In vitro effects of malathion and O,O,S-trimethyl phosphorothioate on cytotoxic T-lymphocyte responses

Oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in technical formulations of malathion, has been shown to be associated with a high incidence of pneumonia in rats and to be highly immunosuppressive in mice. Based on these findings, an in vitro model was established to study the effect of this and other organophosphorus compounds on murine cytotoxic T-lymphocyte (CTL) responses. The organophosphorus compounds were tested for their ability to block in vitro generation of CTL responses to alloantigen and/or the expression of these cytotoxic responses. Responses were generated in C57Bl/6 (H-2^b) spleen cells to mitomycin C-blocked P815 (H-2^d) tumor cells. The cytotoxicity of the cultured splenocytes to P815 target was measured using a 4-hr chromium release assay. These data demonstrated that malathion was able to block the ability of splenocytes to sensitize to P815 at concentrations as low as 25 μg/ml, but was not able to block the expression of cytotoxicity by mature killer T cells. The same was true for OOS which had been activated by preincubation with rat liver postmitochondrial supernatant (PMS). Activated OOS blocked the generation of CTL responses at concentrations as low as 75 μg/ml while having no effect on mature cytotoxic cells. In fact, both malathion and activated OOS were no longer able to suppress CTL responses if treatment was performed as early as 24 hr after exposure to antigen. Additionally, it was demonstrated that when malathion was preincubated with PMS it was no longer suppressive and that OOS without activation failed to suppress CTL responses.     

The mode of action and neurotoxicity of mirex,chlordecone, and four hydrogenated mirex analogs

The toxicity and neurological effects of mirex, chlordecone, and four hydrogenated mirex analogs were evaluated on the American cockroach. The severity of poisoning symptoms correlated with the ability of each compound to increase spontaneous activity and prolong synaptic afterdischarge in ganglia of the ventral nerve cord. Afterdischarge across the metathoracic ganglion was consistent with a characteristic wing splaying symptom in mirex-poisoned cockroaches. The actions of hemicholinium-3 and nicotine on nerve cords from mirex-poisoned cockroaches are described and are consistent with a hypothesis that the increased spontaneous activity and afterdischarge are the result of enhanced transmitter release in ganglia of poisoned animals.     

Sulfur in pesticide action and metabolism: Edited by J. D. Rosen,P. S. Magee,and J. E. Casida,American Chemical Society,Washington, D.C., 1981. 172 pp., $33.00

The distribution of ¹⁴C-acid-, ¹⁴C-alcohol-, and ¹⁴C-cyano-labeled deltamethrin and selected metabolites were followed in the liver, blood, cerebrum, cerebellum, and spinal cord after iv administration of a toxic, but nonlethal dose (1.75 mg/kg) to rats. Approximately 50% of the dose was cleared from the blood within 0.7–0.8 min, after which the rate of clearance decreased. 3-Phenoxybenzoic acid (PBacid) was isolated from the blood in vivo, and was also the major metabolite when ¹⁴C-alcohol-labeled deltamethrin was incubated with blood in vitro. Deltamethrin levels in the liver peaked at 7–10 nmol/g at 5 min and then decreased to 1 nmol/g by 30 min. In contrast, peak central nervous system levels of deltamethrin were achieved within 1 min (0.5 nmol/g), decreasing to 0.2 nmol/g at 15 min, and remaining stable until 60 min. peak levels of deltamethrin did not correspond to the severity of toxicity, although the levels of non-pentane-soluble radiolabel did appear to correlate with motor signs of toxicity. Experiments with brain homogenates, using in vivo concentrations of deltamethrin, failed to reproduce the pentane-unextractable radioactivity in vitro nor was any metabolism demonstrated.     

Pyrethroid-hydrolyzing esterases in southern armyworm larvae: Tissue distribution,kinetic properties,and selective inhibition

Esterase activity hydrolyzing both [1RS,trans]- and [1RS,cis]-permethrin was detected in crude homogenates of the following southern armyworm (Spodoptera eridania Cramer) larval tissues: cuticle, gut, fat body, head capsule, Malpighian tubules, and silk gland. Neither substrate was detectably hydrolyzed by hemolymph. The highest esterase activities per insect equivalent of tissue were found in cuticle, gut, and fat body for the trans isomer and in cuticle and gut for the cis isomer. Each preparation hydrolyzed the trans isomer more rapidly, but the degree of specificity varied greatly between tissues. Differences in apparent K_m and V_max values between the three most active tissues were threefold or less for trans isomer hydrolysis, but differences between tissues of up to 100-fold were found for K_m and V_max values for cis isomer hydrolysis. Hydrolysis of the trans isomer in cuticle, gut, and fat body homogenates was only partially inhibited by α-naphthyl N-propylcarbamate (NPC). Concentrations of NPC giving maximal inhibition of trans isomer hydrolysis had little effect on the hydrolysis of the cis isomer. These results demonstrate that pyrethroid-hydrolyzing activity is broadly distributed in insect tissues and results from the combined activity of several esterases with different properties. It is likely that the trans and cis isomers of permethrin are hydrolyzed by separate enzymes in this insect.     

The effect of diflubenzuron [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea] on soybean [Glycine max (L.) Merr.] photosynthesis,respiration, and leaf ultrastructure

The effect of the insecticide diflubenzuron [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea] on photosynthesis, respiration, and leaf ultrastructure of soybean [Glycine max (L.) Merr., cv. Swift] was examined on plants treated at the second trifoliate leaf stage with 0, 0.067, and 0.269 kg of active ingredien/ha of diflubenzuron. Photosynthesis and respiration were measured with an infrared CO₂ analyzer in an open flow system prior to diflubenzuron application and at 4, 24, 48, and 96 hr after treatment with diflubenzuron. Diflubenzuron had no effect on soybean photosynthesis at any rate examined. Respiration was stimulated by the high rate (0.269 kg/ha) in a transitory manner. Tissue samples removed from both old and new leaves, 9 days after diflubenzuron application, were used for the ultrastructure study with the transmission electron microscope. The lower trifoliate leaves contained more starch grains than the upper ones being formed after treatment, but no aberrations or degradation of leaf ultrastructure due to diflubenzuron treatment were evident.     

Structure-activity relationships of DDT-type analogs based on in vivo toxicity to the sensory nerves of the cockroach, Periplaneta americana L

Forty-three DDT-type compounds were applied in saline suspension to the crural nerve of Periplaneta americana L. and the threshold concentration (ED₅₀) to produce trains of impulses was determined together with the frequency of appearance of repetitive afterdischarge. These quantitative neurological measures were evaluated in multiple regression analyses of structural parameters including van der Waal's volume, the F and R components of Hammet's σ, and the hydrophobic constant Π. This structure-activity analysis provides an accurate estimation of the intrinsic toxicity of the DDT analogs. The results affirm previous working theories that the bulk of the functional groups within the DDT framework is the primary factor relating to activity. However, conformation is also an important parameter.     

Rates of sulfide oxidation in cotton,carrot, and tobacco cultured plant cells measured with a model aromatic alkyl-sulfide

p-Chlorophenylmethylsulfide (PCPMS) was enzymatically sulfoxidized to p-chlorophenylmethylsulfoxide (PCPMSO) in aerobic cotton, carrot, and tobacco cell suspension cultures. Neither boiled nor freeze-killed cell cultures were competent to sulfoxidize PCPMS to PCPMSO. The sulfone, p-chlorophenylmethylsulfone (PCPMSO₂), was not produced in any of the three species. The rates of PCPMS sulfoxidation, were cotton > carrot > tobacco. The apparent K_m for carrot cells was 88 μM PCPMS (14 μg/ml), while the apparent V_max was 7 nmol PCPMSO/mg whole cell protein/hr. These rates of sulfide oxidation are higher than previously reported in intact plants.     

Pyrethroid action on the nicotinic acetylcholine receptor/channel

The interactions of natural pyrethrins and nine pyrethroids with the nicotinic acetylcholine (ACh) receptor/channel complex of Torpedo electric organ membranes were studied. None caused significant reduction in [³H]ACh binding to the receptor sites, but all inhibited [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) binding to the channel sites in presence of carbamylcholine. Allethrin inhibited [³H]H₁₂-HTX binding noncompetitively, but [³H]imipramine binding competitively, suggesting that allethrin binds to the receptor's channel sites that bind imipramine. The pyrethroids were divided into two types according to their actions: type I, which included pyrethrins, allethrin, bioallethrin, resmethrin, and tetramethrin, was more potent in inhibiting [³H]H₁₂-HTX binding and acted more rapidly (i.e., in <30 sec). Type II, which included permethrin, fluvalinate, cypermethrin and fenvalerate, was less potent and their potency increased slowly with time. Also, inhibition of the initial rate of [³H]H₁₂-HTX binding by type I compounds increased greatly by the presence of the agonist carbamylcholine, but this was not so with type II compounds. The receptor-regulated ⁴⁵Ca²⁺ flux into Torpedo microsacs was inhibited by pyrethrins and pyrethroids, suggesting that their action on this receptor function is inhibitory. There was very poor correlation between the potencies of pyrethrins and pyrethroids in inhibiting [³H]H₁₂-HTX binding and their toxicities to house flies, mosquitoes, and the American cockroach. However, the high affinities that several pyrethroids have for this nicotinic ACh receptor suggest that pyrethroids may have a synaptic site of action in addition to their well known effects on the axonal channels.     

Effects of DDT on electrical properties of lecithin-decane bilayer membranes

Lecithin-decane bilayer membranes were treated with DDT and valinomycin, either by adding the compounds to the electrolyte around the membrane or by adding them directly to the lecithin-decane. Membrane capacitance was calculated using the dc transient technique. Specific capacitance was determined from capacitance versus area regressions and was not significantly altered by DDT. Specific K⁺ conductance was decreased by DDT, but only when DDT and the K⁺ carrier, valinomycin, were administered to the electrolyte where they may have interacted hydrophobically. It is concluded that the valinomycin-induced conductance of artificial membranes is inappropriate as a model for investigating the effects of DDT on the electrical properties of excitable membranes.     

Influence of pyrethroid ester,oxime ether,and other central linkages on insecticidal activity,hydrolytic detoxification,and physicochemical parameters

The relative potency to target and nontarget insects of ester pyrethroids and the analogous oxime ethers, and the degree of synergism of the esters with tributyl phosphorotrithioate, provide a useful guide to the importance of esterase detoxification in species specificity. These criteria indicate that esterase detoxification is a more important component of pyrethroid tolerance in Chrysopa carnea and Cryptolaemus montrouzieri larvae than in Exochomus flavipis larvae and Musca domestica adults. Studies with 3-phenoxybenzyl 2-(4-chlorophenyl)-2-cyclopropylacetate and the corresponding 2,3,4,5,6-pentafluorobenzyl ester, their oxime ether analogs, and permethrin and its thiol ester and amide analogs provide evidence that the high insecticidal activity of some ester and E-oxime ether pyrethroids, relative to that of the corresponding thiol ester and amide, is more closely associated with the dipole moment and polarizability of the central linkage and its resistance to esterase detoxification than with bond lengths or lipophilicities conferred by a specific linkage. Pentafluorobenzyl tetramethylcyclopropanecarboxylate is very effective in the vapor state for house fly control.     

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.     

Differential inhibition responses caused by DDT on oligomycin-sensitive Mg2+-ATPase activity: Nature of the requirements for DDT sensitivity

Earlier communications from this laboratory have shown that DDT inhibited oligomycin-sensitive Mg²⁺-ATPase (EC 3.6.1.3) but that its active component, F₁, was not affected. In the present investigation evidence has been obtained to determine the nature of the requirements for DDT sensitivity. The results showed that DDT sensitivity was conferred to F₁ from pig heart mitochondrial preparations when it was bound to F₀ from the same preparation. The F₁ from house fly (Musca domestica L) thorax was able to bind to F₀ from pig heart. This combination showed similar sensitivity to that of the original F₁-F₀ combination from pig heart mitochondria. However, when F₁ from pig heart mitochondria was incorporated into F₀ depleted in oligomycin sensitivity-conferring protein (OSCP) from the same source, the resulting ATPase activity was insensitive to DDT. Addition of crude (50–200 μg) or purified (5–20 μg) OSCP in the above preparation restored DDT sensitivity. Presence of dioleyl or dipalmitoyl phosphatidyl choline or Triton X-100 in the reaction medium antagonized the DDT inhibitions. Depletion of phospholipids from submitochondrial membrane preparations (SMP) decreased ATPase activity. Addition of dioleyl or soybean phosphatidyl choline to this lipid-depleted preparation restored DDT sensitivity. Evidence presented suggests that DDT acted on F₁ in association with one or more membrane components and that OSCP and phospholipid were essential for DDT sensitivity.     

Pesticide action: Different response of erythrocyte membrane acetylcholinesterase to inhibition by organophosphorus compounds under varied dietary conditions

The inhibitory effect of malathion in conjunction with maloxon or other derivatives on the activity of rat erythrocyte membrane-bound acetylcholinesterase has been investigated after feeding diets known to affect the lipid composition of the membrane. The inhibition of AchE by preincubation of membrane preparations with this organophosphorus compound, possibly with contaminants, differed with the diet: the curve was sigmoidal after supplementation with corn oil and hyperbolic after supplementation with lard. In the latter case the kinetics of the inhibition became sigmoidal by assaying in the presence of cortisol or isoproterenol or by previous solubilization of the enzyme. These results indicate that some organophosphorus compounds can differentiate conformational states of acetylcholinesterase present in the erythrocyte membrane.     

The toxicological significance of paraoxon deethylation

The in vivo formation of deethylation and hydrolytic products of paraoxon degradation after parathion or paraoxon administration was nearly equal in control male rats, and the relative abundance of metabolites was not appreciably altered by pretreatment of rats with enzymeinducing agents. However, pretreatment with inducers dramatically increased the oxidative paraoxon O-deethylase of male rat liver while having little effect on hydrolytic enzymes. Prior to induction, the hepatic O-deethylase activity was greatly inferior to the various hydrolytic enzymes, but nearly equal levels of both enzyme systems were found after induction. These results indicate that a large portion of the hepatic hydrolases detected in vitro is not active in vivo. It also appears that the majority of the induced hepatic deethylase was not involved in vivo at the dosage levels employed. The in vivo metabolism of monoethyl paraoxon was also demonstrated. The predominant metabolite of ethyl-[1-¹⁴C]monoethyl paraoxon is ¹⁴CO₂, while phenyl-[1-¹⁴C]monoethyl paraoxon yielded 4-nitro[1-¹⁴C]phenol. Paraoxon deethylation was also shown to be an important detoxication mechanism in female rats and male mice and must be considered in interpreting the toxicological properties of parathion and paraoxon.     

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.     

Herbicidal interference with the photochemical activities of chloroplasts (in vivo and in vitro) of certain crop and weed species

The independent modes of action of diuron and atrazine on the photochemical activities of chloroplasts (In vivo and in vitro) from the leaves of crop plants Pisum sativum and Pennisetum typhoides and the weeds Amaranthus viridis and Cyperus rotundus were investigated. Hill reaction activity (DCPIP photoreduction) of in vivo chloroplasts (chloroplasts isolated from herbicide-sprayed plants) was unaffected by treatment at sublethal or intermediate levels of diuron or atrazine while that of in vitro chloroplasts (chloroplasts incubated in the required herbicidal concentration) was severely inhibited. The ferricyanide catalyzed noncyclic photophosphorylation was markedly reduced in both the in vivo and in vitro chloroplast systems. N-Methyl phenozonium sulfate (PMS)-mediated cyclic photophosphorylation was inhibited in the in vivo system while a pronounced enhancement of activity was noticed in the in vitro chloroplasts. The rate of NADP⁺ photoreduction was severely inhibited in the in vitro chloroplasts. The unaffected in the in vivo system. The herbicidal effects on the photoreactions of isolated chloroplasts were compared with chloroplasts isolated from herbicide-sprayed plants.     

Structure-activity correlations of the insecticide Prolan and its analogs

Structure-activity correlations for 45 insecticidal diaryl nitropropanes (Prolan analogs) were analyzed by multiple regression analysis. Molecular bulk constants including van der Waal's radii, molar attraction constants, parachor, steric constants such as Taft's E₈ and Verloop's dimensional steric constants, hydrophobic constants such as II, and electronic parameters such as σ, F, and R were evaluated. It was concluded that the diaryl nitropropanes like the diaryl trichloroethanes fit into a receptor site which has an optimum volume for maximum interaction. The interaction between the insecticide and the receptor shows high correlation with steric constants for the aryl substituents and with intermolecular attractive forces. Highly asymmetrical compounds such as 1-(p-fluorophenyl)-1-(p-hexoxyphenyl)-2-nitropropane were surprisingly effective insecticides.     

In vitro activity and binding characteristics of the hydroxybenzonitriles in chloroplasts isolated from Matricaria inodora and Viola arvensis

The electron transport inhibition, uncoupling, and binding of ioxynil and bromoxynil salts is compared in chloroplast fragments isolated from two weed species with contrasting responses to the hydroxybenzonitriles. Ioxynil Na was three to four times more inhibitory than bromoxynil K towards DCPIP and SiMo reduction in both Matricaria inodora and Viola arvensis. Ioxynil Na was also a more potent uncoupler of PSI-dependent electron transport from ascorbate/DCPIP to methyl viologen. Uncoupling occurred at concentrations higher than those that inhibited electron transport. Binding studies with [¹⁴C]bromoxynil K and [¹⁴C]ioxynil Na salts revealed slightly biphasic curves with no significant difference in the amounts of the two herbicides bound at a given concentration. The ratios of inhibition constant (K_i) and binding constant (K_b) were approximately one for ioxynil Na and three for bromoxynil K. Radiolabelled herbicide displacement studies revealed that ioxynil Na could partially displace bound [¹⁴C]bromoxynil K, but bromoxynil K could not displace bound ioxynil Na at biochemically active concentrations. Ioxynil Na may be a more effective inhibitor than bromoxynil K because it binds more strongly to the thylakoid membrane.     

Membrane damage in human erythrocytes caused by captan and captafol

Treatment of human erythrocytes with 5 × 10^?5M captan or captafol caused a rapid increase in the efflux of intracellular potassium. Captafol had a more pronounced effect than captan on cation permeability. Captafol also decreased anion permeability whereas captan did not affect this process. Glutathione (5 × 10^?4M) had little effect in reducing potassium efflux when added to the cells after they were incubated for 1 h with captan or captafol, but it was effective in reducing the potassium loss when added to the cells prior to their treatment with the fungicides. Captafol caused an increase in osmotic fragility of the cells. Incubation of the cell membranes with captafol resulted in the liberation of a small fraction of membrane phospholipids, whereas captan produced no effect. Both the fungicides readily reacted with the sulfhydryl groups in the isolated membrane; 31.5 and 45.7% of the membrane sulfhydryl groups had disappeared following treatment with captan and captafol, respectively. It is suggested that the reaction of captan or captafol and/or their reaction products with the sulfhydryl and amino groups of the red cell membrane protein produces changes in the structure of the membrane with consequent alteration in its permeability.     

Uptake of dieldrin,lindane, and DDT by isolated rat hepatocytes

Studies of the uptake of dieldrin, lindane, and DDT by isolated rat hepatocytes in Krebs-Henseleit salt solution containing 2.34% bovine serum albumin have shown that the partition equilibrium of these hydrophobic organochlorine compounds is established very rapidly between the hepatocytes and the extracellular medium by reversible uptake and release processes. In the hepatocyte suspension system, these compounds are in dynamic partition equilibrium between the hepatocytes and the Krebs-Henseleit salt solution, and also between the bovine serum albumin molecules and the solution. The respective rate constants for the uptake and release processes were 0.83 and 2.52 × 10^?2 sec^?1 in a 2-ml suspension containing 10⁶ cells. It has thus been demonstrated that the transport of hydrophobic organochlorine compounds between the hepatocytes and the extracellular medium is a much faster process than the metabolic transformation reaction in hepatocytes.