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.     

Comparative metabolism of six ethoxychlor analogs and DDT in DDT-resistant and susceptible strains of the house fly Musca domestica L

The metabolic fate of six ³H-ring-substituted ethoxychlor analogs with altered aliphatic moieties and [¹⁴C]p,p′-DDT was investigated in susceptible and DDT-resistant strains of the house fly Musca domestica Linnaeus. The chloroalkane analogs, dichloroethane, chloropropane, and dichloropropane were primarily metabolized to the corresponding dehydrochlorinated products. This pathway was relatively more prominent in the resistant strain than in the susceptible strain. Biotransformation and detoxication of the isobutane, nitropropane, and neopentane derivatives was through microsomal oxidation (O-deethylation) of aryl ethoxy degradophores, and oxidation of the aliphatic moieties to produce the corresponding benzophenones, with no substantial differences between the resistant and susceptible strain. There was a strong correlation between the Taft ($\text{σ}{}^1$) values for the altered aliphatic moieties of chloroalkane analogs and their rate of dehydrochlorination in both the strains. These results suggest the importance of altered aliphatic moieties in developing resistance-proof DDT derivatives.     

Cytochrome P-450 content and aldrin epoxidation to dieldrin in isolated rat hepatocytes

A rat hepatocyte suspension effectively epoxidized aldrin to dieldrin with a V_max of 7.19 mol/mol P-450/min and a K_m of 9.27 μM. Viability and metabolic activity were stable for 6 hr after isolation when cells were maintained at room temperature (20°C) with the gentle introduction of $\text{O}{}_2\text{CO}{}_2$ onto the surface of the suspension. The cytochrome P-450 content of the suspension was 303 pmol/10⁶ cells. Primary maintenance culture of the cells also epoxidized aldrin. During culture for 3 days, metabolic activity decreased slowly day by day. Metabolic activity of microsomal fraction from rat liver was also examined. Microsomes epoxidized aldrin with a V_max of 5.11 mol/mol P-450/min and a K_m of 1.64 μM. Significant loss of some subspecies of cytochrome P-450 during fractionation of liver homogenate was indicated.     

Deuterium isotope effects on the formation of mercapturic acids from lindane in rats

Metabolism experiments with rats showed that significant isotope effects ($\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 2.4}\text{to}\text{3.5}$) were associated with the in vivo formation of dichloro and trichlorophenylmercapturic acids from a 1:1 mixture of normal and hexadeuterated lindane. This is evidence that rate-determining dehydrogenation and dehydrochlorination, both of which proceed with significant isotope effects, are essential in the pathway of dichloro- and trichlorophenylmercapturic acid formation from lindane. No significant primary isotope effects were associated ($\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 1.31 ± 0.17}$) with the formation of monochlorophenylmercapturic acid. This suggests that the 1,2-dechlorination to tetrachlorocyclohexene followed by glutathione conjugation is the probable pathway that produces this metabolite from lindane.     

The metabolism of p,p′-DDT by the feral pigeon (Columba livia)

Male feral pigeons were dosed with ring-labeled $\text{[}{}^{14}\text{C}\text{]p,p′-}\text{DDT}$ and the tissues and droppings analyzed for total ¹⁴C, extractable ¹⁴C, and metabolites. Only 16% of an intraperitoneal dose of 1.5–2.2 mg kg^?1 was voided in the droppings over 28 days; the rate of loss reached a maximum on the 14th day and then fell quickly away. The rate of removal of ¹⁴C in droppings was low in comparison to that found in the rat and the Japanese quail. When pigeons were dosed with 32–38 mg kg^?1 DDT per bird, and killed after 77 days, 5.4% of the dose was eliminated in droppings and 87% was recovered in the body. The tissues and droppings from this experiment were analyzed for DDT and its metabolites. Of the ¹⁴C remaining in tissues 88% was accounted for as the apolar compounds DDE, DDT, and DDD. Approximately half of the ¹⁴C in droppings was present as DDE, DDT, and DDD, whereas 27–35% was apparently in conjugated form, extractable from aqueous solutions by ethyl acetate after prolonged acid hydrolysis. Two polar metabolites were isolated from the acid-released material. One was p,p′-DDA; the other was extractable from aqueous solution at pH 8 and was tentatively identified as a monohydroxy derivative of p,p′-DDT. DDE accounted for 93% of the ¹⁴C present as metabolites in tissues and droppings, clearly indicating the importance of this intermediate in this study. The metabolism of DDT in the feral pigeon is discussed in relation to its metabolism by other species.     

Induction of glutathione S-transferase by phenobarbital and pesticides in various house fly strains and its effect on toxicity

The effect of phenobarbital and certain pesticides on glutathione S-transferase activity was investigated. The maximum amount of enzyme induction occurred 96 hr after phenobarbital treatment. Chlorinated hydrocarbons were more effective inducers than the other pesticides evaluated. Phenobarbital treatment did not alter the apparent K_m value but altered the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value of glutathione S-transferase to 3,4-dichloronitrobenzene. The amount of reduced glutathione was not increased by phenobarbital treatment. Pretreatment of house flies with phenobarbital provides some protection against methyl parathion, methyl paraoxon, azinphosmethyl, and methidathion toxicity.     

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.     

Interaction of insecticides with the Ca2+-pump activity of sarcoplasmic reticulum

The organophosphorus insecticides, parathion and azinphos (10^?5-10^?4M), significantly stimulate the Ca²⁺-pump activity of sarcoplasmic reticulum, while malathion has a limited effect. The rates of Ca²⁺ translocation and ATP hydrolysis are both stimulated and, apparently, the $\text{Ca}{}^{\mathrm{2+}}\text{ATP}$ ratio is improved. Parathion and azinphos maximally increase this ratio by 26 and 14%, respectively. The organochlorine compounds, DDT and aldrin, also stimulate the Ca²⁺ pump, and lindane has a reduced effect. These effects are smaller than those observed for parathion and azinphos. The order of effectiveness is similar to the toxicity of the compounds to mammals and can be described as follows: parathion > azinphos > DDT ≈ aldrin > malathion ≈ lindane.     

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.     

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.     

Inhibition of Penicillium duponti carboxylesterase by the fungicides captan and folpet

Captan, folpet, and perchloromethylmercaptan were effective inhibitors of Penicillium duponti p-nitrophenylpropionate esterase activity (I₅₀ = 0.5 – 2 μM) whereas α-naphthyl acetate esterase activity was not affected by the presence of these compounds. Captan and folpet are both equally effective at pH 7.3 and 8.3. The ionic composition of the medium had strong effects on the degree of inhibition produced by all inhibitors but did not alter esterase activity. Neither succinamide nor phthalimide caused inhibition of the p-nitrophenylpropionate esterase activity: The trichloromethylmercaptan portion of these fungicides appears to be responsible for the observed inhibition. The rapidity of captan and folpet inhibition of esterase activity (complete in < 1 min) compared to the rates of spontaneous decomposition ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{> 1}\text{min}$) and the insensitivity of captan and folpet inhibition to hydrogen ion concentration suggest that generation of spontaneous decomposition products is not required for inhibition. The results are consistent with a mechanism in which the entire fungicide molecule binds to the protein followed by enzyme-promoted reactions of captan and folpet which result in loss of esterase activity.     

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.     

Picrotoxinin receptor in the central nervous system of the American cockroach: Its role in the action of cyclodiene-type insecticides

The nature of the picrotoxinin receptor was studied using the central nervous system (CNS) of the American cockroach. It first was confirmed by using an electrophysiological technique that the abdominal nerve cord of the American cockroach was sensitive to picrotoxinin. By using a $\text{[}{}^3\text{H]α-dihydropicrotoxinin}$ binding test it was determined that the picrotoxinin receptor in CNS of this insect had a higher affinity toward picrotoxinin and heptachlor epoxide than the corresponding receptor in the rat brain. Also, the cockroach brain preparation had a higher percentage of specific binding in the total binding, making this material suitable for receptor studies. By using a sucrose density centrifugation technique, it was determined that the fraction sedimented at the interphase of 1.0 to 1.2 M sucrose at 100,000g contained the highest level of specific binding site. The receptor showed a sensitivity to all insecticidal cyclodienes tested, namely photodieldrin, oxychlordane, endrin, heptachlor epoxide, γ-chlordane, dieldrin, aldrin, heptachlor, and isodrin (expressed in the order of potency). Among four BHC isomers, the γ-isomer showed the highest potency to bind with this receptor.     

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.     

Carotenogenic inhibition by norflurazon in wheat

Wheat (Triticum aestivum L. cv Holley) seedlings were exposed to [N-¹⁴CH₃]norflurazon in nutrient solution studies. The ¹⁴CH₃ group was incorporated into a compound eluting on GLC at a relative retention temperature R_f equivalent to n-C₂₁ H₃₆ and mass spectrometry validated a 295 MW. The concentration of [N-¹⁴CH₃]norflurazon and/or Rl[¹⁴C]norflurazon which resulted in carotenogenesis inhibition was 0.07 μM in the water contained in the leaves. The concentration of norflurazon required for phytoene accumulation as a mode-of-action was ca. 140 × the concentration of norflurazon required for geranylgeraniol accumulation. Geranylgeraniol accumulated at 1 ppbw (3.2 nM) norflurazon and phytofluene accumulated throughout the norflurazon concentration series (1 to 1000 ppbw). Carotene content was increased by 1 to 16 ppbw norflurazon but was decreased by 64 ppbw norflurazon. Thus, two modes-of-activity for norflurazon are documented that depend upon concentration of the toxicant in the tissue. Norflurazon demethylation in prephytoenepyrophosphate synthesis resulted in a C₂₁ conjugate and increased concentrations of GGPP and phytoene in the tissue. At approximately 31 ppbw norflurazon, an inhibition of phytoene dehydrogenation occurred and phytoene accumulated. At 62 ppbw norflurazon, phytofluene hydrogenation inhibition occurred and phytofluene accumulated while β-carotene synthesis was inhibited. These inhibitions may possibly be reversible when substrate concentrations are in excess.     

Metribuzin metabolism in tomato: Isolation and identification of N-glucoside conjugates

Metribuzin [4-amino-6-tert-butyl-3-(methylthio)-1,2,4-triazin-5(4H)-one] metabolism was studied in tomato (Lycopersicon esculentum Mill. “Sheyenne”). Pulse-treatment studies with seedlings and excised leaves showed that [5-¹⁴C]metribuzin was rapidly absorbed, translocated (acropetal), and metabolized to more polar products. Foliar tissues of 19-day-old seedlings metabolized 96% of the root-absorbed [¹⁴C]metribuzin in 120 hr. Excised mature leaves metabolized 85–90% of the petiole-absorbed [¹⁴C]metrubuzin in 48 hr. Polar metabolites were isolated by solvent partitioning, and purified by adsorption, thin-layer, and high-performance liquid chromatography. A minor intermediate metabolite (I) was identified as the polar β-d-(N-glucoside) conjugate of metribuzin. The biosynthesis of (I) was demonstrated with a partially purified UDP-glucose: metribuzin N-glucosyltransferase from tomato leaves. A possible correlation between foliar UDP-glucose: metribuzin N-glucosyltransferase activity levels and differences in the tolerance of selected tomato seedling cultivars to metribuzin was suggested. The major polar metabolite (II) was identified as the malonyl β-d-(N-glucoside) conjugate of metribuzin.     

Oxidative metabolism of tetrachlorocyclohexenes,pentachlorocyclohexenes, and hexachlorocyclohexenes with microsomes from rat liver and house fly abdomen

Microsomal mixed-function oxidase systems from rat liver and house fly abdomen effectively metabolized isomers of 3,4,5,6-tetrachlorocyclohexene, 1,3,4,5,6-pentachlorocyclohexene, and 1,2,3,4,5,6-hexachlorocyclohexene to tetrachlorocyclohexenol isomers, 2,4,5,-trichlorophenol, and 2,3,4,6-tetrachlorophenol, respectively. The $\text{(}\text{346}\text{5}\text{)-isomer}$ of pentachlorocyclohexene gave also an abundant amount of pentachlorocyclohexenol isomers. As the metabolites of ($\text{36}\text{45}$)-, ($\text{35}\text{46}$)-, and $\text{(}\text{34}\text{56}\text{)-hexachlorocyclohexene}$, some compounds such as 1,2,4-trichlorobenzene, 1,2,3,4-tetrachlorobenzene, and pentachlorobenzene were more abundantly formed, respectively, than 2,3,4,6-tetrachlorophenol. These oxidative metabolic reactions were shown to mainly proceed via “ene-like” hydroxylation accompanied by double bond migration. Inhibition by CO, piperonyl butoxide, and SKF 525-A suggested that the “ene-like” hydroxylating enzyme was cytochrome P-450 dependent. The formation of an isomer of pentachlorocyclohexenol from $\text{(}\text{36}\text{45}\text{)-hexachlorocyclohexene}$ was also observed, and this reaction was activated by SKF 525-A.     

Importance of pH in assessing the potential for nitrosocarbamate formation in the stomach

Formation of the nitroso derivatives of ¹⁴C-labeled carbaryl, carbofuran, and p-chlorophenyl methylcarbamate (PCMC), and their subsequent stabilities were investigated using low levels of carbamate (0.5 μmol) and aqueous HCl concentrations encompassing those considered maximum for the gastric contents of humans (pH 1–2) and of rats (pH 3–4). Reacting the carbamates with excess sodium nitrite for 10 min at 37°C in a pH 1.0 HCl solution gave nitrosocarbamate yields of 42 to 64%, while only trace amounts were formed at pH 2 and above. The nitrosocarbamates were most stable at pH 3–5 with half-lives ranging from 114 to 470 min. Stability of all three nitroso derivatives was considerably less at pH 1.5 ($\text{t}\text{1}\text{2}$, 25–34 min), but at pH 7 the stability varied: nitroso-PCMC $\text{t}\text{1}\text{2}$, 6 min; nitrosocarbofuran $\text{t}\text{1}\text{2}$, 70 min; nitrosocarbaryl $\text{t}\text{1}\text{2}$, 139 min. Denitrosation to the parent carbamate was the predominant degradation pathway at pH 1.5, but at pH 3–7 degradation was primarily by hydrolysis of the carbamate ester linkage. Each of the nitrosocarbamates was directly mutagenic in the S. typhimurium assay system. Since the data show that nitrosation of residue levels of carbamate pesticides occurs readily at pH 1 but not at pH 2 and above, it is critical that gastric contents of any animal model used for assessing nitrosocarbamate formation have a pH approaching 1 as may occur in the human stomach.     

Effects of alkyl groups attached to the carbamoyl nitrogen atom of an N-arylcarbamoylpyrazoline on insecticidal activity

N-Alkylated analogs (C₁–C₃) of an N-arylcarbamoylpyrazoline were prepared. The introduction of these alkyl groups completely changed the crystal structure in respect of the torsion angle of the amide C–N bond of the non-alkyl compound. The introduction of methyl and ethyl groups slightly, and that of an isopropyl group markedly, decreased insecticidal activity against American cockroaches and house flies. Conformational analyses of the compounds suggested that the insecticidally active conformer of this class of compounds is in the anti-position regarding the N′–C(=O) and N-aryl bonds in which the non-alkyl compound is energetically the most stable. The most stable conformers of the alkylated compounds are in the syn-position, and these compounds would interact with target sites in the less stable anti-form. © 1999 Society of Chemical Industry     

Mitochondrial changes during storage of untreated or CIPC-treated potatoes

During storage of potato tubers (Solanum tuberosum L., var. Bintje), important changes appear which affect respiratory control, $\text{ADP}\text{O}$, intensity of O₂ consumption in the presence of different substrates, and NAD⁺ dependence, In mitochondria extracted under strictly similar conditions, from patato tubers stored at 4°C, the respiratory control (RC) maintains a value near 4 for 4 to 5 months. It then declines progressively to low values. At 20°C, a stable RC of 4 can be observed for several months, which then decreases at the end of dormancy. Then, the RC increases sharply; at this stage, $\text{ADP}\text{O}$ are abnormally low, and, some time later, NAD⁺ dependence disappears. Mitochondria treated with 250 μM chlorpropham show a 50% inhibition of the electron transfer with exogenous NADH as substrate. After tuber treatment with 1% chlorpropham, sprouting is inhibited for several months. The activities of mitochondria extracted from such tubers remain unaffected by the treatment. The use of this phenylcarbamate for potato tuber treatment permits obtaining functional mitochondria from tubers after a slightly longer period of storage.