The action of insecticides on neurosecretory neurons in the stick insect, Carausius morosus

The action of insecticides on the spontaneous electrical activity of neurohemal tissue in the stick insect, Carausius morosus, has been studied using extracellular electrodes. The pyrethroid, permethrin, causes a massive increase in the frequency of the spontaneously generated action potentials at concentrations between 5 × 10^?5 and 5 × 10^?8M. Concentrations as low as 5 × 10^?11M are still effective in producing bursting activity.DDT, at concentrations between 5 × 10^?5M and 5 × 10^?6M, produces an overall increase in activity although the bursting activity is less violent than that shown with permethrin. DDT, 5 × 10^?7M, is ineffective at altering the resting pattern.Carbaryl and coroxon cause a transitory increase in electrical activity at 1 × 10^?4M, but are ineffective at 1 × 10^?5M.It is concluded that insecticides could have a direct effect upon the neurohormonal balance in insects.     

Effects of herbicidal carbamates on mitochondria and chloroplasts

At concentrations near 2 × 10^?4M, barban, chlorpropham, and phenmedipham are inhibitors of the electron transfer in potato and mung bean mitochondria. The inhibition seems to be localized in the flavoprotein region. It affects preferentially the exogenous NADH dehydrogenation, in potato mitochondria (I₅₀, 10^?4M). Succinate dehydrogenation is less inhibited. At noninhibiting concentrations, the studied carbamates cannot uncouple the oxidative phosphorylations. Photosynthesis is completely inhibited by 2.10^?7M phenmedipham, 5 × 10^?5M barban, and 2 × 10^?4M chlorpropham. The inhibition takes place at the PS II level. Moreover, barban and chlorpropham are uncouplers of the photophosphorylations for concentrations between 5 × 10^?5 and 5 × 10^?4M. The effects observed on mitochondrial respiration can also be found on respiration of Acer cultured cells. The effects on isolated chloroplast photosynthesis are also observed for slightly higher concentrations on cultured Chlorella and on pea and oat leaf fragments.     

Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds

In apterous adults of the spirea aphid, Aphis citricola van der Goot, the optimum conditions for determining acetylcholinesterase (AChE) activity consist of reaction mixture of 0.1 M phosphate buffer (pH 7.5), 10^?3M acetylthiocholine (ASCh), and enzyme extract equivalent to 80 ± 3 μg protein incubated for 15 min at 30°C. The K_m value for ASCh (6.7 × 10^?5M) was much lower than that of butyrylthiocholine (BuSCh) (1.25 × 10^?2M). The enzyme activity was almost completely inhibited by 10^?6M paraoxon or 10^?5M eserine and was 84% inhibited by 10^?5M BW284C51 (a specific AChE inhibitor). DTNB was found to inhibit the enzyme activity and was therefore added at the end of the reaction. AChE activity of A. citricola was inhibited in vitro and in vivo by dimethoxon > dimethoate, and aldicarb sulfoxide > aldicarb > aldicarb sulfone. The in vivo effect correlates well with the toxicity level of the various toxicants. A neurotoxicity index which combines both mortality and in vivo inhibition of the aphid AChE activity is suggested as a measure for determining the toxicity of organophosphorus and carbamate compounds toward aphids.     

Pyrethroid insecticides: Potent,stereospecific enhancers of mouse brain sodium channel activation

Deltamethrin and NRDC 157, pyrethroid insecticides that produce different poisoning syndromes in mammals, enhanced veratridine-dependent, sodium channel-mediated ²²Na⁺ uptake in mouse brain synaptosomes. Concentrations producing half-maximal enhancement were 2.5 × 10^?8M (deltamethrin) and 2.2 × 10^?7M (NRDC 157). This effect was stereospecific: The nontoxic 1S enantiomers had no significant effect on veratridine-dependent activation. At high deltamethrin concentrations, enhancement was maximal at 5 × 10^?5?1 × 10^?4M veratridine. Pyrethroid enhancement was completely blocked by 5 × 10^?6M tetrodotoxin, and neither pyrethroid affected ²²Na⁺ uptake in the absence of veratridine at concentrations up to 1 × 10^?5M. The relative potencies of deltamethrin and NRDC 157 in the synaptosomal sodium channel assay agree well with their relative acute toxicities to mice when administered by intracerebral injection. These findings demonstrate that pyrethroids exemplifying both characteristic poisoning syndromes are potent, stereospecific modifiers of sodium channel function in mammalian brain.     

Structure-activity relationships of 1,2,3-benzothiadiazole insecticide synergists as inhibitors of microsomal oxidation

The activities of 47 substituted 1,2,3-benzothiadiazoles as inhibitors of microsomal epoxidation and/or hydroxylation in enzyme preparations from rat liver or armyworm (Spodoptera eridania) gut have been evaluated. Many were found to be effective inhibitors of microsomal oxidation, the most active being the 6-butyl and 6-propoxy derivatives with I₅₀ values of 4.9 × 10^?7 and 7.0 × 10^?7M, respectively, for the epoxidation reaction. Regression analyses have established that activity of the 5-, 6-, and 5,6-substituted compounds can be satisfactorily described in equations in terms of π², π, and σ whereas that of the 4-substituted derivatives depends on π and the steric parameter E₈.     

The effect of DDT and its analogs on the fragility of human erythrocytes

The antihemolytic actions of DDT and eight analogs were examined with human erythrocytes. Apparent aqueous concentrations to produce 60% of control hemolysis ranged from 3.7 × 10^?4 to 2.4 × 10^?6M, with DDT being one of the least active. No correlation was found between antihemolytic potency and neurotoxicity, and it was concluded that the findings did not illuminate the toxic or neural actions of these compounds.     

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.     

Interference of benomyl and methyl-2-benzimidazolecarbamate (MBC) with DNA metabolism and nuclear divisions in Fusarium oxysporum

Methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) severely decreased DNA synthesis when applied at 3.5 × 10^?6M during the G1 phase of germinating conidia of Fusarium oxysporum; nuclear divisions were completely inhibited at a fungicide concentration of 10 × 10^?6M. The same concentration applied only after the S phase also completely inhibited the nuclear divisions. This dual interference of benomyl with DNA formation and mitosis might be related to a disturbed phosphorus metabolism.     

Effects of U-40481 and formetanate on the isolated rabbit central ear artery

Formetanate, a formamidine-type pesticide, and U-40481 (N-methyl-N′-2,4-xylylformamidine), a metabolite of amitraz, also a formamidine pesticide, contract the rabbit central ear artery with their maximal contractions being 22 ± 8% and 49 ± 6% of norepinephrine contractions, respectively. Maximal contractions were obtained with 10^?3M formetanate and 10^?4M U-40481, and cumulatively added higher concentrations caused a decrease in tension from that maximum. Their contractions were antagonized by 10^?6M and 3 × 10^?6M phentolamine. U-40481 reversibly antagonized contractions induced by serotonin, norepinephrine, and histamine, and to some extent potassium. Formetanate had little antagonist activity. Neither compound altered the resting rate of washout of radioactivity from [³H]norepinephrine preloaded strips. Both reduced electrically induced release, which may be related to local anesthetic-like actions on sympathetic neurones. Thus both compounds are partial agonists at the α-adrenergic receptor, and reduce electrically induced norepinephrine release, and U-40481 antagonizes contractions induced by certain other vasoactive agents.     

Potassium uptake in atrazine-treated roots of sensitive and resistan plants

The action of atrazine and its biodegradation products on the membrane transport of potassium in roots was evaluated in both sensitive and resistant plants. Excised roots of maize and oat showed inhibition of potassium uptake efficiency in the presence of 1.4 × 10^?4M atrazine and 1.4 × 10^?4M deethylated atrazine. Other biodegradation products such as 2-chloro-4-amino-6-ethylamino-1,3,5-triazine,2-chloro-4,6-,bisamino-1,3,5-triazine, and 2-chloro-4-amino-1,3,5-triazine showed no inhibitory effect on the K⁺ uptake capacity. Two maize hybrids showing different uptake efficiency were inhibited differently by atrazine. We suggest that atrazine and deethylated atrazine inhibited the K⁺ transport interacting directly with the plant cell membranes without discerning between resistant and sensitive plants.     

The mechanism of fungistatic action of sec-butylamine: II. The effect of sec-butylamine on pyruvate oxidation by mitochondria of Penicillium digitatum and on the pyruvate dehydrogenase complex

sec-Butylamine at 5 mM inhibited the oxidation of pyruvate by mitochondria isolated from hyphae of Penicillium digitalum, but had little effect on the oxidation of citrate, isocitrate, succinate, malate, acetyl-coenzyme A, or reduced nicotinamide adenine dinucleotide. sec-Butylamine did not interfere with oxidative phosphorylation, as evidenced by similar $\text{P}\text{O}$ ratios in treated and control mitochondria. The pyruvate dehydrogenase complex (EC 1.2.4.1) isolated from young hyphae of P. digitatum was inhibited strongly by 20 mM sec-butylamine, whereas other tricarboxylic acid cycle enzymes were only slightly affected at most. Inhibition of the pyruvate dehydrogenase complex by sec-butylamine was competitive with respect to pyruvate. The K_i for sec-butylamine in the reaction was 1.38 × 10^?2M, and the K_m for pyruvate was 2.28 × 10^?4M. These observations and other evidence derived from studies with intact hyphae support the hypothesis that the pyruvate dehydrogenase complex is the primary site of the fungistatic action of sec-butylamine.     

Influence of monuron on photosystem II and light-dependent 14CO2 fixation in isolated cells of C3 and C4 plants

Cells were isolated from the developing leaves of Ipomoea aquatica (water spinach), a C₃ plant, and three kinds of C₄ plants, namely, Digitaria sanguinalis (NADP⁺-specific malate dehydrogenase type), Panicum miliaceum (NAD⁺-specific malic enzyme type), and Panicum texanum (phosphoenopyruvate carboxy kinase type), to study the effect of monuron on light-dependent ¹⁴CO₂ fixation and oxygen evolution. Bundle sheath cells, except for those of D. sanguinalis, and mesophyll cells of all plants fixed approximately the same amount of ¹⁴CO₂. Monuron, at the range used (2 to 10 × 10^?7M), showed strong inhibition in the mesophyll cells of water spinach and in bundle sheath cells of P. miliaceum and P. texanum and moderate inhibition in the mesophyll cells of all C₄ plants. In the bundle sheath cells of D. sanguinalis the low rate of ¹⁴CO₂ fixation was stimulated to some extent by the addition of malate and ribose 5-phosphate. The I₅₀ value was 6 × 10^?7M for the sensitive cells. Monuron inhibited the oxygen evolution of all seven cell types and their I₅₀ values varied between 3 × 10^?7 to 6 × 10^?7M. The differential response of isolated plant cells from different species to light-dependent CO₂ fixation in the presence of monuron may also be involved in urea herbicide selectivity and undoubtedly is due to the differential photosynthetic pathways present nn them.     

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.     

The effects of pyrethroids on the electrical activity of neurosecretory cells from the brain of Rhodnius prolixus

The effects of pyrethroids on the on-going electrical activity of the axons of neurosecretory cells from the brain of fifth instar Rhodnius prolixus have been studied using extracellular electrodes. Low concentrations of the pyrethroids decamethrin, bioresmethrin, permethrin, and bioallethrin all produce dramatic increases in the overall frequency and dramatic changes in the pattern of electrical activity when applied directly to the exposed brain and corpora cardiaca in an otherwise intact insect. This change in activity was brought about by a recruitment in active units and the production of phasic acivity. A doubling of frequency over that of controls was brought about by low doses of the pyrethroids, namely decamethrin, 1 × 10^?10M; bioresmethrin, 2 × 10^?10M; permethrin, 1 × 10^?9M; and bioallethrin, 2 × 10^?7M. Similar hyperactivity of this system occurred during intoxication of intact insects following topical application of LD₉₅ bioresmethrin. The enhanced sensitivity shown by neurosecretory cells over that of other cell types is discussed, as is the possibility that the increases in electrical activity of neurosecretory axons may result in massive neurohormonal release and thereby contribute to the eventual poisoning of the insect.     

Trifluoromethylketones as possible transition state analog inhibitors of juvenile hormone esterase

A series of compounds containing the trifluoromethylketone group have been synthesized utilizing either a modified Grignard procedure or by reacting selected aliphatic bromides or tosylates with the Collman reagent [Na₂Fe(CO)₄]. When tested in vitro as inhibitors of crude juvenile hormone esterase from the hemolymph of the cabbage looper, Trichoplusia ni (Noctuidae), the most active compounds were trifluoromethylketones possessing either a juvenoid-like structure or a straight aliphatic chain. The logarithm of the inhibitory potency of the aliphatic compounds was proportional to their chain length, up to 1,1,1-trifluorotetradecan-2-one (I₅₀ = 1 × 10^?7M). This powerful inhibition was found to be highly selective for JHE, reversible, competitive by Lineweaver-Burk analysis, and was characterized by high affinity of the inhibitor for the esterase (K_i = 3.2 × 10^?9M, K_m JH III = 2 × 10^?7M). Other trifluoromethylketones were shown to be inhibitors of T. ni α-naphthylacetate esterase and bovine trypsin. By analogy with the mechanism of trypsin action, trifluoromethylketones are probably potent inhibitors due to their resemblance to a tetrahedral transition state on the reaction coordinate to the acylated enzyme.     

Inhibition of two acetylcholinesterases by the 4-nitrophenyl esters of methyl-, ethyl-, and isopropyl(phenyl)phosphinic acid

The inhibition of eel acetylcholinesterase and bovine erythrocyte acetylcholinesterase by the 4-nitrophenyl esters of methyl-, ethyl-, and isopropyl(phenyl)phosphinic acid (MPP, EPP, and IPP, 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 a homologous series of this class of organophosphorus compounds. The largest k₁ value (29,428 M^?1 sec^?1) was observed for the reaction of eel acetylcholinesterase with 4-nitrophenyl methyl(phenyl)phosphinate. The smallest k_i value (9.6 M^?1 sec^?1) was observed for the reaction of bovine erythrocyte acetylcholinesterase with 4-nitrophenyl isopropyl(phenyl)phosphinate.     

Inhibition of fatty acid biosynthesis in isolated bean and maize chloroplasts by herbicidal phenoxy-phenoxypropionic acid derivatives and structurally related compounds

The effects of nine phenoxy-phenoxypropionic acid derivatives and structurally related compounds on the incorporation of [¹⁴C]-acetate into free fatty acids in isolated bean and maize chloroplasts were studied. The compounds tested were esters and the corresponding free acids, OH-diclofop, a nonherbicidal metabolite of diclofop in plants, and d and l enantiomers of diclofop. Fatty acid biosynthesis in bean chloroplasts was not affected by all compounds. OH-Diclofop had a weak inhibitory effect on fatty acid synthesis in maize chloroplasts, while free acids were stronger inhibitors than the corresponding esters in the same system. Uptake studies with diclofop-methyl and diclofop indicated that the esters showed higher uptake rates in chloroplasts suspension. d-Diclofop (I₅₀, 9 × 10^?8M) was a more potent inhibitor than l-diclofop (I₅₀, 4 × 10^?6M). This agrees with the low herbicidal activity of the l enantiomer in vivo. The results suggest that the mode of action in this type of herbicide may be closely linked with the inhibition of fatty acid biosynthesis. The tolerance of beans could be based on an insensitivity of the target site.     

Cholinesterase inhibition by methamidophos and its subsequent reactivation

Methamidophos (O,S-dimethylphosphoramidothioate, Monitor) is an organophosphorus, cholinesterase-inhibiting insecticide. The rate constant (k_i) for inhibiting rat plasma cholinesterase (ChE) was 1.57 ± 0.03 × 10³M^?1 min^?1, for rat erythrocyte ChE was 8.86 ± 1.10 × 10³M^?1 min^?1, and for rat brain ChE was 6.58 ± 0.42 M^?1 min^?1. Brain and plasma cholinesterases spontaneously recovered from over 90% inhibition at 30 min to 50% inhibition in 4 and 14 hr, respectively. Pralidoxime increased the rate of reactivation in vitro. In vivo, rats poisoned with methamidophos exhibited signs of cholinergic stimulation. The LD₅₀ of ip methamidophos in male rats was 15 ± 0.7 mg/kg. Pralidoxime (60 mg/kg) and atropine (10 mg/kg) given with the methamidophos increased the LD₅₀ to 52 ± 4.9 mg/kg and 60 ± 0.4 mg/kg, respectively. In rats given 12.5 mg methamidophos (an LD₂₀), ChE activity was depressed 95 ± 12.5% in plasma, 92 ± 0.6% in stomach, and 88 ± 1% in brain at 1 hr after injection. At 48 hr after injection ChE activity had returned to 60% or more of control values in each of the tissues. Administration of a single dose of 60 mg/kg of pralidoxime along with methamidophos did not increase ChE activities at the times and places it was measured.     

The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates

A range of compounds were tested as inhibitors of the enzyme epoxide hydrase, using a cyclodiene epoxide (HEOM) as substrate. Rat and rabbit liver microsomes and pupal homogenates of the blowfly (Calliphora erythrocephala) and the yellow mealworm (Tenebrio molitor) were compared as sources of the enzyme. Only minor differences were found between the four enzyme preparations, when considering I₅₀ values and percentage inhibition at standard concentration. The simple epoxide 1,1,1-trichloropropane-2,3-epoxide and two glycidyl ethers p-nitrophenyl glycidyl ether and p-ethylphenyl glycidyl ether tended to have lower I₅₀ values (1.8×10^?6 to 8.0×10^?5M) than triphenyl phosphate and SKF 525A (4.5×10^?5 to 1.4×10^?4M). Triphenyl phosphate and SKF 525A were competitive inhibitors for both the rat and Tenebrio enzymes. The only clear difference found between these two epoxide hydrase preparations was with respect to their inhibition by 1,1,1-trichloropropane-2,3-epoxide, which was an uncompetitive inhibitor with the rat enzyme, but showed kinetics of mixed inhibition with the insect preparation.     

Kinetic properties of the inhibition of juvenile hormone esterase by two trifluoromethylketones and O-ethyl,S-phenyl phosphoramidothioate

Some inhibition kinetic properties and in vivo inhibition of the plasma juvenile hormone esterase from the cabbage looper (Trichoplusia ni Hübner) by one phosphoramidothioate and two trifluoromethylketones were examined. O-ethyl,S-phenyl phosphoramidothioate was shown to react irreversibly with the enzyme in a time-dependent manner, and the inhibition reaction can be factored into a reversible step with a dissociation constant, K_d, of 4.55 × 10^?5M followed by a phosphorylation step with a rate constant, k₂, of 1.98 min^?1. The phosphorylated enzyme did not show spontaneous recovery after 48 hr of dialysis. On the other hand, the two trifluoromethylketones were shown to act as reversible inhibitors, as their inhibited enzyme was regenerated completely after dialysis. However, 1,1,1,-trifluoro-3-thiooctylpropan-2-one, in contrast to 1,1,1-trifluorotetradecan-2-one, showed progressive time-dependent inhibition, and its reaction with the enzyme followed characteristic bimolecular second-order kinetics with a rate constant, k_i, of 3.37 × 10⁷M^?1 min^?1. The in vivo inhibition data of topically treated larvae at equimolar amounts of the tested compounds indicated rapid penetration, and the stability of the inhibition was higher for the phosphoramidothioate than for the trifluoromethylketones. The relationship of the mechanism of inhibition and the in vivo inhibition of these compounds to the understanding of the interactions between juvenile hormone and juvenile hormone esterase is discussed.