Spindle disturbances in mammalian cells. I. changes in the quantity of free sulfhydryl groups in relation to survival and c-mitosis in V79 chinese hamster cells after treatment with colcemid,diamide, carbaryl and methyl mercury

Asynchronously growing V79 Chinese hamster cells were treated with colcemid, diamide, carbaryl and methyl mercury, which are all known to be spindle disturbing agents. For each compound the dose response for c-mitosis, survival and level of free sulfhydryl groups was investigated under comparable conditions. Diamide, carbaryl and methyl were all found to give a significant increase of c-mitosis at a dose giving a decrease of non-protein sulfhydryl groups (NPSH, mainly glutathione) of 30–40% suggesting that a decrease of this magnitude may have a predictive value for spindle disturbances. Despite this similarity at concentrations close to the respective thresholds it was found that the c-mitotic activity at higher concentrations was not a simple function of average NPSH decrease. Diamide, which rapidly oxidizes glutathione to glutathione disulfide, was a less efficient c-mitotic agent than carbaryl and methyl mercury in relation to average NPSH decrease at higher concentrations. Protein bound sulfhydryl groups (PSH) were not significantly affected with diamide and carbaryl at their lowest c-mitotic concentrations while methyl mercury caused a significant decrease already at concentrations below the lowest c-mitotic concentration. With colcemid a significant decrease of average NPSH (14%) and PSH (12%) was observed only with concentrations giving close to 100% c-mitotic cells. Concentrations giving more than 20% c-mitosis gave a pronounced decrease of survival with carbaryl, diamide and methyl mercury while no toxic effects were obtained with colcemid, not even with concentrations giving close to 100% c-mitosis. Carbaryl, diamide and methyl mercury caused increased glutathione peroxidase activity indicating that these compounds cause increased lipid peroxidation. The possible connection between peroxidative damage of membranes and c-mitosis is discussed.     

Physiological and morphological alterations induced by carbaryl and 1-naphthol combinations inNostoc linckia isolated from soil

Carbaryl and its major hydrolysis product, 1-naphthol, were tested singly and together at concentrations ranging from 2 to 20 g/ml towards photosynthesis, in vivo nitrate reductase activity and nitrogen fixation, and ultrastructure of a diazotrophic cyanobacterium,Nostoc linckia, isolated from soil. All three physiological processes tested were drastically affected and correlated well with the alterations in the ultrastructure of the organism. Combination of carbaryl and 1-naphthol, at different concentrations, interacted significantly, yielding additive, antagonistic, and synergistic responses.     

Sister-chromatid exchanges and thioguanine resistance in V79 Chinese hamster cells after treatment with the aneuploidy-inducing agent carbaryl +/- S9 mix

Carbaryl induced sister-chromatid exchanges (SCEs) but no thioguanine resistance in V79 Chinese hamster cells. Addition of S9 from Aroclor-pretreated rats, or glutathione, reduced the toxic effects of carbaryl. Glutathione or S9 mix reduced the effect of carbaryl on SCE. However, the latter result indicates that carbaryl's effect may be enhanced at a certain compound/S9 ratio. Since treatment with microsomes alone, but not S9 mix, was clastogenic it cannot be excluded that this enhancement of SCE was due to perturbations in the S9 mix by carbaryl rather than to formation of some particular SCE-inducing metabolite from the compound. The effects of carbaryl on chromosomes and chromosomal distribution are comparable to those sometimes reported for TPA. This, in conjunction with the weak indications on carcinogenic activity of carbaryl, makes it of interest that the compound be tested for promotion or co-carcinogenicity in vivo.     

Acetylcholinesterase activity in the earthworm Eisenia andrei at different conditions of carbaryl exposure

Recent reports have stressed the need for a better understanding of earthworm biomarker responses. We aimed at investigating acethylcholinesterase (AChE) activity in the earthworm Eisenia andrei after exposure to carbaryl or its commercial formulation Zoril 5 under different in vitro and in vivo experiments. In addition, lysosome membrane stability was assessed by neutral red retention assay in the same experimental conditions. AChE basal Km and Vm values were about 0.16 mM and 41 nmol min(-1) mg protein(-1), respectively. Carbaryl dose-dependently decreased Vmax, while not affecting Km values. Carbaryl reduced earthworm AChE activity within 1 day of in vivo exposure to contaminated filter paper. Tested on soil, carbaryl inhibited AChE with the maximum effect after 3 days; in contrast, lysosome membrane stability of coelomocytes indicated a maximum toxicity after one day, followed by a recovery. AChE inhibition by Zoril 5 was highest after one day, while lysosome membrane stability declined progressively. In all cases, carbaryl dose-dependently decreased Vmax while not affecting Km values. In conclusion, E. andrei AChE activity assessed in vitro is dose-dependently inhibited by the carbamate compound carbaryl, which acts as a pure competitive inhibitor. In vivo experiments suggested that pure and co-formulated carbaryl have different time and/or dose dependent effects on earthworms. Our results further support the use of AChE inhibition as an indicator of pesticide contamination, to be included in a battery of biomarkers for monitoring soil toxicity.     

Mechanism of inhibition of cyclo-oxygenase in human blood platelets by carbamate insecticides.

Carbamates are a widely used class of insecticides and herbicides. They were tested for their ability to affect human blood platelet aggregation and arachidonic acid metabolism in platelets. (1) The herbicides of the carbamate type have no, or only little, influence up to a concentration of 100 microM; the carbamate insecticides, however, inhibit both aggregation and arachidonic acid metabolism in a dose- and time-dependent manner. (2) Carbaryl, the most effective compound, inhibits platelet aggregation and cyclo-oxygenase activity completely at 10 microM. The liberation of arachidonic acid from phospholipids and the lipoxygenase pathway are not affected, whereas the products of the cyclo-oxygenase pathway are drastically decreased. (3) By using [14C]carbaryl labelled in the carbamyl or in the ring moiety, it could be proved that the carbamyl residue binds covalently to platelet proteins. In contrast with acetylsalicylic acid, which acetylates only one protein, carbaryl carbamylates a multitude of platelet proteins. (4) One of the carbamylated proteins was found to be the platelet cyclo-oxygenase, indicating that carbaryl resembles in this respect acetylsalicylic acid, which is known to inhibit this enzyme specifically by acetylation.     

Arsenite-induced lipid peroxidation in Saccharomyces cerevisiae

The ability of sodium arsenite at concentrations of 10(-2), 10(-4), and 10(-6) M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10(-2) and 10(-4) M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10(-6) M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

Actions of carbaryl on the ionic transport across the isolated skin of Rana esculenta

1. Carbaryl, a carbamate used as a pesticide, increases the short-circuit current (SCC) across the isolated frog skin in a dose-dependent manner. 2. This effect is due to the stimulation of sodium absorption and chloride secretion. 3. Carbaryl action on short-circuit current is unrelated to its inhibitory power on cholinesterase; this statement is supported by two experimental results: (a) carbaryl is equally active on both sides of the skin, (b) atropine pretreatment does not inhibit the carbaryl action on SCC.     

Interactions of bullfrog tadpole predators and an insecticide: predation release and facilitation

The effect of a contaminant on a community may not be easily predicted, given that complex changes in food resources and predator-prey dynamics may result. The objectives of our study were to determine the interactive effects of the insecticide carbaryl and predators on body size, development, survival, and activity of tadpoles of the bullfrog (Rana catesbeiana). We conducted the study in cattle tank mesocosm ponds exposed to 0, 3.5, or 7.0 mg/l carbaryl, and no predators or two red-spotted newts (Notophthalmus viridescens), bluegill sunfish (Lepomis macrochirus), or crayfish (Orconectes sp.). Carbaryl negatively affected predator survival by eliminating crayfish from all ponds, and by eliminating bluegill sunfish from ponds exposed to the highest concentration of carbaryl; carbaryl exposure did not effect survival of red-spotted newts. Because crayfish were eliminated by carbaryl, bullfrogs were released from predation and survival was near that of predator controls at low concentrations of carbaryl exposure. High concentrations of carbaryl reduced tadpole survival regardless of whether predators survived carbaryl exposure or not. Presence of crayfish and newts reduced tadpole survival, while bluegill sunfish appeared to facilitate bullfrog tadpole survival. Presence of carbaryl stimulated bullfrog tadpole mass and development. Our study demonstrates that the presence of a contaminant stress can alter community regulation by releasing prey from predators that are vulnerable to contaminants in some exposure scenarios.Due to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.     

Relative Efficacy of Some Insecticides Against Brinjal Fruit and Shoot Borer,Leucinodes orbonalis Guen., and Their Impact on Fruit Yield

Six insecticides and their eight combinations were tested for their efficacy against brinjal fruit and shoot borer, Leucinodes orbonalis. Endosulfan + deltamethrin (0.07%, 0.0025%) and endosulfan + fenvalerate (0.07% + 0.005%) were highly effective against fruit borer that recorded only 13.3% damage as compared to 69.8% in control. The other promising treatments which significantly reduced the fruit damage over the control were in the order: carbaryl + fenvalerate = dichlorvos + fenvalerate (14.9%) > malathion + fenvalerate (16.4%) > fenvalerate + deltamethrin (16.6%) > dichlorvos = carbaryl + deltamethrin = malathion = dichlorvos + deltamethrin = malathion + deltamethrin (18.3%) > endosulfan (20.0%) > carbaryl (21.6%) with mean percentage of damage 14.9, 16.4, 18.3, 20.0, 21.6 and 69.8%, respectively. Carbaryl was least effective, but its combinations with pyrethroids were proved superior over carbaryl alone. Cost - benefit ratio ranged from a minimum of 1: 5.10 (carbaryl) to a maximum of 1: 20.44 (fenvalerate). Dichlorvos + fenvalerate combination gave the highest yield of 263.45 q/ha, whereas carbaryl was least effective giving 225.7 q/ha. with a net gain of Rupees 42,443.00 (US$ 886.00) and 28,141.00 (US$ 587.49), respectively. The other treatments were intermediate between the two insecticide regimes. However, all the treatments were superior over the control which produced 113.58 q/ha with a net gain of Rupees 340.00 only.     

In vitro metabolism of carbaryl by human cytochrome P450 and its inhibition by chlorpyrifos

Carbaryl is a widely used anticholinesterase carbamate insecticide. Although previous studies have demonstrated that carbaryl can be metabolized by cytochrome P450 (CYP), the identification and characterization of CYP isoforms involved in metabolism have not been described either in humans or in experimental animals. The in vitro metabolic activities of human liver microsomes (HLM) and human cytochrome P450 (CYP) isoforms toward carbaryl were investigated in this study. The three major metabolites, i.e. 5-hydroxycarbaryl, 4-hydroxycarbaryl and carbaryl methylol, were identified after incubation of carbaryl with HLM or individual CYP isoforms and analysis by HPLC. Most of the 16 human CYP isoforms studied showed some metabolic activity toward carbaryl. CYP1A1 and 1A2 had the greatest ability to form 5-hydroxycarbaryl, while CYP3A4 and CYP1A1 were the most active in generation of 4-hydroxycarbaryl. The production of carbaryl methylol was primarily the result of metabolism by CYP2B6. Differential activities toward carbaryl were observed among five selected individual HLM samples with the largest difference occurring in the production of carbaryl methylol. Co-incubations of carbaryl and chlorpyrifos in HLM greatly inhibited carbaryl metabolism. The ability of HLM to metabolize carbaryl was also reduced by pre-incubation of HLM with chlorpyrifos. Chlorpyrifos inhibited the generation of carbaryl methylol, catalyzed predominately by CYP2B6, more than other pathways, correlating with an earlier observation that chlorpyrifos is metabolized to its oxon primarily by CYP2B6. Therefore, carbaryl metabolism in humans and its interaction with other chemicals is reflected by the concentration of CYP isoforms in HLM and their activities in the metabolic pathways for carbaryl. (Supported by NCDA Environmental Trust Fund)     

Arsenite as an Inducer of Lipid Peroxidation in Saccharomyces cerevisiae Cells

The ability of sodium arsenite at concentrations of 10^–2, 10^–4, and 10^–6 M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10^–2 and 10^–4 M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10^–6 M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of -ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

Carbaryl-induced behavioural and reproductive abnormalities in the earthworm Metaphire posthuma: a sensitive model

Carbaryl, an N-methyl carbamate insecticide, is used in India to control foliar insects, but, due to soil contamination, it also adversely affects non-target organisms such as earthworms. This paper deals with the toxic effects of carbaryl on the behavioural and reproductive profiles of the earthworm, Metaphire posthuma. Locomotion and geotaxis were significantly affected, even after a 20-minute exposure to 0.125ppm carbaryl. The hatching of cocoons was altered at 0.5ppm, whereas cocoon production was retarded even at 0.125ppm carbaryl. No cocoon production was observed at 2.0ppm carbaryl. Sperm head abnormalities were reported even at the lowest test concentration of 0.125ppm. Wavy head abnormalities were observed at 0.125ppm carbaryl, whereas at 0.25ppm and 0.5ppm, the sperm heads became amorphous and the head nucleus was turned into granules deposited within the wavy head. It is concluded that the earthworm could be used as an ecosystem model for the initial toxicity testing of environmental pollutants.     

Purification and characterization of a novel carbaryl hydrolase from Aspergillus niger PY168

A fungus capable of using carbaryl as the sole source of carbon and energy was isolated from a soil enrichment, and characterized as Aspergillus niger and designated strain PY168. A novel carbaryl hydrolase from cell extract was purified 262-fold to apparent homogeneity with 13.6% overall recovery. It had a monomeric structure with a molecular mass of 50,000 Da and a pI of 4.6, and the enzyme activity was optimal at 45 degrees C and pH 7.5, The activities were strongly inhibited by Hg(2+), Ag+, rho-chloromercuribenzoate, iodoacetic acid, diisofluorophosphate and phenylmethylsulfonyl fluoride but not EDTA and phenanthroline. The purified enzyme hydrolyzed various N-methylcarbamate insecticides. Carbaryl is the preferred substrate.     

Cyclopentenone isoprostanes are novel bioactive products of lipid oxidation which enhance neurodegeneration

Oxidative stress and subsequent lipid peroxidation are involved in the pathogenesis of numerous neurodegenerative conditions, including stroke. Cyclopentenone isoprostanes (IsoPs) are novel electrophilic lipid peroxidation products formed under conditions of oxidative stress via the isoprostane pathway. These cyclopentenone IsoPs are isomeric to highly bioactive cyclopentenone prostaglandins, yet it has not been determined if these products are biologically active or are formed in the brain. Here we demonstrate that the major cyclopentenone IsoP isomer 15-A2t-IsoP potently induces apoptosis in neuronal cultures at submicromolar concentrations. We present a model in which 15-A2t-IsoP induced neuronal apoptosis involves initial depletion of glutathione and enhanced production of reactive oxygen species, followed by 12-lipoxygenase activation and phosphorylation of extracellular signal-regulated kinase 1/2 and the redox sensitive adaptor protein p66shc, which results in caspase-3 cleavage. 15-A2t-IsoP application also dramatically potentiates oxidative glutamate toxicity at concentrations as low as 100 nm, demonstrating the functional importance of these molecules in neurodegeneration. Finally, we employ novel mass spectrometric methods to show that cyclopentenone IsoPs are formed abundantly in brain tissue under conditions of oxidative stress. Together these findings suggest that cyclopentenone IsoPs may contribute to neuronal death caused by oxidative insults, and that their activity should perhaps be addressed when designing neuroprotective therapies.     

Novel and potent biological antioxidants on membrane phospholipid peroxidation: 2-hydroxy estrone and 2-hydroxy estradiol

Catechol estrogens, 2-hydroxy estrone, 2-hydroxy estradiol and 2-hydroxy estriol, were tested as possible antioxidants of phospholipid peroxidation induced by Fe3+-ADP-adriamycin, using phospholipid liposomes as lipid source and alpha-tocopherol or other steroids as reference compounds. The parameters of antioxidant activities were: elongation of induction period, inhibition of O2 consumption required for lipid peroxidation and inhibition of peroxidative cleavage of unsaturated phospholipid. Of the tested compounds, 2-hydroxy estradiol or 2-hydroxy estrone had more potent activity than that of tocopherol.     

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with alpha-linolenic acid

Lipid hydroperoxide (LOOH)-dependent lipid peroxidation was induced in alpha-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 microM. The effects of structurally related flavonoids at concentrations from 10 to 500 microM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid-metal complexes.     

Relative efficacies of indole antioxidants in reducing autoxidation and iron-induced lipid peroxidation in hamster testes

Increased iron stores are associated with free radical generation and carcinogenesis. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of tumor initiation. Melatonin and structurally related indoles are effective in protecting against oxidative stress. The aim of the study was to compare the relative efficacies of melatonin, N-acetylserotonin (NAS), indole-3-propionic acid (IPA), and 5-hydroxy-indole-3-acetic acid (5HIAA) in altering basal and iron-induced lipid peroxidation in homogenates of hamster testes. To determine the effect of the indoles on the autoxidation of lipids, homogenates were incubated in the presence of each agent in concentrations of 0.0, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 2.5, or 5.0 mM. To study their effects on induced lipid peroxidation, homogenates were incubated with FeSO(4) (30 microM + H(2)O(2) (0.1 mM) + each of the indoles in the same concentrations as above. The degree of lipid peroxidation was expressed as concentrations of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. The indoles decreased both basal and iron-related lipid peroxidation in a concentration-dependent manner. Melatonin reduced basal MDA + 4-HDA levels when used at the concentrations of 0.25 mM or higher, and prevented iron-induced lipid peroxidation at concentrations of 1.0, 2.0, 2.5, or 5.0 mM. The lowest effective concentrations of NAS required to lower basal and iron-related lipid peroxidation were 0.05 mM and 0.25 mM, respectively. IPA, only when used in the highest concentrations of 2.5 mM or 5 mM inhibited basal lipid peroxidation levels and it was ineffective on the levels of MDA + 4-HDA due to iron damage. 5HIAA reduced basal lipid peroxidation when used at concentrations of 0.25 mM or higher, and it prevented iron-induced lipid peroxidation only at the highest applied concentration (5 mM). In conclusion, melatonin and related indoles at pharmacological concentrations protect against both the autoxidation of lipids as well as induced peroxidation of lipids in testes. In doing so, these agents would be expected to reduce testicular cancer that is initiated by products of lipid peroxidation.     

Influence of histidine on lipid peroxidation in sarcoplasmic reticulum.

The free amino acid, histidine, which exists at high concentrations in some muscle systems, has previously been demonstrated to both inhibit and activate lipid peroxidation in membrane model systems. This study sought to characterize the specificity of histidine's effect on iron-catalyzed enzymatic and nonenzymatic lipid peroxidation. Under conditions of activation (histidine added to the reaction mixture after ADP and ferric ion), alpha-amino, carboxylate, and pyrrole nitrogen were demonstrated to be involved by kinetic techniques in the activation of the enzymatic system. It is hypothesized that a mixed ligand complex (iron, ADP, and histidine) formed may allow rapid redox cycling of iron. While increasing concentrations of histidine led to increasing levels of stimulation in the enzymatic system, the maximum stimulation of a nonenzymatic lipid peroxidation system of ascorbate and ferric ion occurred at histidine concentrations near 2.5 mM. Inhibition of a nonenzymatic system (ferrous ion), on the other hand, occurred at all concentrations of histidine when the ferrous ion was exposed to ADP prior to histidine. In enzymatic systems, under conditions when the ferric ion was exposed to histidine prior to ADP, inhibition of lipid peroxidation by histidine also occurred. The inhibitory effect of histidine was ascribed to the imidazole group and may arise from the formation of a different iron complex or the acceleration of polymerization, dehydration, and insolubilization of the ferric ion by the imidazole nitrogen. The demonstrated ability of histidine to affect in vitro lipid peroxidation systems raises the possibility that this free amino acid may modulate lipid peroxidation in vivo.