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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Metabolic fate of [14C]endrin in bluegill fish

Bluegill absorbed 85% of 1 ppb of endrin from water within 48 hr under static exposure conditions. The absorbed radiocarbon was eliminated linearly with a half-life of about 4 weeks. Analyses of eliminated radioactivity revealed only conjugated metabolites. 12-anti-Hydroxyendrin and 12-syn-hydroxyendrin were tentatively identified by cochromatography using thin-layer chromatography/autoradiography and gas chromatography. These metabolites were also present as conjugates in the fish organs. Seventy-three percent of the absorbed radioactivity recovered from fish extracts was in the form of unchanged endrin.     

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.     

Effects of herbicides and herbicide analogs on [14C]leucine incorporation by suspension-cultured Solanum nigrum cells

Assays of [¹⁴C]leucine incorporation were used to measure effects of herbicides on suspensioncultured heterotrophic Solanum nigrum cells. Most herbicidal vs. nonherbicidal chemicals in a set of 47 compounds could be distinguished from each other based on their extent of inhibition of leucine incorporation by S. nigrum cells. Herbicides which failed to inhibit leucine incorporation were photosynthetic inhibitors. Both phytotoxic and nonphytotoxic thiocarbamate analogs (as determined by whole-plant studies) tended to inhibit leucine incorporation. It was concluded that the leucine incorporation screen could detect a majority of compounds tested which are herbicidal, and that it may also be useful to detect compounds which have cellular toxicity which is not observed in the whole plant.     

Binding of organophosphate insecticides to lecithin: A proton magnetic resonance study

The interaction of four vinyl-type organophosphate insecticides with a phospholipid lecithin has been studied by proton magnetic resonance technique. The vinyl proton of organophosphate showed a low field chemical shift change when lecithin was added to a CCl₄ solution of the former. When organophosphate insecticide was added to a dilute solution of lecithin the choline protons peak also showed small chemical shift changes. These chemical shift changes suggest that the most probable binding site in the interaction involves a hydrogen bond formation between the vinyl proton and oxygen atom of phosphatidyl group of lecithin. Chemical shift data has been used to calculate the equilibrium constant of the interaction.     

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.     

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.     

Disposition and metabolism of [14C]chlorpyrifos in the black imported fire ant, Solenopsis richteri Forel

The short-term disposition and metabolism of topically administered [¹⁴C]chlorpyrifos was assessed in the black imported fire ant (Solenopsis richteri Forel) in the presence and absence of the mixed-function oxidase inhibitor piperonyl butoxide. Chlorpyrifos is readily absorbed into an internal organosoluble fraction which was quickly converted into a water-soluble fraction. The radioactivity was slowly excreted over a 24-hr period. Piperonyl butoxide slowed the conversion of the internal organosoluble radioactivity to the water-soluble fraction. Thin-layer chromatography indicated that piperonyl butoxide slowed the conversion of chlorpyrifos to material remaining at the origin, presumably water-soluble metabolites. The results of acid hydrolysis studies indicated that the water-soluble radioactivity was comprised mainly of conjugates. Although very little chlorpyrifos oxon was recovered in the metabolism experiments, in vitro studies on fire and head homogenates showed the compound to be an extremely potent anticholinesterase, with an I₅₀ of 4.6 × 10^?10M, while a major metabolite, 3,5,6-trichloropyridinol, was an ineffective acetylcholinesterase inhibitor.     

The effect of phosphine on the absorption and circular dichroic spectra of cytochrome c and cytochrome oxidase

The interaction of phosphine with purified cytochrome c and cytochrome oxidase was studied by absorption and circular dichroic spectrometry. Treatment of cytochrome oxidase with phosphine yielded absorption spectra in both the visible and Soret region that were similar to that produced by dithionite. Striking changes in the circular dichroic spectra were also recorded, sugesting that a valence change of the heme iron was accompanied by a conformation change of the prosthetic group.Phosphine treatment of cytochrome c also yielded spectra similar to those produced by reduction with dithionite suggesting a valence change of the heme iron. However, both the time necessary for complete reduction and the concentration of phosphine necessary for this effect suggest that, in comparison with cytochrome oxidase, cytochrome c was relatively insensitive. Reduction of cytochrome oxidase by phosphine had only a very minor effect on the intrinsic dichroic spectrum suggesting little effect on the polypeptide chains.The persistence of a component after reduction by phosphine and the subsequent reduction of this component by dithionite suggests that phosphine primarily reduced only one comonent of the cytochrome aa₃ complex. The failure of EDTA and fluoride, which are known ligands of cytochrome a₃ to affect the reduction of cytochrome aa₃ by phosphine, as well as the occurrence of a 436-nm isosbestic point of the ferric-ferrous absorption spectra suggests that phosphine reduced primarily cytochrome a. However, it is argued that a slow reduction of cytochrome a₃ cannot be ruled out.     

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.     

Cytochrome P-450 content and aldrin epoxidation to dieldrin in isolated rat hepatocytes

A rat hepatocyte suspension effectively epoxidized aldrin to dieldrin with a V_max of 7.19 mol/mol P-450/min and a K_m of 9.27 μM. Viability and metabolic activity were stable for 6 hr after isolation when cells were maintained at room temperature (20°C) with the gentle introduction of $\text{O}{}_2\text{CO}{}_2$ onto the surface of the suspension. The cytochrome P-450 content of the suspension was 303 pmol/10⁶ cells. Primary maintenance culture of the cells also epoxidized aldrin. During culture for 3 days, metabolic activity decreased slowly day by day. Metabolic activity of microsomal fraction from rat liver was also examined. Microsomes epoxidized aldrin with a V_max of 5.11 mol/mol P-450/min and a K_m of 1.64 μM. Significant loss of some subspecies of cytochrome P-450 during fractionation of liver homogenate was indicated.     

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.