Interstrain comparison of glutathione-dependent reactions in susceptible and resistant houseflies

Glutathione S-alkyl- and S-aryltransferase activities and the glutathione-dependent reactions involved in the metabolism of diazinon, parathion, DDT and γ-BHC were determined in two susceptible and three resistant housefly strains. The relative rate of formation of desethyl diazinon and desethyl parathion and the degradation of γ-BHC paralleled the activities of the alkyl and aryltransferases in the various strains of houseflies suggesting that a single enzyme might be involved. DDT-dehydrochlorinase showed different relative rates among the strains indicating that the dechlorination was catalyzed by a different enzyme. The enzyme responsible for the conjugation of the pyrimidinyl moiety of diazinon appears to be different from the one which catalyzes the conjugation of the p-nitrophenyl moiety of parathion. The dearylation reactions were not mediated by the glutathione S-aryltransferase in the various housefly strains.     

Genetic studies on glutathione-dependent reactions in resistant strains of the house fly,Musca domestica L

Genetic studies of glutathione-dependent reactions were conducted with a diazinon-resistant house fly strain in which resistance is controlled primarily by genes on chromsome II. The resistant strain was crossed with a susceptible strain which had mutant markers on chromosomes II, III, and V, and the F₁ was backcrossed to the susceptible strain. Glutathione transferase activities of the resultant eight phenotypes were measured using 3,4-dichloronitrobenzene, methyl iodide, and γ-benzene hexachloride as substrates. High levels of all these activities are controlled by gene(s) on chromosome II. Further analysis was made by introducing diazinon resistance into a susceptible strain via genetic crossing-over. Intermediate activity levels for 3,4-dichloronitrobenzene and methyl iodide conjugations were introduced along with intermediate levels of resistance. Assays of individual flies of the synthesized strain revealed they were heterogeneous for glutathione-dependent activities, consisting of individuals with low, intermediate, and high transferase activity. Based on these results, high levels of the glutathione-dependent enzymes are not a major biochemical mechanism responsible for diazinon resistance. It was also demonstrated that glutathione S-aryltransferase and S-alkyltransferase in the house fly, as measured with 3,4-dichloronitrobenzene and methyl iodide, are inseparable genetically and may, therefore, be the same enzyme.     

Host plant induction of glutathione S-transferase in the fall armyworm

The influence of various host plants on glutathione S-transferase activity was studied in the fall armyworm, Spodoptera frugiperda (J. E. Smith). Fall armyworm larvae were maintained on a semidefined artificial diet until the end of the fifth instar. The newly molted sixth instar larvae were then fed fresh leaves of various host plants for 2 days prior to glutathione S-transferase assays using 3,4-dichloronitrobenzene as substrate. The order of the midgut glutathione S-transferase activity of larvae after the worms fed on these plants was: mustard > turnip > cowpeas > peanuts > cotton > corn > cucumber > potato > Bermudagrass > millet > sorghum > soybeans. The difference in the transferase activity between soybean- and mustard-fed larvae was 10-fold. Kinetic study revealed a quantitative, but no qualitative difference in the glutathione S-transferase between soybean- and cowpea-fed larvae. Monoterpenes, such as α-pinene, β-pinene, menthol, and peppermint oil, had no effect on the enzyme. Cowpea-fed larvae were more tolerant of the insecticides diazinon, methamidophos, and methyl parathion than soybean-fed larvae were. These new observations help explain what has been happening in the field and might be of use in the development of pest management programs.     

Acute effects of methyl parathion and diazinon as inducers for oxidative stress on certain biomarkers in various tissues of rainbowtrout (Oncorhynchus mykiss)

Present study aimed mainly to assess oxidative stress pesticides such as methyl parathion (MP) and diazinon, which are widely used insecticides and contaminate aquatic ecosystems, on certain biomarkers in various tissues of rainbowtrout (Oncorhynchcus mykiss). Biomarkers selected for stress monitoring were malondialdehyde (MDA) and antioxidant defense system (ADS) mainly reduced glutathione (GSH), glutathione reductase (GR), peroxidase (GSH-PX), transferase (GST) and superoxidedismutase (SOD) activities in the liver, gills and muscle of fishes exposed to 0.5 and 1 ppm dosages of MP and diazinon for 24, 48 and 72 h. According to these results, after the administrations of MP and diazinon promote MDA content in some of the tissues of fishes treated with both dosages of MP and diazinon. With regard to the ADS, GSH-Px, GST, SOD, GR activities and GSH levels fluctuated after 24, 48 and 72 h in all the treatment groups compared with controls. Collective results demonstrated that exposure of fish to pesticides induced an increase in MDA joined with fluctuated ADS. This may reflect the potential role of these parameters as useful biomarkers for assessment of water pollution.     

Characterization of multiple glutathione transferases from the house fly, Musca domestica (L)

Glutathione transferases have been purified to a high degree of homogeneity from three strains of house fly by a procedure involving affinity chromatography on glutathione-sulfobromophthalein conjugate immobilized on Sepharose 4B, followed by preparative isoelectrofocusing. The affinity chromatography yielded purifications of between about 10- and 100-fold, depending on the strain and the substrate with which activity was measured. Each strain was shown to possess several proteins with glutathione S-transferase activity which fell into two clearly defined groups. The first group, of relatively low isoelectric point, showed activity with CDNB but little with DCNB, p-nitrobenzylchloride, or 1,2-epoxy-3-(p-nitrophenoxy)propane, whereas the second group, of higher isoelectric points, showed substantial activity with all substrates tested. Studies on the subunit structure of these enzymes demonstrated the existence of three different sized subunits of M_r 20,000, 22,000, and 23,500. From the experimental evidence recorded here, the existence of at least three functionally different glutathione transferases is inferred.     

Photometric determination of methyl parathion GSH S-methyl transferase

Two methods are described for the assay of the enzyme that transfers a methyl group from methyl parathion (O,O-dimethyl-O, p-nitrophenyl phosphorothionate) to glutathione. A colorimetric assay depends on the measurement of alkali-soluble, chloroform-insoluble aromatic nitro compounds in the reaction mixture and a more rapid and sensitive direct spectrophotometric assay is described that uses the double wavelength Perkin-Elmer 356 Spectrophotometer. The utility of both methods has been demonstrated by the measurement of some kinetic constants and the distribution of the enzyme among some insects and vertebrates.     

Glutathione Transferase Isozymes Involved in Insecticide Resistance of Diamondback Moth Larvae

In addition to the three glutathione transferase (GST) isozymes already identified in diamondback moth larvae, Plutella xylostella (L.), a fourth one, GST-4, was purified from a teflubenzuron (TFB)-resistant strain. This GST isozyme was similar to GST-3 in terms of biochemical and toxicological properties. GST-4, a homodimer with a subunit molecular mass of 26.6 kDa and a pI of ca. 8.9, displayed even stronger substrate preference than GST-3 for 1,2-dichloro-4-nitrobenzene and several organophosphorus insecticides, i.e., parathion, methyl parathion, and paraoxon. These two proteins were highly immunorelated and shared at least the first eight amino acids at the N-terminus. Immunoblotting analysis indicated that polyclonal antiserum raised against GST-3 cross-reacted with GST-1 and GST-2 at least 40-fold less intensely than with the antigen. Using this antiserum as probe, higher amounts and greater variations of GST-3/GST-4 were observed in larvae of a methyl parathion- and a TFB-resistant strain compared with a susceptible and a fenvalerate-resistant strain. Among the six lepidopterous insects examined, only Spodoptera exigua larvae clearly had proteins immunorelated to GST-3/GST-4 of diamondback moth. No such cross-reactivity was observed in Musca domestica, Drosophila melanogaster, and Aedes aegypti.     

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.     

The effect of phenobarbital induction on glutathione conjugation of diazinon in susceptible and resistant house flies

The relationship between glutathione S-transferase activity toward 3,4-dichloronitrobenzene and O-alkyl or O-aryl conjugation of diazinon was investigated in eight strains of house flies. No significant difference was found in the amount of O-aryl conjugation. In contrast, house flies which had higher glutathione S-transferase activity toward 3,4-dichloronitrobenzene also had higher O-alkyl conjugating activity toward diazinon. The glutathione S-transferase(s) in phenobarbital-pretreated flies degraded diazinon faster than those in the nontreated ones. The present results showed that the formation of the O-alkyl conjugate was enhanced by phenobarbital pretreatment, while the formation of the O-aryl conjugate was not affected by induction. Based on these findings, it would appear that one of the multiple forms of glutathione S-transferase is specifically induced and responsible for the increase in O-alkyl conjugation.     

in vitro metabolism of azinphosmethyl in susceptible and resistant houseflies

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

Eel acetylcholinesterase inhibition studies with heteroarylphosphinates

The inhibition of eel acetylcholinesterase by the 4-nitrophenyl esters of 2-furyl(methyl)-, methyl(2-thienyl)-, di-2-furyl-, and di-2-thienylphosphinic acid (I, II, III, and IV, respectively) was investigated at pH 6.90 in 0.067 M phosphate buffer (25.0°C) using stopped-flow instrumentation and automated data processing. Our evaluation of the dissociation constant, K_d, the unimolecular bonding rate constant, k₂, and the bimolecular reaction constant, k_i, are the first reported values for these constants for alkyl/heteroaryl and diheteroaryl esters of phosphinic acids. The largest k_i value (19,330 M^?1 sec^?1) was observed for the reaction of I with the enzyme. The order for the remaining three is II > IV > III. There is no direct relationship between the hydrolysis rates of the esters and their anticholinesterase activities on eel acetylcholinesterase. Likewise, there is no direct relationship between their anticholinesterase activities and the LD₅₀ values in rats.     

Metabolism and absorption of methyl parathion by tobacco budworms resistant or susceptible to organophosphorus insecticides

Factors involved in insecticide resistance were evaluated by using ¹⁴C-labeled methyl parathion and aldrin to compare rates of absorption and metabolism by Heliothis virescens (F.) larvae that were resistant (R) and susceptible (S) to methyl parathion. Tests with third-stage R and S larvae suggested that the rate of insecticide absorption from the cuticular surface was not a major resistance factor. Further evidence for this assumption was demonstrated by the resistance of R larvae to injected and orally administered doses of methyl parathion. Smaller amounts of unmetabolized methyl parathion and aldrin were recovered from S larvae, an indication that differences in metabolism were probably related to the resistance.     

Comparative effects of organophosphorus insecticides on the activities of acetylcholinesterase,diacylglycerol kinase,and phosphatidylinositol phosphodiesterase in rat brain microsomes

The activities of acetylcholinesterase, diacylglycerol kinase, and phosphatidylinositol phosphodiesterase in rat brain microsomes were measured in the presence and absence of the organophosphorus insecticides, parathion and diazinon, and their respective oxon analogs, paraoxon and diazoxon. Marked inhibition of acetylcholinesterase (by 45–99%) was observed in the presence of paraoxon (10^?2–10^?6M) and diazoxon (10^?2–10^?4M). Reduction of acetylcholinesterase activity (by 22–33%) was achieved with the parent insecticides at high concentrations only (10^?2M). In most cases, diacylglycerol kinase was insensitive to the pesticides. Marked stimulation of phosphatidylinositol phosphodiesterase (by 10–57%) was observed in the presence of all pesticides (10^?2–10^?3M). The phosphodiesterase exhibited slightly greater sensitivity to the parent compounds compared to the oxon derivatives. Stimulation of the phosphodiesterase by the insecticides was not correlated with acetylcholinesterase inhibition. Accordingly, the increase in phosphodiesterase activity was judged not to be acetylcholine mediated, but rather represented a direct effect of the pesticides on the enzyme or its microenvironment. Based on the present in vitro observations, it is proposed that certain organophosphorus pesticides may interfere with the normal process of synaptic transmission through both the inhibition of acetylcholinesterase and the stimulation of phosphatidylinositol phosphodiesterase. In view of the high concentrations of pesticides required to elicit the latter effect, interpretation of its physiological significance must await results from further studies performed in vivo.     

Metabolism of substituted diphenylether herbicides in plants. I. Enzymatic cleavage of fluorodifen in peas (Pisum sativum L.)

A “soluble” glutathione S-transferase that catalyzes the cleavage of the herbicide, 2,4′-dinitro-4-trifluoromethyl diphenylether (fluorodifen), was isolated and partially characterized from epicotyl tissues of pea seedlings. A 32-fold purification of the enzyme was achieved by differential centrifugation, ammonium sulfate precipitation, Sephadex gel filtration, and DEAE-cellulose ion exchange chromatography. The enzyme had a pH optimum of 9.3–9.5 and was specific for reduced glutathione, with an estimated apparent K_m value of 7.4 × 10^?4M. Limited specificity studies with four substituted ¹⁴C-labeled diphenylether compounds indicated that fluorodifen was the only effective substrate, with an estimated apparent K_m value of 1.2 × 10^?5M. Differences and similarities between the pea epicotyl enzyme and other plant and animal glutathione S-transferases were discussed from the standpoint of substrate specificity, pH optima, distribution, stability, and inhibitor studies.     

Comparative toxicity,absorption, and metabolism of chlorpyrifos and its dimethyl homologue in methyl parathion-resistant and -susceptible tobacco budworms

Chlorpyrifos (Dowco 179) and its dimethyl homologue, chlorpyrifosmethyl (Dowco 214), were used to study the influence of the O,O-dialkyl group of organophosphorus insecticides on toxicity, absorption, and metabolism among larvae of the tobacco budworm [Heliothis virescens (F.)] from strains that were resistant (R) and susceptible (S) to methyl parathion. In toxicity tests, chlorpyrifos and chlorpyrifosmethyl were more toxic than methyl parathion to 3rd-stage R larvae but less toxic to S larvae. Chlorpyrifosmethyl was more toxic (3–4 ×) than chlorpyrifos to both strains of larvae, and the results of absorption studies indicated that the toxicity differential of the homologues may be explained in part by the more rapid absorption of the dimethyl form. Studies of the in vivo metabolism of both Dowco compounds indicated that each was degraded mainly by the cleavage of the pyridylphosphate linkage. In vitro tests demonstrated that the NADPH-dependent microsomal oxidases were of primary importance in detoxification, while glutathione (GSH)-dependent mechanisms (aryl- and alkyltransferases) present in the soluble cell fractions were of lesser importance. O-dealkylation occurred only with chlorpyrifosmethyl. The R larvae demonstrated greater capability in detoxifying both compounds in the comparative in vivo and in vitro studies of metabolism, but the differences were more apparent during the 5th instar than during the 3rd instar.     

Effects of diazinon on biochemical parameters of blood in rainbow trout (Oncorhynchus mykiss)

Existence of diazinon, an organophosphorous pesticide, in river waters of Iran near rice paddy fields has been reported by some authors. The present research aimed to determine the acute toxicity and evaluate the effect of sub-lethal concentrations of diazinon on some biochemical parameters of rainbow trout, Oncorhynchus mykiss after 7, 14 and 28 days. No significant differences were observed in the plasma levels of creatinine among the treatment groups at different sampling intervals. Acetylcholinesterase activity and the levels of total protein, albumin as well as globulin in plasma were significantly reduced at both concentrations tested (p < 0.05). Lactate dehydrogenase activity was only decreased on 7th day in 0.1 mg/L diazinon treatment (p < 0.05). Creatine kinase activity was significantly lower in 0.1 mg/L diazinon group at 14th and 28th sampling periods, whereas its activity significantly increased in fishes exposed to 0.2 mf/L diazinon only on 7th day (p < 0.05). Aspartate aminotransferase, alanine aminotransferase activities and glucose levels in diazinon treated groups were significantly higher than the controlled group at experimental periods (p < 0.05). In conclusion, long-term exposure to diazinon at sub-lethal concentrations induced biochemical alterations in rainbow trout, and offers a simply tool to evaluate toxicity-derived alterations.     

Growth regulator inhibition of tobacco callus growth

The activity of two groups of growth regulators, substituted dinitroanilines and nitrophenylhydrazines, were evaluated in a tobacco (Nicotiana tabacum L. “X-73”) callus tissue bioassay. Molar concentrations required to inhibit fresh weight gain by 50% (I₅₀) was determined by using linear regression analysis on data obtained by testing a range of five concentrations of each chemical. All chemicals tested were inhibitory to callus tissue grown in the dark. Cell division seemed to be the primary activity inhibited. The most active of the dinitroaniline series was α,α,α-trifluoro-2,6-dinitro-N-ethyl-N-2′,6′-dichlorobenzyl-p-toluidine (I) (I₅₀ = 1.5 × 10^?10M). I and two other N-(o-halobenzyl) dinitroanilines were more active than α,α,α-trifluoro-2,6-dinitro-N-ethyl-N-2′-chloro-6′-fluorobenzyl-p-toluidine (IV), which is being developed commercially for suppression of axillary buds in tobacco. The two most active nitrophenylhydrazines tested were 1,1-dimethyl-2-(2′,6′-dinitro-3′-n-propylamino-α,α,α-trifluoro-p-tolyl)hydrazine (XVIII) and 3′,5′-dinitro-p-(2,2-diethylhydrazino)-N-methoxy-N-methylbenzamide (XIX) (I₅₀ values of 7.9 × 10^?9 and 9.3 × 10^?9M, respectively). Factors such as electronic distribution, steric hindrance, and lipid solubility were considered to influence the biological activity of the compounds tested.     

Glucose metabolism in hepatopancreas and gill of Lamellidens marginallis during methyl parathion toxicity

Changes in glucose metabolism were studied in hepatopancreas and gill of freshwater mussel, Lamellidens marginalis, exposed to a sublethal concentration (8 ppm) of methyl parathion. A slight decrease in glycogen and pyruvate and an increase in lactate levels were observed. An increase in phosphorylase and aldolase suggested increased formation of trioses during methyl parathion toxicity. The decrease in lactate dehydrogenase activity and increase in lactate content indicated reduced mobilization of pyruvate into the citric acid cycle. Glucose-6-phosphate dehydrogenase activity was increased, suggesting enhanced oxidation of glucose by the HMP shunt pathway. Citric acid cycle enzymes such as isocitrate, succinate, and malate dehydrogenases were found to be decreased, suggesting abnormality in mitochondrial oxidative metabolism as a consequence of methyl parathion toxicity. The decreased cytochrome c oxidase and Mg²⁺-ATPase, apart from citric acid cycle enzymes, indicated impaired energy synthesis as a result of reduced aerobic oxidation of glycose. The increase in acid and alkaline phosphatase activities suggested enhanced breakdown of phosphate to release energy in view of inhibition of the ATPase system during methyl parathion stress. The changes were more pronounced in hepatopancreas as compared to gill of mussel exposed to methyl parathion.     

Control of wireworms with organophosphorus and carbamate insecticides

Carbaryl, chlorfenvinphos, diazinon, disulfoton, parathion, phorate, fenitrothion, thionazin and trichlorphon were tested for their effectiveness in killing wireworms by broadcast treatments. Each insecticide was tested once or more in two field trials cropped with wheat and two cropped with potatoes. Phorate and parathion were very lethal to wireworms at 4 Ib active ingredient per acre (4–48 kg/ha). Thionazin and diazinon were intermediate in effectiveness and the other insecticides ineffective.     

The effect of phosphine on electron transport in mitochondria

The effects of phosphine on electron transport and on some partial reactions of oxidative phosphorylation of mitochondria from mouse liver, housefly flight muscles and granary weevils has been studied. Phosphine was a strong inhibitor of respiration of mitochondria in the “active” state (state 3), uncoupled state, and ion-pumping state on glutamate, pyruvate plus malate, succinate, α-glycerophosphate, and ascorbate-cytochrome c as substrates. Respiration of mitochondria in state 3 was completely inhibited by about 250 μM phosphine. By contrast, the respiration of mitochondria in state 4 was much less sensitive. This inhibition could not be released by uncouplers suggesting that it is due to a direct effect on electron transport. Only site III was inhibited to any significant extent. Kinetic studies show that the inhibition was noncompetitive with Ki ranging from 1.6×10^?5 to 7.2×10^?5 depending on the source and purity of cytochrome oxidase. The inhibition of site III was also more pronounced in sonicated particles than in intact mitochrondria. The significance of this is discussed in relation to membrane sideness and topology of the components of the respiratory chain.Phosphine was unable to activate the “latent” ATPase nor did it have any inhibition of the Mg²⁺-simulated ATPase and only high levels (1.1 mM) showed modest inhibition (41%) of uncoupler-stimulated ATPase. Phosphine had no effect on the ATP-Pi exchange and on the ATP-ADP exchange reaction at concentrations causing strong respiratory inhibition.