Insecticides and electrophysiological symptoms: A comparative study on a tarsal motor nerve of Locusta migratoria

Spontaneous discharges of a tarsal motor nerve preparation of the jumping leg of Locusta migratoria were recorded extracellularly under the influence of various insecticides applied to the in situ metathoracic ganglion of the insect. Insecticides from different chemical classes were found to exhibit rather specific electrophysiological symptoms: cholinesterase inhibitors produced groups of action potentials with highly increased frequencies, and led to the appearance of new units which were not present in untreated control insects. They also caused transitory phases of conduction blocks, the length of which increased as intoxication proceeded. The DDT analog tested could be characterized by pronounced repetitive dischanges, whereas the neuroactivity of pyrethroids was typically associated with a very regular spike pattern in continuous phases of extremely high frequencies. Also chlordimeform, a formamidine insecticide, was found to increase the spike frequency, but to a much lesser extent than that observed for the other insecticides tested.     

Induction of the mixed-function oxidase system in the liver of the barn owl Tyto alba by PCBs

Injection of 30 mg/kg body wt of polychlorinated biphenyl (Aroclor 1254) into liver parenchymal tissue of nestling and adult barn owls Tyto alba resulted in increases in the level of cytochrome P-450. Concomitantly, there were increases in catalytic activity of the microsomal enzyme system as measured by aldrin epoxidation and aminopyrine N-demethylation. However, the ratio $\text{455}\text{430}\text{nm}$ in the ethylisocyanide-difference spectrum remained unchanged. Of particular interest is the sudden drop in the level and catalytic activity of cytochrome P-450 in nestling owls at age 40 days. Treatment with Aroclor 1254 produced small hemorrhages in the liver of nestling owls and the liver appeared much enlarged (hepatomegaly), indicating a toxic effect and resulting in little induction of microsomal enzymes. In adult owls the inductive effect was much greater. Aroclor 1254 produced a spectral shift in the cytochrome P-450-difference spectrum from 450 to 448 nm and in the ethylisocyanide-difference spectrum from 455 to 453 nm and from 430 to 427 nm.     

Differential inhibition of potassium ion absorption by difenzoquat in wild oat and cereals

Over a concentration range of 5.0 × 10^?6?7.5 × 10^?4M, the selective herbicide difenzoquat (1,2-dimethyl-3,5-diphenyl-1H-pyrazolium) caused more pronounced inhibition of potassium ion (K⁺) absorption by excised seedling roots of susceptible wild oat (Avena fatua L.) compared to those of tolerant barley (Hordeum vulgare L. cv. Bonanza) or wheat (Triticum aestivum L. cv. Neepawa). At 2.5 × 10^?5M difenzoquat, the relative inhibition of K⁺ (⁸⁶Rb) absorption by wild oat root segments inceased from 30% with a 10-min uptake period to 75% with an uptake period of 90 min, whereas no inhibition at all was evident for wheat root segments even after a 90-min exposure to the herbicide. An ion efflux compartmental analysis procedure demonstrated that difenzoquat did not affect the passive permeability properties of the plasma membrane of wild oat root cells. The experimental findings indicated that difenzoquat interfered directly with the process of active ion transport across the plasma membrane of root cells.     

Effects of chlordimeform on mitochondria from eggs of Spodoptera littoralis

Mitochondria were isolated from eggs of Spodoptera littoralis. With succinate (+pyruvate) as substrate, respiratory control ratios between 1.70 and 2.54 were obtained. Uncouplers and the energy transfer inhibitor oligomycin influenced these mitochondria in the well-known manner. The uncoupling activity of chlordimeform in vitro was very weak and decreased with increasing age of the eggs. Electron transport in mitochondria from chlordimeform-treated eggs was not uncoupled. Therefore, it is concluded that the ovicidal effect of this pesticide is not due to its uncoupling effect.     

S-Ethyl dipropylthiocarbamate, N,N-Diallyl-2-chloroacetamide,and 2-chloroallyl diethyldithiocarbamate influence on sulfhydryl-dependent sucrose accumulation

l-[U-¹⁴C]sucrose accumulation by phloem sieve tube members (PSTM) of wheat (Triticum aestivum L. ‘Holley’) and sorghum (Sorghum bicolor L. ‘G522 DR’) was inhibited by the nonpermeant sulfhydryl inhibitor p-chloromercuribenzenesulfonic acid (PCMBS), and this inhibition was reversed by the permeant sulfhydryl protectants dithiothreitol (DTT) and dithioerythritol (DTE). S-Ethyl dipropylthiocarbamate (EPTC) (≤0.1 mM) did not inhibit [¹⁴C]sucrose accumulation by wheat or sorghum PSTM. N-N-Diallyl-2-chloroacetamide (CDAA) (1 mM) inhibited [¹⁴C]sucrose accumulation by sorghum but not by wheat PSTM. The inhibition of [¹⁴C]sucrose accumulation in sorghum PSTM by the membrane permeant CDAA was reversed by DTT. Sorghum growth was inhibited by <1 μM CDAA. Membrane permeant 2-chloroallyl diethyldithiocarbamate (CDEC) (0.1 mM) inhibited [¹⁴C]sucrose accumulation by PSTM of sorghum but not wheat. The inhibition of sucrose accumulation in sorghum PSTM by 0.1 mM CDEC was reversed by DDT.     

Biochemical identification of putative GABA/benzodiazepine receptors in house fly thorax muscles

[³H]Flunitrazepam ([³H]Flu) was used to identify benzodiazepine binding sites in house fly thorax muscle membranes using a filter assay. [³H]Flu bound to a finite number of sites in a concentration- and time-dependent manner, reaching equilibrium in 10 min. Scatchard plots of the binding indicated a high-affinity site at 0.2 pmol/mg protein (K_d 24.3 nM) and a low-affinity site at 8.2 pmol/mg protein (K_d994nM). Binding of [³H]Flu to the high-affinity binding site was inhibited by several benzodiazepine analogs, with Flu, diazepam, and Ro 5-4864 being more potent than β-CCE, Ro 5-3027, and Ro 5-2180. Clonazepam was least potent in inhibiting [³H]Flu binding. Thus, the drug specificity of these insect muscle benzodiazepine binding sites was quite different from both the mammalian central and peripheral benzodiazepine receptor sites, though closer to the peripheral ones. GABA (γ-aminobutyric acid) and its agonists enhanced the specific binding of [³H]Flu in a dose-dependent manner, and this effect was inhibited with the GABA antagonist bicuculline. The effect was biphasic since at high GABA concentrations this stimulation was reduced. The data suggest that house fly muscles have benzodiazepine receptors, which are coupled allosterically to GABA receptors, analogous to the GABA/benzodiazepine receptors of vertebrates, but with some differences in their drug specificities.     

On the mode of action of 3-phenylindole towards Aspergillus niger

3-Phenylindole is an antimicrobial compound active towards many fungi and gram-positive bacteria. At 5 μg/ml it inhibits growth of Aspergillus niger. Higher concentrations (50 μg/ml) also suppress spore germination; they do not kill the fungus. Dry weight of the fungus still increases for 1 or 2 days after fungicide treatment. The toxicant has no effect on O₂ uptake even at higher concentrations (100 μg/ml). The compound markedly affects composition of the lipid fraction of A. niger inducing a decrease in phospholipid concentration with a coincident increase in free fatty acids. Sterol pattern and sterol concentration were not affected. Antifungal activity was reversed by phospholipids added to the medium. 3-Phenylindole induced a slight leakage of ³²P-labeled compounds from the treated cells under growth conditions but not under nongrowth conditions. A strain of A. niger resistant to 3-phenylindole had the same phospholipid and sterol pattern as the wild type, but the level of both components was higher (40–60%). The 3-phenylindole-resistant strain showed resistance to triarimol and pimaricin. The wild type and the resistant strain both took up 3-phenylindole quite rapidly and accumulated it in the mycelium. 3-Phenylindole possibly interferes with phospholipid function in cell membranes, although the specific site of action has not yet been elucidated.     

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.     

Interaction with phospholipids as a possible mode of action of 3-phenylindole on Aspergillus niger

Low concentrations of 3-PI(3-phenylindole) inhibit several uptake and biosynthetic processes in liquid cultures of Aspergillus niger. The incorporation of ³²P_i into phospholipids, [¹⁴C]uridine into nucleic acids, and [¹⁴C]phenylalanine into proteins was inhibited and the uptake of all three precursors was reduced. Studies on an in vitro interaction between 3-phenylindole and phospholipids showed, that at about equimolar concentrations 3-phenylindole prevents phospholipids from swelling in water. At the same ratio a decrease of the absorption intensity of the NH-band of 3-phenylindole was observed in ir spectra. Moreover, the maxima of the uv spectrum of 3-phenylindole shifted after addition of a sonicated phospholipid suspension. It is suggested that binding to phospholipids is the first effect of 3-phenylindole. Apparently this affects, consequently, several membrane-bound reactions, i.e., transport and biosynthetic processes.     

Protective effect of dimercaptosuccinic acid on methylmercury and mercuric chloride inhibition of rat brain muscarinic acetylcholine receptors

The reactivation of the rat brain muscarinic acetylcholine receptor (mACh-R) binding with dimercaptosuccinic acid (DMSA) after in vitro and in vivo inhibition by mercuric chloride (HgCl₂) and methylmercuric chloride (MeHg) was investigated. Receptor binding was estimated by the potent and specific antagonist l-[³H]quinuclidinyl benzilate ([³H]QNB). Rat brain synaptosomal membranes were exposed to HgCl₂ and MeHg. At 1 × 10^?4M. HgCl₂ caused complete inhibition of the [³H]QNB binding. The inhibition of [³H]QNB binding by HgCl₂ was still higher than 50% at 1 × 10^?8M. MeHg caused less inhibition of [³H]QNB binding than HgCl₂. The inhibited receptors showed a significant degree of reactivation when treated with DMSA. The recovery was almost complete after MeHg inhibition or with the lower HgCl₂ concentrations. Generally, the reactivation was dependent on the concentration of DMSA. When rats injected with either early or delayed doses of DMSA following administration with five consecutive daily doses (8 mg/kg body wt, Gavage method) of MeHg or HgCl₂, the inhibition of [³H]QNB binding was less than untreated ones. The early treatment with DMSA decreased the inhibition of [³H]QNB binding due to MeHg or HgCl₂ intoxication. However, DMSA was more effective in reducing HgCl₂ inhibition than MeHg either in vitro or in vivo treatment. The ability of DMSA to reactivate the mACh-R after inhibition with the mercurials emphasizes the involvement of essential sulfhydryl groups in [³H]QNB binding sites, and also shows that these sulfhydryl groups are the primary target for the MeHg and HgCl₂ inhibition of the rat brain muscarinic receptors.     

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.     

Difference in the picrotoxinin receptor between the cyclodiene-resistant and susceptible strains of the german cockroach

As a result of toxicity tests, it was established that all cyclodiene-resistant strains of the German cockroach are also resistant to picrotoxinin, a plant-origin neurotoxicant. Two of the cockroach strains which exhibit a distinct cross-resistance pattern to picrotoxinin (i.e., LPP and FRP) are the ones that have been purified genetically by backcrossing against the susceptible (CSMA) strain. This cross-resistance pattern appears to be specific to picrotoxinin and does not extend to other neuroexcitants such as bicuculline, beta-bungarotoxin, and DDT. The nervous system of the resistant cockroach was found to be less sensitive to picrotoxinin. Furthermore, it was determined that nerve components from the resistant cockroaches have significantly lower binding capacity to [³H]α-dihydropicrotoxinin. The most likely explanation for the above phenomenon is that these cockroaches have developed the cyclodiene resistance by altering the nerve receptor for picrotoxinin.     

Structure assignment to biologically produced malathion monocarboxylic acid isomers with 13C-nuclear magnetic resonance

Chemical synthesis of malathion α- and β-monocarboxylic acid yields a mixture of the two structural isomers. These two isomers were separated by preparative anion-exchange chromatography on QAE-Sephadex. ¹³C-Nuclear magnetic resonance of the pure components shows that the main product is the β-isomer, with the α-isomer being present in much smaller quantities. This result was used for identification of hydrolytic malathion metabolites produced by rat tissues. On incubation of malathion with rat liver fractions in vitro it was found that α- and β-monoacid are formed in a ratio of 3:2, whereas this ratio is 9:2 for the metabolites excreted into the urine after intraperitoneal injection of malathion in rats.     

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 effect of O,S,S,-trimethyl phosphorodithioate on the in vivo metabolism of phenthoate in the rat

The in vivo metabolism of phenthoate (O,O-dimethyl S-[α-(carboethoxy)benzyl]phosphorodithioate) was followed in rats after oral administration of a nontoxic dose of 100 mg/kg. The same metabolic study was conducted following coadministration of 0.5% O,S,S-trimethyl phosphorodithioate (OSS-Me). When administered alone, phenthoate was metabolized principally by carboethoxy ester hydrolysis and cleavage of the PO and CS bonds, resulting in at least six metabolites. The primary urinary metabolite excreted was phenthoate acid. Coadministration of 0.5% OSS-Me did not alter the types of metabolites excreted. However, a reduction of the carboxylesterase-catalyzed product (phenthoate acid) was observed, indicating that the enzyme responsible for the major pathway of phenthoate detoxication was inhibited. Alternate detoxication processes did not compensate for the reduction in carboxylesterase-catalyzed detoxication. It was concluded that inhibition of the carboxylesterase enzymes is the major cause of the potentiation of phenthoate toxicity by OSS-Me.     

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.     

The apparent in vivo neuromuscular effects of the δ-endotoxin of Bacillus thuringiensis var. israelensis in mice and insects of four orders

Alkaline-dissolved crystal δ-endotoxin from Bacillus thuringiensis var. israelensis (serovar H 14) was injected into mice and seven species of insects representing the orders Lepidoptera, Orthoptera, Coleoptera, Hemiptera, and Diptera. High in vivo toxicity, at 1 to 5 ppm (μg toxin/g body wet wt), was observed with mice and some insects, including some that are not sensitive to the toxin when administered orally. Neuromuscular effects were observed when the toxin was injected directly into the body cavity of the test animals. Biochemical studies suggested that different protein fragments within the crystal δ-endotoxin may be responsible for the majority of the mosquito larvacidal activity and the neurotoxic symptoms observed in larvae of Trichoplusia ni.     

The effects of organophosphorus poisoning on dopamine and 5-hydroxytryptamine concentrations in the brain of the cricket, Acheta pennsylvanicus, and the cockroach, Periplaneta americana

Liquid chromatography with electrochemical detection was used to measure dopamine and 5-hydroxytryptamine concentrations in the brains of crickets (Acheta (Gryllus) pennsylvanicus) and cockroaches (Periplaneta america) treated with the organophosphate dicrotophos. Organophosphate treatment did not cause depletion in dopamine and 5-hydroxytryptamine concentrations although reserpine treatment caused a reduction in the concentrations of both amines in the brains of these insects. Fluorescent histochemical studies of cricket brains confirmed these observations.     

Kinetics of sodium channel modification as the basis for the variation in the nerve membrane effects of pyrethroids and DDT analogs

The effects of a wide range of pyrethroids and DDT analogs on the membrane potential and membrane sodium currents were studied in crayfish giant axons. Compounds differed greatly in their ability to produce depolarizing afterpotentials, repetitive firing, and membrane depolarization. The differences observed at the membrane potential level could be explained by differences in the kinetics with which the insecticides interact with the nerve membrane sodium channel. The compounds containing a cyano group at the α position retain sodium channels in a modified open state persistently, depolarize the membrane, and block the action potential without causing repetitive firing. The pyrethroids without an α-cyano group and DDT analogs retain sodium channels in a modified open state only transiently, cause large depolarizing afterpotentials, and evoke repetitive firing with minimal effect on the resting potential. The effects of the phenoxybenzyl pyrethroids were found to be intermediate between these two extremes suggesting that a continuous variation exists in kinetics with which pyrethroids and DDT analogs modify sodium channels. It was not necessary to assume a second site of action to account for the variability observed. The implications of these results to the construction of quantitative structure-activity relationships is discussed.     

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.