Metabolism of lindane in house flies: Metabolic desaturation,dehydrogenation and dehydrochlorination,and conjugation with glutathione

In lindane-treated house flies, a cis-dehydrogenated metabolite, $\text{(}\text{36}\text{45}\text{)-}\text{hexachlorocyclohexene}$, was identified by gas-liquid chromatography and mass spectrometry. The in vitro metabolism study showed that in the presence of NADPH the microsomal fraction of house flies converted lindane to three hexane-soluble metabolites. This conversion was inhibited by piperonyl butoxide, SKF-525A, and carbon monoxide. These metabolites were identified as $\text{(}\text{36}\text{45}\text{)-}\text{hexachlorocyclohexene}$, $\text{(}\text{36}\text{45}\text{)- and (}\text{346}\text{5}\text{)-pentachlorocyclohexene}$ (PCCHE) by gas-liquid chromatography. They, as well as lindane, were excellent substrates for the reaction with the postmicrosomal fraction in the presence of glutathione. While the reaction with lindane-d₆ showed a significant deuterium isotope effect (6.82), that of $\text{(}\text{36}\text{45}\text{)-}\text{PCCHE-d}{}_5$ did not (1.18). Enzymatic conjugation with glutathione probably occurs at the stage of PCCHE.     

Mercapturic acid formation from lindane in rats

4-Chloro-, 2,4-dichloro-, 3,4-dichloro-, 2,3,5-trichloro-, and 2,4,5-trichlorophenylmercapturic acids were identified as main metabolites of lindane, γ-isomer of 1,2,3,4,5,6-hexachloro-cyclohexane, in rat urine. Pathways to these metabolites were shown to include $\text{(}\text{36}\text{45}\text{)-}\text{hexachlorocyclohexene}$ as the most important intermediary metabolite. $\text{(}\text{346}\text{5}\text{)-}\text{Pentachlorocyclohexene}$ and $\text{(}\text{346}\text{5}\text{)-}\text{tetrachlorocyclohexene}$ also seem to be involved in these pathways, while $\text{(}\text{36}\text{45}\text{)-}\text{pentachlorocyclohexene}$ plays a minor role in the pathway. Glutathione conjugation, using the rat liver soluble fraction, occurred directly on the polychlorocyclohexenes, not on their further transformed products. In in vivo biodegradation, $\text{(}\text{36}\text{45}\text{)-}\text{hexachlorocyclohexene}$ may be dechlorinated and dehydrochlorinated at the endoplasmic reticulum before it undergoes the glutathione conjugation in cytosol, although other polychlorocyclohexenes generally react in a manner similar to that in the in vitro reaction.     

Inhibition of microsomal oxidoses from the housefly

The hydroxylation of naphthalene, O-demethylation of p-nitroanisole, and N-demethylation of N,N-dimethyl-p-nitrophenylcarbamate by housefly microsomes were inhibited by CO, sulfhydryl reagents, Cu (II), methylene dioxyphenyl compounds and β-diethylaminoethyldiphenylvaleric acid (SKF 525-A). Kinetic analysis indicates that the inhibitory properties of SKF 525-A and piperonyl butoxide are changed during the incubation period. Bovine serum albumen changes the kinetic constants in a way which suggests binding of an endogenous inhibitor(s). Patterns of inhibition indicate p-nitroanisole oxidation is effected by a system with at least one component distinct from the system which oxidizes naphthalene and N,N-dimethyl-p-nitrophenylcarbamate.     

The metabolism of R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene] in rat hepatocyte suspensions

The metabolism of R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene] by rat hepatocytes has been analyzed and compared with that of juvenile hormone I [methyl-(E,E)-cis-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate] under identical conditions. The metabolism of R-20458 is characterized by the predominance of NADPH-dependent cytochrome P-450 and epoxide hydrolase reactions; whereas, JH I is metabolized mainly by carboxylesterase, epoxide hydrolase, and glutathione S-transferases. The metabolites of R-20458 have been shown to correspond to (E)-6,7-epoxy-1-(4-hydroxyethylphenoxy)-3,7-dimethyl-2-octene; (E)-6,7-epoxy-1-(4-acetylphenoxy)-3,7-dimethyl-2-octene; (E)-6,7-dihydroxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene; and, (E)-6,7-dihydroxy-1-(4-acetylphenoxy)-3,7-dimethyl-2-octene. The production of the α-hydroxyethyl, p-acetylphenoxy, and acetylphenoxy-6,7-diol metabolites is markedly inhibited by SKF 525-A. No dramatic effects are produced by diethylmaleate and 1,2-epoxy-3,3,3-trichloropropane.     

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.     

Selective alterations of membrane properties of cultured human liver cells caused by the insecticide DDT

A variety of membrane-specific parameters was examined in both intact cells and isolated plasma membranes following exposure of cultured human liver cells to the insecticide 1,1-(2,2,2-trichloroethylidene)bis(4-chloro)benzene (DDT). Uptake of DDT was at equilibrium within 6 hr. In contrast, a decrease in the number of β-adrenergic hormone receptors first became significant after 48 hr of cell exposure. Whereas the uptake was largely reversible, the loss in the number of β receptors did not recover after DDT-exposed cells were cultured in fresh medium lacking the insecticide. Experiments in vitro substantiated the time lag of the biological effect. The decrease in receptor proteins was persistent in membranes with increased phospholipid unsaturation. Temperature-activity profiles (“Arrhenius plots”) of $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}\text{-ATPase}$ and 5′-nucleotidase were unchanged. Endogenous tryptophan fluorescence of membrane proteins was lower in membranes from DDT-exposed cells. These selective alterations in membrane parameters suggest a specific interaction of DDT with membrane proteins; interference with cellular protein synthesis is possible. The results indicate that membrane lipid “fluidization” does not play a physiologically important role in the mechanism of DDT action in biomembranes.     

Mechanism of inhibition reaction of acetylcholinesterase by phenyl N-methylcarbamates: Separation of hydrophobic,electronic, hydrogen bonding and proximity effects of aromatic substituents

The association equilibrium constant, $\text{1}\text{K}{}_{\mathrm{d}}$, and the carbamylation constant, k₂, of 53 o-, m-, and p-substituted phenyl N-methylcarbamates with bovine erythrocyte acetylcholinesterase were determined. The $\text{1}\text{K}{}_{\mathrm{d}}$ value varied 1000-fold, whereas the k₂ value did not depend upon the nature and position of substituents. The variation in $\text{log}\text{(}\text{1}\text{K}{}_{\mathrm{d}}\text{)}$ was analyzed using free energy related substituent parameters and regression analyses. The effect of substituents at o-, m-, and p-positions was nicely separated into hydrophobic, electronic, hydrogen bonding, and proximity (steric and field electronic for o-substituents) factors. The physicochemical significance of these factors was established by comparison with those for model organic reactivities. The mechanism of the whole reaction process was elucidated in terms of physical organic chemistry.     

Identification of products arising from the metabolism of cis- and trans-chlordane in rat liver microsomes in vitro: Outline of a possible metabolic pathway

The metabolism of pure cis- and trans-chlordane was studied in vitro. Microsomal preparations from the livers of male rats induced with cis- or trans-chlordane in feed for 10 days were used to metabolize the pure compound corresponding to the inducer. Subsequent extraction, column fractionation, and combined gas chromatography-mass spectroscopy resulted in the characterization of four compounds not previously reported from an in vitro system. In addition to the substrate, trans-chlordane extracts contained species with the following molecular weights and empirical formulas: $\text{m}\text{e}$ 370, C₁₀H₅Cl₇, heptachlor; $\text{m}\text{e}$ 352, C₁₀H₆OCl₆, a hydroxylated chlordene; and $\text{m}\text{e}$ 422, C₁₀H₆OCl₈, a hydroxylated chlordane. Dichlorochlordene, oxychlordane, and 1-chloro-2-hydroxy-dihydrochlordene were also present. With the exception of the hydroxychlordane, cis-chlordane extracts contained all of the metabolites found in the trans incubates. Additionally, a fully saturated compound, $\text{m}\text{e}$ 372, C₁₀H₇Cl₇, a dihydroheptachlor, was present. The 1,2-trans-dihydrodiol of heptachlor found in previous in vitro incubates of cis-chlordane was not present in this extract. This information has been incorporated into a proposed route for the biotransformation of the chlordanes that offers an explanation for the observed differences in the metabolism of cis and trans isomers. The pathway is based on the reductive dechlorination of the chlordanes through dihydroheptachlor to dihydrochlordene. Parallel pathways of hydroxylation, desaturation, and epoxide formation arise at each of these species and at chlordane itself.     

Influence of methabenzthiazuron on ATP-level and protein synthesis in wheat

Spring wheat (Triticum aestivum L. “Kolibri”) was grown in vermiculite treated with the photosynthesis inhibiting herbicide methabenzthiazuron (1-(benzothiazol-2-yl)-1,3-dimethylurea) either before germination or when the plants were 13 days old. The plants were analyzed from 2 days before until 11 days after treatment or up to a plant age of 23 days.The results are interpreted to fit the concept of a “shade adaptation reaction” caused by a herbicidal photosynthesis inhibition. Soluble reducing sugars have been found to decrease 4 hr after treatment and to stay at a low level throught the experiment. The ATP level was decreased 1 day after treatment but increased above control values 4 days later. This increase was accompanied by a decrease of the chlorophyll $\text{a}\text{b}$ ratio. The explanation is given that the ATP level was first decreased because of a lowered carbohydrate supply for substrate and oxidative phosphorylation, and was afterwords increased as a result of an increased cyclic photophosphorylation activity.The soluble protein content and the incorporation of [¹⁴C]leucine into protein were increased 4 days after herbicidal treatment. The total protein was slightly decreased beginning 1 day after treatment. The nitrate concentration and the in vitro nitrate reductase activity were both increased 1 day after treatment. The increase of the nitrate concentration occurred in two phases: a first increase by 50% 1 and 2 days after treatment and a second, much stronger increase beginning on the third day. The first increase is interpreted as the result of a decreased in vivo nitrate reductase activity. The second increase possibly was the result of an increased rate of nitrate uptake.     

Significance of phosphorylation in organophosphate-mediated β-glucuronidase release from the rat liver

The elevation of rat blood β-glucuronidase caused in vivo by O,O-dialkyl O-phenyl phosphates and phosphorothioates correlated well with the electron-withdrawing tendency (σ^?) of leaving group substituents indicating the importance of a phosphorylation mechanism in the enzyme release. Hydrophobic bonding of these compounds may facilitate the phosphorylation since hydrophobicity (π) of substituents also correlated with the enzyme release. SKF 525-A decreased the elevation of β-glucuronidase by parathion through the suppression of paraoxon production. Pretreatment of rats with phenobarbital or DDE resulted in lower and delayed enzyme release caused by parathion.     

The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates

A range of compounds were tested as inhibitors of the enzyme epoxide hydrase, using a cyclodiene epoxide (HEOM) as substrate. Rat and rabbit liver microsomes and pupal homogenates of the blowfly (Calliphora erythrocephala) and the yellow mealworm (Tenebrio molitor) were compared as sources of the enzyme. Only minor differences were found between the four enzyme preparations, when considering I₅₀ values and percentage inhibition at standard concentration. The simple epoxide 1,1,1-trichloropropane-2,3-epoxide and two glycidyl ethers p-nitrophenyl glycidyl ether and p-ethylphenyl glycidyl ether tended to have lower I₅₀ values (1.8×10^?6 to 8.0×10^?5M) than triphenyl phosphate and SKF 525A (4.5×10^?5 to 1.4×10^?4M). Triphenyl phosphate and SKF 525A were competitive inhibitors for both the rat and Tenebrio enzymes. The only clear difference found between these two epoxide hydrase preparations was with respect to their inhibition by 1,1,1-trichloropropane-2,3-epoxide, which was an uncompetitive inhibitor with the rat enzyme, but showed kinetics of mixed inhibition with the insect preparation.     

Isomerization and Beckmann rearrangement reactions in the metabolism of methomyl in rats

Methomyl {S-methyl-N-[(methylcarbamoyl)oxy]thioacetimidate}, also known as Lannate, may exist in two geometric configurations but the more stable syn isomer is the form applied as an insecticide. In the rat, syn[¹⁴CN]methomyl [CH₃S(CH₃)CNOC(O)NHCH₃] was metabolized to respiratory ¹⁴CO₂ and $\text{CH}{}_3{}^{14}\text{CN}$ in a ratio of about 2 to 1. Studies with the anti isomer showed that it was metabolized predominately to $\text{CH}{}_3{}^{14}\text{CN}$. These and other data are presented supporting the contention that syn methomyl is partially isomerized to the anti isomer in the animal prior to the hydrolysis of the ester linkage. After hydrolysis, the syn oxime [CH₃S(CH₃)¹⁴CNOH] is further metabolized to ¹⁴CO₂ while the anti oxime is metabolized to $\text{CH}{}_3{}^{14}\text{CN}$. Proposed immediate precursors to the carbon dioxide and acetonitrile, formed by Beckmann rearrangement of the syn and anti oximes, are CH₃S¹⁴C(O)NHCH₃ and $\text{[}\text{CH}{}_3{}^{\mathrm{14\oplus }}\text{CNSCH}{}_3\text{]x}{}^{\mathrm{?}}$, respectively.     

Insect juvenile hormones: Chemistry and action: Edited by Julius J. Menn and Morton Beroza. Academic Press,New York, 1972, $11.00

Heptachlor metabolism was determined in young male rats fed for 10 days diets containing 18% protein supplied either by gluten or casein. Rats on the casein diet were either pair-fed on an individual basis to the gluten-fed animals or they were fed ad lib. Saline, sodium phenobarbital (80 mg/kg) or diethylaminoethyl 2,2-diphenylvalerate HCl (SKF 525-A, 50 mg/kg) was administered ip to the animals before sacrifice. Weight gain, liver microsomal protein, and heptachlor metabolism (nmole of product/g liver/10 min), assayed in 9000g rat liver supernatants, were found to be significantly reduced in the gluten-fed animals. Animals pair-fed the casein diet had higher heptachlor epoxidase activity than those fed ad lib. Phenobarbital pretreatment significantly enhanced the metabolism of heptachlor in animals in all groups to varying magnitudes. Heptachlor epoxidase activity (units/g liver) was increased 11-fold in the gluten-fed animals, 6-fold in the pair-fed animals on casein diet, and 7-fold in the animals fed casein ad lib. Heptachlor metabolism was less inhibited by SKF 525-A in the rats fed gluten compared to the rats on casein diet, either pair-fed or ad lib. The degree of induction or inhibition of enzyme activity was not influenced by restriction of food intake (pair-fed vs ad lib). The results of these studies suggest an interaction of protein inadequacy with drug metabolism and its induction or inhibition.     

Spectral and inhibitory interactions of methylenedioxyphenyl compounds with southern armyworm (Spodoptera eridania) midgut microsomes

Characteristics of the Type III optical difference spectra of 13 methylenedioxyphenyl compounds in NADPH-fortified armyworm midgut microsomes varied with the nature of the substituents in the aromatic ring. Compounds with electron-donating substituents yielded spectra with large $\text{427}\text{458}\text{nm}$ peak ratios, whereas those with electron-withdrawing groups exhibited low $\text{427}\text{458}\text{nm}$ peak ratios. Small amounts of carbon monoxide were generated during incubation of the 4,5-dihalo derivatives with midgut microsomes, and cis- and trans-methylenedioxycyclohexanes exhibited spectra with a major Soret peak at about 430 nm and a very weak absorbance maximum at about 480 nm. Formation of the Type III spectral complex occurred very rapidly and was associated with a marked decrease (up to 72%) in cytochrome P-450 levels as measured by carbon monoxide binding. Although a 24% reduction of cytochrome P-450 was observed in the absence of any measureable 458-nm spectral complex a linear relationship existed between further decreases in the cytochrome and the increase in Type III complex formation (458 nm). Inhibitory potencies of the compounds towards aldrin epoxidase and benzopyrene hydroxylase activities were not clearly correlated with either spectral complex formation or decrease in cytochrome P-450 and it is apparent that different factors are involved in the inhibition of different monooxygenase reactions.     

Antagonism of iprodione toxicity to Botrytis cinerea by mixed function oxidase inhibitors

The fungitoxicity of iprodione to a sensitive strain of Botrytis cinerea was antagonised by a variety of cytochrome P-450 mixed function oxidase inhibitors. Piperonyl butoxide, metyrapone and N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide (MGK-264) at non-fungitoxic concentrations were strongly antagonistic, whereas sesamex, nuarimol, fenarimol, etaconazole and 6-nitro or 6-methoxy 1,2,3-benzothiadiazole were moderately antagonistic. Phenobarbital and 2-diethylaminoethyl 2,2-diphenylvalerate (SKF 525-A) were slightly antagonistic. The results suggest that fungitoxicity of iprodione may be dependent on an activation catalysed by a cytochrome P-450 mixed function oxidase.     

Hydrolysis of malathion by rabbit liver oligomeric and monomeric carboxylesterases

The hydrolysis of malation by rabbit liver oligomeric and monomeric carboxylesterases (CE's) (EC 3.1.1.1) results in the formation of a mixture of α- and β-monoacids. A new chromatographic procedure was utilized to investigate the formation of α- and β-monoacids. The oligomeric carboxylesterase (oCE) produced an $\text{α}\text{β}$ ratio of monoacids of 4.55, and the monomeric carboxylesterase (mCE) produced an $\text{α}\text{β}$ ratio of monoacids of 2.33. The ratios of α- and β-monoacids were independent of the initial concentration of malathion and remained constant over the time course of the reaction. Kinetic studies demonstrated that the K_m values were the same for the corresponding reactions which produced either α-monoacid or β-monoacid with the same enzyme. Since both carboxylesterases are electrophoretically pure, the kinetic data strongly supports the theory that the reactions which produced α- and β-monoacids are catalyzed by the same active site. Comparison of the k_cat and K_m values governing the hydrolysis of malathion by the two esterases, together with their relative abundance in liver, indicated that the oCE would be responsible for about 80 to 98% of the hydrolytic detoxication of malathion by rabbit liver.     

Studies on the mechanisms of accumulation of phorate by the free-living nematode Panagrellus redivivus

Accumulation and metabolism of the organophosphate pesticide, phorate by the free-living nematode Panagrellus redivivus was studied under anaerobic conditions and in the presence of carbon monoxide or SKF 525A, and compared with results obtained under normal, aerobic conditions and using heat-killed nematodes. Both phorate hydrolysis and side-chain oxidation were inhibited under anaerobic conditions and with heat-killed nematodes, but only hydrolysis was affected in the presence of carbon monoxide. The much greater phorate accumulation in the nematodes under anaerobic conditions and with carbon monoxide could be explained by this metabolic inhibition. SKF 525A had no significant effect on phorate metabolism or accumulation. The possible mechanisms involved in phorate breakdown by P. redivivus are discussed.     

Steric,electronic, and polar parameters that affect the toxic actions of O-alkyl,O-phenyl phosphonothionate insecticides

The toxic action of a series of O-alkyl, O-substituted-phenyl alkyl- and aryl-phosphonates and phosphonothionates have been evaluated by correlating the linear free energy parameters for steric (E_s), electronic (σ), and polar ($\text{σ}{}^1$) effects with topical LD₅₀ to the house fly and oral LD₅₀ to the white mouse. In molecules free from major steric interactions with the reactive P atom, variations in these linear free energy parameters account for >90% of the variations in the LD₅₀ values, and the degree of correlation with LD₅₀ is at least as precise as that with the biomolecular rate constants for inhibition of the target-site enzyme acetylcholinesterase. The value of correlations of linear free energy parameters with LD₅₀ in understanding quantitative structure-activity relationships is illustrated.