the diet of mice for 14 days caused an increase in liver cytochrome P-450 and blood B-esterase. Liver A-esterase was not significantly increased. Under in vitro conditions, phenobarbital and dieldrin induced the oxidative as well as hydrolytic metabolism of dicrotophos, dimethoate, and phosphamidon by liver homogenates or combined microsomes plus 105,000g supernatant fractions. The concentration of dimethoxon was increased more than fourfold by the pretreatments after incubation for 4 hr at 37.5°C with NADPH added. The organophosphorus insecticides used in this study were not metabolized as well by the liver microsomes alone or 105,000g supernatant alone, as by the combination of microsomes and 105,000g supernatant. Under in vivo conditions in mice, phenobarbital and dieldrin treatments increased the urinary recovery of metabolites in the initial 6 hr after [¹⁴C]carbonyl-dimethoate or [¹⁴C]N-ethyl-phosphamidon administration. Analysis of urine showed that the inducers caused a more than sixfold increase in dimethoxon recovered and twofold increase in water-soluble nontoxic metabolites within 6 hr after dimethoate administration. With phosphamidon both inducers increased the rate of metabolism, and the total recovery in aqueous and chloroform fractions was decreased. These results suggest that increased dimethoate toxicity after phenobarbital and dieldrin treatments in whole animals results from stimulation of the activation of dimethoate to dimethoxon, while the increase in hydrolytic products after both pretreatments results in decreased toxicity of the direct acetylcholinesterase inhibitors, dicrotophos and phosphamidon.     

Paraoxon-induced release of β-glucuronidase from rat hepatocytes in vitro

A suspension culture of isolated rat hepatocytes was used to reproduce in vitro the paraoxon-induced release of hepatic β-glucuronidase observed in vivo. After a short latent period, exposure of hepatocytes to paraoxon at 10^?7 to 10^?4M resulted in a typical dose-dependent response, with highest release occurring at 10^?4M paraoxon. With 10^?3M paraoxon, however, response was anomalous with a much-decreased enzyme release. As expected from earlier results in vivo, SV₁-oxon exhibited less effect than paraoxon.     

Metabolism of trifluralin,profluralin, and fluchloralin by rat liver microsomes

Three structurally related [¹⁴C]dinitroaniline herbicides, trifluralin, profluralin, and fluchloralin, were extensively metabolized in vitro by both normal and phenobarbital-induced rat liver microsomes. Identification of the metabolites in the ethyl acetate extracts indicated that aliphatic hydroxylation, N-dealkylation, reduction of a nitro group, and cyclization were the predominant metabolic routes for these herbicides in vitro. Of particular interest was the formation of a benzimidazole metabolite.     

Microbial metabolism of propanil and 3,4-dichloroaniline

A Pseudomonas sp. which grew on 4-chloroaniline as a sole source of carbon and nitrogen was able to degrade 15% of 0.05 mM [¹⁴C]3,4-dichloroaniline to ¹⁴CO₂ within 10 days in presence of 1.5 mM 4-chloroaniline. The catabolic enzymes which degraded 3,4-dichloroaniline to CO₂ were inducible by 4-chloroaniline and by 3,4-dichloroaniline. However, their activity was much lower on 3,4-dichloroaniline than on 4-chloroaniline. The strain showed no significant growth on 3,4-dichloroaniline as a sole source of carbon and nitrogen. Soils supplemented with [ring-¹⁴C]propanil and the Pseudomonas sp. evolved 25–50% ¹⁴CO₂ within 5 days. The ¹⁴CO₂ evolution remained below 1% in absence of the Pseudomonas sp.     

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.     

Microsomal oxidation in the house cricket, Acheta domesticus (L.)

Optimal in vitro assay conditions for the microsomal enzymes effecting aldrin epoxidation and dihydroisodrin hydroxylation have been established for the house cricket Acheta domesticus (L.). Both enzymes require NADPH and oxygen and are completely inhibited by carbon monoxide. Tissue fractionation studies have revealed that these enzymes are found primarily in the tissues of the Malpighian tubules. Enzyme activity was found to be greater in female crickets than in male, the absolute levels being dependent on age in both the adult insect and in the final nymphal instar.     

in vitro metabolism of azinphosmethyl in susceptible and resistant houseflies

The in vitro metabolism of [¹⁴C-methoxy] or [³²P]azinphosmethyl by subcellular fractions of abdomens from a resistant and a susceptible strain of houseflies was studied. The degradative activity in both strains was associated with the microsomal and soluble fractions and required NADPH and glutathione, respectively. The resistant strain possessed higher activity for both the mixed-function oxidases and the glutathione transferase than the susceptible strain, and both systems appear to be important in the resistance mechanism. The mixed-function oxidases were involved in the oxidative desulfuration as well as the dearylation of azinphosmethyl. A glutathione transferase located in the soluble fraction catalyzed the formation of desmethyl azinphosmethyl and methyl glutathione. This enzyme also demethylated azinphosmethyl oxygen analog. Although the soluble fraction exhibited both glutathione S-alkyltransferase and S-aryltransferase activity against noninsecticidal substrates, no evidence of the transfer of the benzazimide moiety from azinphosmethyl to glutathione was obtained. Sephadex G-100 chromatography of the soluble enzymes revealed a common eluting fraction responsible for both types of transferase activity.     

Influence of glyphosate on selected plant processes

Biochemical effects of glyphosate have been examined with a variety of plant materials using mostly merislematic or actively growing tissues. The accumulation of chlorophyll was severely retarded and photosynthetic CO₂ uptake was inhibited to a lesser extent. These inhibitory effects could not be alleviated by the simultaneous admixture of divalent cations. Glyphosate enhanced the initial substrate-induction of nitrate reductase, but repressed induction of nitrite reductase, in Ihe latter case correlating with both inhibition of chlorophyll accumulation and CO₂ uptake. Inhibition of macromolecule synthesis in single node buds of Agropyron repens(L.) Beauv. was due partly to inhibition of ¹⁴C-precursor uptake. The specific activity of soluble acid phosphatase was enhanced as was the evolution of ethane. Ethylene production was not greatly affected. A marked decrease in microsomal protein was observed but the specific activities of several microsomal enzymes did not decline. Glyphosate had little inhibitory effect on the activity of microsomal ATPases in vitro.     

Effects of the synergist piperonyl butoxide on metabolism of pesticides in green sunfish

The effects of piperonyl butoxide on metabolism of ¹⁴C-labeled methoxychlor, aldrin, and trifluralin were investigated in green sunfish, Lepomis cyanellus. Piperonyl butoxide inhibited epoxidation of aldrin to dieldrin, O-dealkylation of methoxychlor, and N-dealkylation of trifluralin, resulting in higher levels of total radioactivity in animals exposed to the combination compared to those exposed to pesticide alone. Where piperonyl butoxide was present a greater proportion of the total radioactivity in the fish extract occurred as parent compound compared to metabolites than in fish exposed to pesticide alone. After 16 days of exposure piperonyl butoxide increased the proportion of parent compound eight times for methoxychlor, 17 times for aldrin, and 15 times for trifluralin.     

Hydrolysis of paraoxon and malaoxon in three strains of Myzus persicae with different degrees of parathion resistance

Homogenates of three strains of Myzus persicae, A, R, and E, with an LD₅₀ for topically applied parathion of 9, 93, and 263 ng per aphid, showed an in vitro hydrolytic degradation of paraoxon of 2.3, 4.7, and 8.6 pmol/mg aphid/h, respectively. These values represent V_max; K_m was <10^?7M. The three strains showed a malaoxon degradation of 2.4, 11.9, and 18.8 pmol/mg/h at 10^?6M substrate concentration. V_max for R and E was 21 and 27 pmol, respectively and K_m 7 and 4 × 10^?7M. Activity in strain A was too low to estimate these entities. The breakdown product of paraoxon was mainly diethyl phosphoric acid, that of malaoxon mainly dimethyl phosphoric acid. No hydrolysis of the carboxylester groups of malaoxon was found. Hydrolysis of paraoxon and malaoxon was inhibited by isopropyl and n-propyl paraoxon and by the salioxon-analog K₂. The two latter compounds were shown to act as synergists with parathion when added in amounts that caused little mortality when given alone. The hydrolytic enzyme is soluble and retains its activity during incubations of several hours. It is likely that it is responsible for at least part of the resistance. Resistance was maintained without selection over a period of three years. There was no correlation between degree of resistance and carboxylesterase activity of the strains.     

Metabolism of O,O,S-trimethyl phosphorothioate in rats after oral administration of a toxic dose,and the effect of coadministration of its antagonistic thionate isomer

The in vivo metabolism of [¹⁴CH₃S]- and [¹⁴CH₃O]O,O,S-trimethyl phosphorothioate (OOS) was followed in rats after oral administration of threshold or LD₅₀ toxic doses of 20 or 60 mg/kg. Similar metabolic studies were conducted with coadministration of 1% O,O,O-trimethyl phosphorothionate (OOO), which prevented all signs of delayed toxicity, including weight loss. When administered alone, OOS was metabolized mainly (50–60%) via removal of the CH₃S moiety, which was largely converted to expired CO₂. Approximately 20% of the compound was O-demethylated, presumably by conjugation with glutathione, and then further metabolized to CO₂. Major urinary products were identified as O,O-dimethyl phosphoric acid (50–60%) and O,S-dimethyl phosphorothioic acid (～20%). Coadministration of OOO caused a slight decrease (～5%) in the cleavage of the CH₃S moiety, indicated by a reduction in ¹⁴CO₂ from [¹⁴CH₃S]OOS and a quantitatively similar increase in the formation of O,S-dimethyl phosphoric acid. Limited pharmacokinetic studies indicated that OOS was rapidly absorbed and distributed throughout the body. Coadministration of 1% OOO caused a slight increase in the blood half-life of parent OOS when administered at 60 mg/kg. It was concluded that a small proportion of the cleavage of the CH₃S moiety from OOS is involved in the intoxication process, and that this intoxication reaction is specifically inhibited by OOO.