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.     

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.     

Effects of chlordimeform on vascular smooth muscle

The effects of chlordimeform on agonist-induced contractions of rabbit aortic strips and on calcium flux in the deadventitiated strips have been studied. Chlordimeform antagonizes contractions induced by several vasoactive agents, with the order of antagonism being potassium . histamine > serotonin > norepinephrine, and decreases the rate of contraction of strips exposed to each of the four agonists. Muscles contracted by each of the agonists are rapidly relaxed by application of chlordimeform, although in the case of norepinephrine the relaxation only proceeds to approximately one-half of the maximal tension. The rate of washout of ⁴⁵Ca from the media-intimal layer of the rabbit aorta was increased by chlordimeform, but the norepinephrine-induced decrease in the rate of washout of ⁴⁵Ca was not affected by chlordimeform. Chlordimeform did not affect ⁴⁵Ca uptake by the media-intimal strips. The results of this study indicate that chlordimeform probably interferes with loosely bound calcium to a greater extent than it does with more tightly bound stores.     

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.     

Mechanism of neuromuscular block by chlordimeform

The mechanism of action of chlordimeform on the frog sciatic nerve-sartorius muscle preparation has been studied by means of microelectrodes. Chlordimeform (0.1 mM) suppresses the amplitude of spontaneous miniature end-plate potentials without much changing their frequency. At 1 mM, it completely blocks the miniature end-plate potentials. The resting membrane potential is not affected. The end-plate potential evoked by nerve stimulation is also suppressed, while the action potential from the nerve terminal is not impaired. The sensitivity of the end-plate membrane to the transmitter substance as examined by iontophoretic applications of acetylcholine is effectively decreased by chlordimeform. The quantal content of transmitter release is not affected. It is concluded that the block of neuromuscular transmission by chlordimeform is due primarily to a depression of the end-plate sensitivity to the transmitter.     

Metabolism of fenitrothion in red-backed voles (Clethrionomys gapperi)

To estimate the impact of fenitrothion on small mammals, a breeding colony of wild southern red-backed voles (Clethrionomys gapperi) was successfully maintained. The red-backed vole is a nontarget species inhabiting coniferous forests that are subjected to aerial spraying of the pesticide for the control of spruce budworm infestations. The voles were used to examine toxicity and the metabolic pathways involved in the elimination of fenitrothion after intraperitoneal injection of the purified, undiluted organophosphate. The 96-hr LD₅₀ was 1330 mg/kg. Toxicities toward males and females were not significantly different. All observed symptoms of intoxication and 99% of the mortality occurred within 48 hr of dose application. All major metabolites of fenitrothion metabolism in nonruminant mammals were identified and quantified by gas chromatography. At all doses, fenitrothion was rapidly metabolized and excreted. No body accumulation of residues was implied. The toxicity of fenitrothion was not correlated with increasing proportion of the dose converted into fenitrooxon. The low toxicity to red-backed voles is probably due, in part, to rapid metabolic detoxication by the two mechanisms cleaving fenitrothion at the P-O-aryl and PO-alkyl bonds, the latter becoming more prominent at high dose levels.     

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.     

Correlations in phytotoxicity between white and green calli of Rumex obtusifolius,Nicotiana tabacum and Lycopersicon esculentum

The extent that green and white calli reflect the phytotoxicities of various compounds on seedlings was measured. Green calli, which were found to photosynthetically fix ¹⁴CO₂ but which were not autotrophic, better mirrored the effects of the same compounds tested on seedlings for two reasons. (a) Inhibitors of photosynthesis inhibited green calli more strongly than white calli; (b) compounds which were inactive in foliar sprays on seedlings but are known to be active through the soil more strongly inhibited white calli than green calli. Thus it is suggested that green cell cultures have potential use in studies of mode of action and for preliminary phytotoxicity screening.     

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.     

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.     

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.     

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.     

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.     

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.     

In vitro effects of DDT analogs on trout brain Mg2+-ATPases: I. Specificity and physiological significance

A continuous steady-state assay procedure was used to investigate the effects of DDT and several analogs on the in vitro Mg²⁺-stimulated adenosinetriphosphatase of a trout brain mitochondrial fraction. Pharmacological dissection of the enzyme with oligomycin, dicyclohexyl-carbodiimide, and azide failed to yield a fraction specifically sensitive to the organochlorines. At 25°C, low doses of DDT (≤1.35 μmol/mg of protein) stimulated enzyme activity, while methoxychlor was stimulatory at all doses. Higher doses of DDT and of several analogs caused only 45.5% or less inhibition at 25°C, but inhibition increased at lower temperatures. The physiological significance of these effects is discussed.     

Structure-related effects of pyrethroid insecticides on the lateral-line sense organ and on peripheral nerves of the clawed frog, Xenopus laevis

The effects of seven different pyrethroid insecticides on the lateral-line sense organ and on peripheral nerves of the clawed frog, Xenopus laevis, were investigated by means of electrophysiological methods. The results show that two classes of pyrethroid can be clearly distinguished. (i) Pyrethroids without an α-cyano group (permethrin, cismethrin, and bioresmethrin). These noncyano pyrethroids induce short trains of nerve impulses in the lateral-line sense organ. In peripheral nerve branches they induce a depolarizing afterpotential and repetitive firing. These effects are very similar to those previously reported for allethrin. (ii) Pyrethroids with an α-cyano-3-phenoxybenzyl alcohol (cypermethrin, fenpropathrin, deltamethrin, and fenvalerate). In the lateral-line sense organ these α-cyano pyrethroids induce very long trains of nerve impulses which may last for seconds and may contain hundreds or even thousands of impulses. The α-cyano compounds do not cause repetitive activity in peripheral nerves. Instead they induce a quickly reversible, stimulus frequency-dependent suppression of the action potential. Since the chemical structure of cypermethrin differs from that of permethrin only in the α-cyano group and because all four α-cyano compounds act in a very similar way, it is concluded that the α-cyano substituent is responsible for the large differences in neurotoxic effects. In the lateral-line sense organ the duration of nerve impulse trains induced by the noncyano as well as the α-cyano pyrethroids increases dramatically when the temperature is lowered. Further, in sensory fibers the effects of both classes of pyrethroid on the nerve action potential are more pronounced compared to their effects on motor fibers. It is argued that the different neurotoxic effects reported here originate from a common mechanism of action of pyrethroids, which is a prolongation of the transient increase of sodium permeability of the nerve membrane associated with excitation.It is concluded that the sodium channel in the nerve membrane is the major target site of noncyano and α-cyano pyrethroids.     

In vitro effects of DDT analogs on trout brain Mg2+-ATPases: Inhibition kinetics

A continuous in vitro steady-state assay procedure was used to investigate the dependence of trout brain mitochondrial Mg²⁺-stimulated adenosinetriphosphatase specific activity on temperature and substrate concentration. The inhibition of enzyme activity by DDT was independent of substrate concentration. DDT and several analogs caused increases in the experimental activation energy and frequency factor of the enzyme-catalyzed reaction, which gave rise to a negative temperature coefficient of inhibition. It is suggested that DDT and other highly lipophilic compounds have the potential to allosterically affect membrane-bound enzymes by simply becoming a major lipoid component of the membranes.