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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of some herbicides on CO2 fixation,intermediates pattern,and ruDP-carboxylase and FDPase activities of spinach chloroplasts

We have studied the inhibitory effect of the herbicides phenmediphan, chloroxuron, dinoseb, dichlobenil, dicamba, 2,4-D, 2,4-DB, and 2,4-DP on photosynthetic CO₂ fixation and on the level of intermediates of the CO₂ assimilation cycle by isolated chloroplasts, as well as their in vitro activities on the enzymatic systems ribulose-1,5-diphosphate carboxylase and fructose-1,6-bisphosphatase. Phenmedipham showed the strongest inhibition of CO₂ assimilation, with an I₅₀ of 0.05 μM, followed by chloroxuron and dinoseb, with a 50% inhibition in the range of 0.5–1 μM. A weaker inhibitory effect, with an I₅₀ of 50 μM, is promoted by 2,4-DB, whereas dicamba and 2,4-DP showed this inhibition at 100 μM; dichlobenil and 2,4-D were completely ineffective. In the presence of phenmedipham and chloroxuron, the $\text{trioses-}\text{P}\text{P}\text{-glycerate}$ ratio showed a sharp decrease, which means an inhibition of the P-glycerate reduction step by a low NADPH synthesis; a low ratio is also promoted by 2,4-D, but it may be a consequence of induced collateral metabolic pathways of P-glycerate. Dinoseb showed a 25% inhibition of ribulose-1,5-diphosphate carboxylase activity in the concentration range of 10–100 μM and an I₅₀ of 50 μM of the fructose-1,6-bisphosphatase. Thus these effects could contribute, in addition to the photochemical ones, to an explanation of the dinoseb inhibition of CO₂ assimilation by isolated chloroplasts. The other herbicides tested showed a weak or no effect on these enzyme systems.     

Overproduction of reactive oxygen species involved in the pathogenicity of Fusarium in potato tubers

Differences in virulence between Fusarium sulphureum and Fusarium sambucinum were compared. Changes in reactive oxygen species production and metabolism in inoculated slices of potato tubers were also compared. The result showed that Fusarium infection induced significant production of ROS, lipid peroxidation and loss of cell membrane integrity, but low activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Compared to F. sambucinum, F. sulphureum led larger lesion diameters on potato tubers and slices. It resulted in more superoxide anion ($O_2^{-}$) and earlier peak of hydrogen peroxide (H₂O₂), but lower activity of catalase (CAT) and APX, and accompanied with higher malondialdehyde (MDA) content and lower cell membrane integrity. These findings suggested that overproduction of ROS involved in the pathogenicity of Fusarium in potato tubers.     

Components of the electron transport system in the microsomal mixed-function oxidase system in wild birds

Notable differences were found among six species of wild-caught birds in the levels of cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase. Ethyl isocyanide difference spectra showed significant variations among the species in peak height and in the ratios of the $\text{430}\text{455}\text{-}\text{nm}$ peaks. Substantial aldrin epoxidase activity was found in all species, and the amounts of dieldrin produced compared favorably with pigeon and rat liver microsomes. Higher content of cytochrome P-450 was not always accompanied by a similar rise in specific catalytic activity. Thus, no correlation could be established between these two parameters. Aldrin epoxidase activity with NADH as the sole electron donor was 25–49% as effective as with the NADPH-generating system. Addition of both NADH and NADPH-generating systems to the incubation mixture produced a synergistic effect with liver microsomes of two species but not with two other species. DDE and polychlorinated biphenyls residues were found in the heart tissue of all species examined, and this might indicate a possible inductive effect on the microsomal mixed-function oxidase system by environmental contaminants.     

Delayed acute toxicity of O,S,S-trimethyl phosphorodithioate and O,O,S-trimethyl phosphorothioate to the rat

The toxicity and LD₅₀ of O,S,S-trimethyl phosphorodithioate were reexamined in the rat. Animals treated orally (single dose) with this compound exhibited early cholinergic signs followed at approximately 5 hr by delayed toxic signs, with an LD₅₀ of 43 mg/kg. Contamination of O,S,S-trimethyl phosphorodithioate by as much as 5% (w/w) O,O,O-trimethyl phosphorothioate provided only limited antagonism to the dithioate's toxicity. In contrast, the addition of 5% O,O,S-trimethyl phosphorodithioate to O,O,S-trimethyl phosphorothioate gave protection against the toxic effects of the latter compound up to 80 mg/kg of toxicant. Pretreatment of rats with as little as 5% O,O,O-trimethyl phosphorothioate, 24 hr prior to treatment with 200 mg/kg O,O,S-trimethyl phosphorothioate, gave complete protection against the toxic effects of this compound. Conversely, administration of 10% (w/w) O,O,O-trimethyl phosphorothioate 4 or 24 hr after treatment with 60 or 80 mg/kg of O,O,S-trimethyl phosphorothioate provided only partial protection at 4 hr and no protection from the effects of the toxicant at 24 hr. The ability of O,O,O-trimethyl phosphorothioate to antagonize the toxicity of this compound depended markedly on the route of administration (oral, intravenous, or intraperitoneal). At 4 hr past treatment with toxicant, only oral administration of the antagonist provided full protection. Intraperitoneal and intravenous administration of antagonist 4 hr after treatment with toxicant were partially effective and completely ineffective, respectively, in halting the toxic effects of this compound.     

Studies on hydrolases in various house fly strains and their role in malathion resistance

Aliesterase, carboxylesterase, and phosphorotriester hydrolase activities in six house fly strains were studied in relation to malathion resistance. Selection of two susceptible strains with malathion for three generations resulted in an increase in both carboxylesterase activity and LD₅₀ of malathion, indicating that the increased detoxication by the enzyme was the major mechanism selected for malathion resistance. With the highly resistant strains, however, the carboxylesterase activity alone was not sufficient to explain the resistance level, and the involvement of additional mechanisms, including phosphorotriester hydrolase activity, was suggested. The E₁ strain, which had high phosphorotriester hydrolase activity but normal or low carboxylesterase activity, showed a moderate level, i.e., sevenfold resistance. Upon DEAE-cellulose chromatography, two or three esterase peaks were resolved from susceptible, moderately resistant, and highly resistant strains. The substrate specificity, the sensitivity to paraoxon inhibition, and the $\text{α}\text{β}$ ratio of malathion hydrolysis were studied for each esterase peak from the different strains. The results suggested the existence of multiple forms of esterases with overlapping substrate specificity in the house fly.