Studies on the chiral isomers of fonofos and fonofos oxon: II. In vitro metabolism

The in vitro metabolism of the chiral isomers of fonofos and fonofos oxon in the presence of mouse liver mixed-function oxidase and serum esterase was investigated. The metabolism of ³⁵S-labeled phenyl-(S)_P-fonofos mediated by mixed-function oxidase took place stereoselectively, resulting predominantly in (R)_P-fonofos oxon. Similarly, (R)_P-fonofos was converted to (S)_P-oxon. In each case, however, a significant amount of racemization occurred. Other products were diphenyl disulfide and diphenyl disulfide oxide. In addition to stereospecificity, the oxidative metabolism of (R)_P-fonofos proceeded at a rate faster than that of (S)_P-fonofos. Stereoselective rate differences also were observed in mouse or rat serum-catalzyed degradation of the fonofos oxon enantiomers, the (S)_P isomer being degraded about twofold faster than its enantiomer. The differences in toxicities of the isomers of fonofos and fonofos oxon were consistent with the in vitro metabolism data.     

Studies on the chiral isomers of fonofos and fonofos oxon: I. Toxicity and antiesterase activities

The toxicity of the (R)_P and (S)_P chiral isomers and racemates of fonofos and fonofos oxon to insects and white mice were determined. (R)_P-Fonofos and (S)_P-fonofos oxon were 2- to 12-fold more toxic to house flies, mosquito larvae, and mice than were the corresponding enantiomers. The racemates were intermediate in toxicity. Stereoselectivity also was observed in the in vitro inhibition of house fly-head and bovine erythrocyte acetylcholinesterase, horse serum cholinesterase, chymotrypsin, trypsin, and a variety of esterases. In all cases the (S)_P-oxon was a more potent inhibitor than the (R)_P-oxon with k₁ ratios of $\text{(S)}{}_{\mathrm{<mtext>P</mtext>}}\text{(R)}{}_{\mathrm{<mtext>P</mtext>}}$ ranging from 4- to 60-fold. Further, differences in levels of house fly-head, mouse brain, and blood cholinesterase obtained from house flies and mice treated with the enantiomers and racemates of fonofos and fonofos oxon were observed. Differences in toxicity of the enantiomers and racemates to house flies and mice were more closely related to in vivo than to in vitro cholinesterase inhibition.     

Metabolism of O,O-dimethyl S-[α-carboethoxy)benzyl]phosphorodithioate (phenthoate) in the white mouse and house flies

The metabolism of O,O-dimethyl S-[α-(carboethoxy)benzyl]phosphorodithioate (phenthoate), an organophosphorus insecticide of low mammalian toxicity, was investigated in white mice and in susceptible and resistant strains of house flies. Phenthoate was metabolized rapidly in the mouse to a wide variety of detoxication products and only an insignificant amount of phenthoate oxon was detected. The same detoxication products were produced in house flies but, compared to the mouse, substantial amounts of phenthoate oxon also were found. The selective toxicity of phenthoate between insect and mammal is attributable to the difference in the accumulation of the oxon.     

Metabolism of O,S-dimethyl propionyl- and hexanoylphosphoramidothioate in the house fly and white mouse

The metabolism of O,S-dimethyl propionyl- and hexanoylphosphoramidothioate was investigated in the white mouse and house flies. Compared to the hexanoylphosphoramidothioate, the propionyl analog is approximately 35-fold more toxic to house flies and is 10-fold less toxic to mice. On a percentage basis, substantially larger amounts of methamidophos were detected in house flies treated topically with the propionylphosphoramidothioate than in flies treated with the hexanoyl derivative. The reverse was evident in the case of the mouse where much larger amounts of methamidophos were formed after oral treatment with the hexanoylphosphoramidothioate. Minor amounts of other metabolic products also were detected, including an unknown from the hexanoylphosphoramidothioate. Metabolism of the S-methyl moiety to carbon dioxide appeared to be a major pathway for metabolic degradation of both compounds in both the white mouse and house fly. The difference in toxicity of the two acylphosphoramidothioates to the mouse and house fly is attributed to difference in the amounts of methamidophos formed in the animals.     

Metabolism of isofenphos in southern corn rootworm

The metabolism of isofenphos was examined in adult and larval southern corn rootworm (SCR), Diabrotica undecimpunctata howardi Barber. SCR adults and larvae were analyzed at specific time intervals after treatment with [¹⁴C]isofenphos at the LD₁₀ dosage level. Most of the ¹⁴C was recovered from the internal organic extract and excreta fractions. The major metabolic pathways of isofenphos in SCR included oxidative desulfuration of isofenphos to isofenphos oxon and hydrolysis of isofenphos oxon and/or parent isofenphos to isopropyl salicylate. Isofenphos penetrated approximately three times faster into larvae than into adults. A lower accumulation of toxic compounds inside the larvae, as a result of a faster metabolism (1.5 ×) and rapid elimination of the parent isofenphos, may explain why larvae are less susceptible than adults.     

Affinity and phosphonylation constants for the inhibition of cholinesterases by the optical isomers of O-2-butyl S-2-(dimethylammonium)ethyl ethylphosphonothioate hydrogen oxalate

The synthesis of the four optical isomers of known absolute configuration of O-2-butyl S-2-(dimethylammonium)ethyl ethylphosphonothioate hydrogen oxalate is described. Values for the affinity constant (K_a), phosphonylation constant (k_p), and bimolecular inhibition rate constant (k_i) for the inhibition of bovine erythrocyte acetylcholinesterase, housefly-head acetylcholinesterase, and horse serum cholinesterase by the chiral isomers and the racemic mixture are reported. Using a relatively simple spectrophotometric technique, inhibition times as low as 0.5 sec were used. The phosphorus isomers of S_p configuration were more potent inhibitors than their R_p enantiomers by 1630-fold against the bovine enzyme, 9120-fold against the fly-head enzyme, and 40-fold against the horse serum enzyme. The differences in anticholinesterase activity were attributable to differences in the affinity constant, K_a, and the phosphonylation constant, k_p. Small but consistent inhibition rate differences were attributable to asymmetry at carbon. Against horse serum cholinesterase, the S_C isomers indicated the presence of three kinetic forms in this enzyme preparation.     

Metabolism of 2,2-dimethyl-2,3-dihydrobenzofuranyl-7 N-dimethoxyphosphinothioyl-N-methylcarbamate in the house fly,rat, and mouse

The metabolism of a selectively toxic derivative of carbofuran, 2,2-dimethyl-2,3-dihydrobenzofuranyl-7 N-dimethoxyphosphinothioyl N-methylcarbamate (PSC), was examined in the house fly, rat, and mouse. In house flies, PSC is metabolized mainly to carbofuran and related oxidation products containing the intact N-methylcarbamyl ester moiety. Degradation to phenolic products was the principal route of metabolism in rodents. The results indicate that the selective toxicity of PSC between insects and mammals is attributable to differing pathways of metabolism.     

Neurophysiological activity and toxicity of pyrethroids derived by addition of methylene,sulfur or oxygen to the chrysanthemate 2-methyl-1-propenyl substituent

Conversion of chrysanthemates to their cyclopropane, episulfide, and epoxide derivatives by addition of methylene, sulfur, or oxygen, respectively, to the 2-methyl-1-propenyl double bond yields products generally of reduced toxicity but enhanced neurophysiological activity and photostability. The reduced toxicity is established with cis-cyphenothrin derivatives administered intracerebrally to mice and topically to house flies and with cis-phenothrin derivatives applied topically to American cockroaches and house flies, even in the presence of piperonyl butoxide for the house flies. In contrast, cyclopropane, episulfide, and epoxide derivatives of phenothrin are more potent than the parent compound in eliciting repetitive firing following stimulation of a cercal sensory nerve of the American cockroach in vitro. The individual 1′R and 1′S isomers of epoxides derived from (1R,cis,αS)cyphenothrin, (1R,cis)phenothrin, and (1R,trans)tetramethrin differ in potency by up to 20-fold for insecticidal activity, >30-fold for intracerebral toxicity to mice, and ～100,000-fold in the cercal sensory nerve assay. In each case the epoxide isomer of higher R_f is more potent than that of lower R_f when derived from a trans-chrysanthemate and vice versa from a cis-chrysanthemate.     

Pyrethroid toxicology in the frog

Adult Rana pipiens pipiens (Shreber) are highly sensitive to insecticidal α-cyano-3-phenoxybenzyl esters administered subcutaneously, i.e., LD₅₀ 0.13–0.35 mg/kg for deltamethrin and the most potent isomer of each of cis-cypermethrin, fenpropathrin, and fenvalerate and 0.65 mg/kg for (1R,αS)-trans-cypermethrin. Pyrethroids lacking the α-cyano substituent [pyrethrins, S-bioallethrin, kadethrin, and the Cis- and trans-isomers of (1R)-tetramethrin, (1RS)-resmethrin, (1R)-phenothrin, and (1R)-permethrin] vary greatly in their toxicity (LD₅₀ 0.14 to > 60 mg/kg) and the trans isomers are much less toxic than the corresponding cis isomers. The trans/cis specificity is due in large part to relative detoxification rates based on synergism studies with the resmethrin and permèthrin isomers and liver pyrethroid esterase assays with the permethrin and cypermethrin isomers. Poisoning by the noncyano compounds involves hyperactivity and tremors whereas by the cyanophenoxybenzyl esters involves tonic seizures and choreoathetosis, i.e., types I and II syndromes, respectively. Picrotoxinin, t-butylbicyclophosphate, and five other small cage compounds give a third type of syndrome with clonic seizures. Diazepam and its 2′-fluoro-4-methyl-4,5-dihydro analog (RO 5-3636) are more effective than 23 other compounds tested in protecting against deltamethrin toxicity. Diazepam is most effective in alleviating the Type II syndrome, intermediate with the type I syndrome, and is not active with picrotoxinin.     

Studies on the optical isomers of EPN and EPNO

The optical isomers of EPN (O-ethyl O-p-nitrophenyl phenylphosphonothionate) and EPNO (O-ethyl O-p-nitrophenyl phenylphosphonate) have been synthesized. No significant difference in the rate of alkaline hydrolysis of the isomers at the two pH's evaluated was observed. The (+)-isomers of EPN and EPNO were more toxic to house flies than the corresponding (?)-isomers, while the (+)- and (?)-isomers, as well as the racemic mixture of EPN, were almost equally toxic to mice. The (+)-EPNO is more toxic to mice than the corresponding (?)-isomer. Cholinesterase inhibition studies demonstrated that (+)-EPNO has a higher bimolecular rate constant, (k_i) than the corresponding (?)-isomer. This higher inhibitory power was due to a higher affinity (K_a) of the (+)-isomer.     

Acute and delayed neurotoxicity of leptophos analogs

Nineteen O-halogenated-phenyl O-methyl phenylphosphonothionates were evaluated for acute toxicity (LD₅₀) to the female house fly Musca domestica L. and to the male Swiss white mouse, and for delayed neurotoxicity to the White Leghorn hen. The electron-withdrawing power of the phenyl substituents (Σσ^? values) correlate with the LD₅₀ values to house fly and mouse, with departures from linearity attributable to the steric hindrance of di-ortho-Cl substitution and by variations in the accessibility of the anionic site of acetylcholinesterase in the two species. The relationship with delayed neurotoxicity is less predictable although it clearly depends on suitable electron-withdrawing capacity. Delayed neurotoxicity also relates to a high degree of lipophilicity and prolonged residence time of the inhibitor in the nerve axon.     

Metabolism of the 1,2,4-triazolylmethane fungicides,triadimefon, triadimenol,and diclobutrazol,by Aspergillus niger (van Tiegh.)

Metabolism of the triazolylmethane fungicides triadimefon, triadimenol, and diclobutrazol by Aspergillus niger was studied using a replacement culture technique and ¹⁴C substrates. Components of metabolite mixtures were characterized by TLC, GLC, radio-GC, and GC-MS analyses of the free materials and their trifluoroacetate and trimethylsilyl ether derivatives. The three compounds underwent a common metabolic change involving oxidation of C(CH₃)₃ to C(CH₃)₂CH₂OH. In this work the isopropyl analog of triadimefon, previously reported as a metabolite, was an artifact and resulted from nonbiological oxidation of the corresponding primary alcohol. The fungus also reduced triadimefon to triadimenol, giving a mixture of 1R2S, 1S2R and 1R2R, 1S2S diastereoisomers. The less fungitoxic 1R2S, 1S2R triadimenol predominated, so that this conversion may be directly associated with the relative insensitivity of A. niger to triadimefon. Implications of oxidative and reductive metabolism of these fungicides are suggested with particular reference to the differing fungitoxicities of diastereoisomers and enantiomers.     

In vitro metabolism of EPN and EPNO in susceptible and resistant strains of house flies

EPN is twice as toxic as EPNO to house flies from both the Diazinon-resistant strain and the susceptible strain. EPN and EPNO are also eight times more toxic to the susceptible than the resistant strain. This is due to the ability of the resistant strain to metabolize these compounds to a greater extent. Metabolism by the glutathione S-transferases present in the 100,000g supernatant is more extensive than that by the NADPH-dependent microsomal mixed-function oxidases. The glutathione S-transferases are the major route of metabolism for EPN and appear to be the principal mechanism conferring resistance. EPN was metabolized by the microsomal fraction via oxidative desulfuration to the oxygen analog, EPNO, and by oxidative dearylation to p-nitrophenol. EPNO was metabolized by the same system to p-nitrophenol and desethyl EPNO as well as to an unknown metabolite. The soluble fraction metabolized EPN to p-nitrophenol, S-(p-nitrophenyl)glutathione, O-ethyl phenylphosphonothioic acid, and S-(O-ethyl phenylphosphonothionyl)glutathione. The identification of the latter conjugate demonstrates a new type of metabolite of organophosphorus compounds. EPNO was metabolized by the soluble fraction to p-nitrophenol and S-(p-nitrophenyl)glutathione.     

Stereoselective metabolism of fipronil in water hyacinth (Eichhornia crassipes)

The enantioselective metabolism of racemic fipronil in water hyacinth (Eichhornia crassipes) had been investigated. In this study, the degradation data and the enantiomer fraction (EF) were determined by chiral high-performance liquid chromatography (HPLC) with a column cellulose-tri-(3, 5-dimethylphe-nylcarbamate)-based chiral stationary phase (CDMPC-CSP). During the uptake phase, the EF value of plant sample increased from 0.50 at 1st day to 0.72 at 63rd day, while it was almost unchanged in water. For the depuration phase, the S- and R-enantiomer of fipronil in water hyacinth plants were degraded 92.22% and 82.07% after 17 days, respectively. The process of the degradation of the two enantiomers was followed first-order kinetics (R² ? 0.94). Stereoselective behavior was observed in both accumulation and degradation process. In this study, fipronil-sulfone and fipronil-sulfide, the metabolites of fipronil, were detected by GC-MS to show the main metabolic pathway of fipronil in water hyacinth.     

Decreased nerve sensitivity and decreased cuticular penetration as mechanisms of resistance to pyrethroids in a (1R)-trans-permethrin-selected strain of the house fly

A fenthion-resistant strain of the house fly (Musca domestica L.) was selected with bioresmethrin resulting in ca. 90-fold resistance to the selecting agent. This strain was subsequently selected with (1R)-trans-permethrin producing ca. 140-fold resistance to this latter insecticide. The permethrin-resistant (147-R) strain was highly cross-resistant to several other pyrethroids and demonstrated resistance to knockdown by these insecticides as well as by DDT. The sensitivity of the central nervous system to four pyrethroids was investigated. The 147-R strain was 2.6-fold less sensitive to (1R)-trans-ethanoresmethrin than the susceptible (NAIDM-S) strain, and >43-fold and >67-fold less sensitive to (1R,S)-cis, trans-tetramethrin and (1R)-trans-permethrin, respectively. It also displayed decreased penetration of (1R,S)-trans-[¹⁴C]permethrin when compared to the NAIDM-S strain. Lower nerve sensitivity and decreased cuticular penetration are potential mechanisms of resistance to pyrethroids in house flies in the United States.     

Metabolism of fenitrothion by organophosphorus-resistant and -susceptible house flies, Musca domestica L

The metabolism of fenitrothion was investigated in highly resistant (Akita-f) and susceptible (SRS) strains of the house fly, Musca domestica L. The Akita-f strain was 3500 times more resistant to fenitrothion than the SRS strain. Fenitrothion, topically applied to the flies, was metabolized in vivo far faster in the Akita-f strain than in the SRS strain. In vitro studies revealed that fenitrothion was metabolized by a cytochrome P-450-dependent monooxygenase system and glutathione S-transferases. The former oxidase system metabolized fenitrothion in vitro into fenitrooxon and 3-methyl-4-nitrophenol as major metabolites, and into 3-hydroxymethyl-fenitrothion and 3-hydroxymethyl-fenitrooxon as minor metabolites. Glutathione S-transferases metabolized fenitrothion into desmethylfenitrothion. The cytochrome P-450-dependent monooxygenase system and glutathione S-transferases of the resistant Akita-f strain had 1.4 to 2.2 times and 9.7 times, respectively, as great activities as those of the susceptible SRS strain. These results suggest the importance of glutathione S-transferases in fenitrothion resistance in the Akita-f strain.     

Comparative insecticidal activities and metabolic rates of lindane and its hexadeuterio-analog

Hexadeuterio-lindane(γ-BHC-d₆) was several times as toxic as lindane against the mosquito (Culex pipiens pallens), house fly (Musca domestica), German cockroach (Blattella germanica) and American cockroach (Periplaneta americana). The neuroexcitatory activity of these two compounds did not differ. Lindane was considerably synergized by piperonyl butoxide, but lindane-d₆ was not. A large isotope effect was observed in the in vivo breakdown of lindane-d₆. Thus, the intrinsic toxicities of both compounds are equivalent. The difference in insecticidal activity seems to be due to the different rates of detoxifying biodegradation caused by the kinetic deuterium isotope effect.     

Studies on the metabolism of vamidothion and its thioanalog in insecticide-resistant and susceptible house fly strains

The in vivo and in vitro metabolism of vamidothion [O,O-dimethyl S-[2-(1-methylcarbamoyl)-ethylthio] ethylphosphorothiolate] as well as the in vitro metabolism of thiovamidothion [O,O-dimethyl S-[2-(1-methylcarbamoyl)ethylthio] ethylphosphorodithioate] was investigated in insecticide-resistant and susceptible house fly strains. Vamidothion was converted in vivo to the sulfoxide, the principle metabolite, and subsequently to the sulfone at a slower rate. Vamidothion and vamidothion sulfoxide were hydrolyzed at the PS and SC bond. The resulting primary alcohol metabolite was further oxidized to a carboxylic acid followed by decarboxylation. No metabolism of vamidothion or thiovamidothion occurred in vitro without the addition of NADPH. The addition of NADPH resulted in rapid conversion of vamidothion to the sulfoxide, and thiovamidothion was oxidatively metabolized to six metabolic products. No qualitative differences were found between resistant and susceptible strains, but there were signficant quantitative differences. The metabolism was highest in the Rutgers strain followed by Cornell-R, Hirokawa, and then CSMA strain. The route of vamidothion and thiovamidothion metabolism was via the cytochrome P-450-dependent monooxygenase system, and none of the resistant strains showed glutathione S-transferase activity toward vamidothion or thiovamidothion. No further oxidation of vamidothion sulfoxide to the sulfone was observed and also no hydrolysis products were formed, in vitro.     

Enantioselective degradation kinetics of metalaxyl in rabbits

Metalaxyl [methyl-N-(2′-methoxyacetyl)-N-(2,6-dimethylphenyl)-d,l- alaninate] is a potent phenylamide fungicide. The (−)-(R)-isomer accounts for most of the fungicidal activity. A possible stereo and/or enantioselective kinetics of metalaxyl in rabbits was investigated by intravenous injection. The concentrations of (−)-(R)- and (+)-(S)-metalaxyl in plasma, liver, and kidney tissue were determined by HPLC with a cellulose-Tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase and gas chromatography-mass spectroscopy. After intravenous administration of racemic metalaxyl (40 mg/kg), the (+)-(S)-enantiomer levels in plasma, liver, and kidney decreased more rapidly than the (−)-(R)-isomer. The area ratio of the (−)-(R)-/(+)-(S)-enantiomer under the concentration-time curve (AUC_0 → ∞) in plasma after drug application was 1.62. The total plasma clearance value of the (+)-(S)-enantiomer was 1.53 and higher than that of the (−)-(R)-enantiomer. The [R]/[S] ratio in plasma was >1 for standard rac-metalaxyl at each time point. The other pharmacokinetic parameters of the enantiomers were also different. The results indicate substantial stereoselectivity in the degradation of metalaxyl enantiomers in rabbits.     

Mode of action of sulfenylated carbamates: Rapid conversion of N,N′-thiodicarbamates to parent carbamate measured by neurophysiological bioassay

Four N,N′-thiodicarbamate derivatives of carbofuran induced uncoupled convulsions with similar latencies following topical application to the cuticle of tethered S_NAIDM house flies, despite considerable differences in their lipophilic properties. There was a small but statistically significant delay in the latency to response to the derivatives compared to carbofuran, but when perfused directly on the exposed thoracic ganglia to assess intrinsic activity, carbofuran acted up to 4.4 times faster than the derivative. It is suggested that the NS bond of the derivatives is cleaved soon after application to the house fly cuticle and carbofuran is released from each derivative in sufficient quantities to accumulate toxic concentrations in the central nervous system at similar rates.