Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels.

BACKGROUND AND PURPOSE: Docking studies predict that the insecticides, lindane and fipronil, block GABA(A) receptors by binding to 6' pore-lining residues. However, this has never been tested at any Cys-loop receptor. The neurotoxic effects of these insecticides are also thought to be mediated by GABA(A) receptors, although a recent morphological study suggested glycine receptors mediated fipronil toxicity in zebrafish. Here we investigated whether human α1, α1ß, α2 and α3 glycine receptors were sufficiently sensitive to block by either compound as to represent possible neurotoxicity targets. We also investigated the mechanisms by which lindane and fipronil inhibit α1 glycine receptors. EXPERIMENTAL APPROACH: Glycine receptors were recombinantly expressed in HEK293 cells and insecticide effects were studied using patch-clamp electrophysiology. KEY RESULTS: Both compounds completely inhibited all tested glycine receptor subtypes with IC(50) values ranging from 0.2-2 µM, similar to their potencies at vertebrate GABA(A) receptors. Consistent with molecular docking predictions, both lindane and fipronil interacted with 6' threonine residues via hydrophobic interactions and hydrogen bonds. In contrast with predictions, we found no evidence for lindane interacting at the 2' level. We present evidence for fipronil binding in a non-blocking mode in the anaesthetic binding pocket, and for lindane as an excellent pharmacological tool for identifying the presence of ß subunits in αß heteromeric glycine receptors. CONCLUSIONS AND IMPLICATIONS: This study implicates glycine receptors as novel vertebrate toxicity targets for fipronil and lindane. Furthermore, lindane interacted with pore-lining 6' threonine residues, whereas fipronil may have both pore and non-pore binding sites.

Potency of andrographolide as an antitumor compound in BHC-induced liver damage.

RELEVANCE: The present investigation relates to the influence of andrographolide, an active compound of Andrographis paniculata Nees. It reverses an experimental liver carcinogenic condition of mice to normal and might be a potential therapeutic/preventive agent for human liver cancer. OBJECTIVE: A. paniculata (Kalmegh) is extensively used in the Indian traditional system of medicine as a hepatoprotective and hepatostimulative agent and has been reported to have protective effect against different hepatotoxins. MATERIALS AND METHODS: Histomorphological, ultrastructural, and biochemical studies were performed for the effect of the andrographolide on control mice, mice treated with hexachlorocyclohexane (BHC) only and BHC + andrographolide. Enzymes for liver function tests were analyzed by spectrophotometric method. RESULTS: The BHC experimental model forms an irreversible liver tumor in male mice. The histological and ultrastructural changes observed in andrographolide supplementation emphasize the recovery of the damaged liver. This recovery was also reflected in the neoplastic nodule formation. The activity of phosphorylase and glucose-6-phosphatase in the liver of the andrographolide-supplemented group suggests improved glycogenolysis in liver. Serum glutamate pyruvate transaminase, serum glutamate oxalate transaminase, alkaline phosphatase, acid phosphatase, and gamma-glutamyl transpeptidase showed a significant decrease in andrographolide-supplemented animals as compared with BHC-treated animals, suggesting regenerative effects elicited by andrographolide. CONCLUSION: The study indicates that the regenerative capability elicited by andrographolide is possibly due to its ability to reactivate liver function enzymes that catalyze the reaction of several biochemical and synthetic processes and that it may be useful for severe liver damage conditions.

Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes.

Ligand-gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate-gated chloride channel (MdGluCl-alpha) and a gamma-aminobutyric acid (GABA)-gated chloride channel (MdRdl), were cloned from house-flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand-gated channels using two-electrode voltage-clamp electrophysiology. It was found that L-glutamate and GABA activated the MdGluCl-alpha homo-oligomers with an EC(50) value of 30 microM and the MdRdl homo-oligomers with an EC(50) value of 101 microM, respectively. Both channels were chloride ion-permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as gamma-hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin, than the MdGluCl-alpha channel. MdGluCl-alpha required only 1-2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4-7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl-alpha was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl-alpha assists in the expression of MdRdl when the two are coexpressed.

Control de calidad de residuos de biocidas en frutos de Silybum marianum L / Quality control of organochlorine biocide residues in fructus from Silybum marianum L

Los estudios analíticos de los biocidas organoclorados demuestran que su nivel de concentración en las raíces de plantas es mayor que la concentración en el suelo, y especialmente en las plantas medicinales utilizadas en las industrias farmacéutica y medicinal. Los fármacos búlgaros más utilizados (Carsil, Tribestan y Nivalin) contienen extractos de plantas y deben ser analizados de acuerdo con las directivas de la Comunidad Europea EEC/76/895 y EEC/90/642 1998; Por ello, se ha estudiado el método de cromatografía de gases para la determinación cuantitativa de biocidas organoclorados (HCH e isómeros, DDT y metabolitos). Se han analizado conforme a las regulaciones de la Pharmacopoeia Europea materiales procedentes de plantas de distintas regiones de Bulgaria (frutos de Silybum marianum, L) y grageas de Carsil producidas por Sopharma Ltd. El método ha sido validado y se han estudiado parámetros analíticos de especificidad, linealidad, precisión, replicabilidad y rango

Analytical studies of organochlorine biocides show that their concentration in the plants (roots) is higher than in the ground and particularly in the medicinal plants used in the therapy and the pharmacy industry. The most favorite Bulgarian drugs (Carsil, Tribestan and Nivalin Nivalin) contain plant extracts and they have to be analyzed in respect to European Community Directives EEC/76/895 and EEC/90/642 1998; In view of that the gas chromatography method for quantitative determination of organochlorine biocides (HCH and isomers, DDT and metabolites) was studied. Plant material from different regions of Bulgaria (fructus from Silybum marianum, L) and Carsil dragee produced by Sopharma Ltd. were analyzed in respect to European Pharmacopoeia regulations. The method was validated and analytical parameters specificity, linearity, precision, repeatability and range were studied

Expression of CONNEXIN43 is highly sensitive to ionizing radiation and other environmental stresses.

To gain a greater understanding of the mechanisms underlying the cellular responses to low-dose ionizing radiation, gene expression profiles were examined by microarray analysis of cDNA from confluent human diploid fibroblast cultures exposed to very low fluences of alpha-particles. The data, supported by Northern and Western analyses, indicate that radiation induces a significant up-regulation of CONNEXIN43 expression. This phenomenon was observed in a variety of irradiated cell types. These findings are consistent with our previous observations that connexin43 (cx43)-mediated gap-junction intercellular communication is involved in the bystander response observed in cell cultures exposed to fluences of alpha-particles by which only a very small fraction of the cell nuclei is traversed by a particle track (E. I. Azzam et al., Proc. Natl. Acad. Sci. USA, 98: 473-478, 2001). Increased mRNA levels in cells from irradiated cultures correlated with increased cx43 protein levels by approximately 4 h after irradiation. The induction of cx43 was observed by mean alpha-particle doses as low as 0.16 cGy, and also in cells exposed to gamma-rays, t-butyl hydroperoxide, and hyperthermia. Exposure to these stresses also resulted in post-translational modification of cx43; increased phosphorylation and hyperphosphorylation of the protein was observed. Up-regulation of cx43 expression in ionizing radiation exposed cells correlated with functional communication through gap junctions, as evidenced by dye transfer from irradiated to nonirradiated cells. In contrast, the response after UV radiation varied and was cell type-dependent. Overall, these data suggest a critical role for genes involved in intercellular communication in mediating the cellular responses to a variety of stresses.

Aromatase activity modulation by lindane and bisphenol-A in human placental JEG-3 and transfected kidney E293 cells.

Aromatase is the cytochrome P-450 involved in converting androgens to estrogens. The cytochrome P-450 family plays a central role in the oxidative metabolism of compounds including environmental pollutants. Since lindane and bisphenol-A (BPA) are two well-characterized endocrine disruptors that have been detected in animals and humans, it was important to learn whether they could affect aromatase activity and consequently estrogen biosynthesis. The present study investigates the effects of BPA and lindane on cytotoxicity, aromatase activity and mRNA levels in human placental JEG-3 cells and transfected human embryonal kidney 293 cells. Both cell lines were exposed to increasing concentrations of lindane (25, 50 and 75 microM) and bisphenol-A (25, 50 and 100 microM) over different time periods (10 min-18 h). As a result, none of these concentrations showed cytotoxicity. After short pre-incubation times (10 min-6 h), aromatase activity was enhanced by both compounds. Longer time incubation (18 h), however, produced dose-related inhibition. Lindane and BPA had no significant effects on CYP19 mRNA levels. Therefore, lindane and BPA modulate aromatase activity suggesting an interaction with the cytochrome P-450 aromatase. This study highlights the endocrine-modulating properties of lindane and bisphenol-A.

[A comparative study on the sensitivity and specificity of cholinesterase and glutathione s-transferase in Gammarus pulex L].

Studies on the influences of lindane, pirimiphos methyl, permethrin, zinc and dodecyl linear alkybenzene sulfonate (LAS) on the activity and toxicity of cholinesterase (ChE) and glutathione s-transferase (GST) in Gammarus pulex L. showed that only pirimiphos methyl caused a change in ChE activity in Gammarus, with a significant reduction in enzyme activity after 24 h and 48 h exposure. Both lindane and permethrin caused a change in GST activity in Gammarus, with a significant increase in enzyme activity after 48 h exposure. Lindane alos caused a significant increase in GST activity after 24 h exposure. Biomarkers ChE and GST were demonstrated a high degree of specificity and sensitivity in comparison to the lethality assay, but GST activity was less specific than ChE activity.

Studies in vitro on the relative efficacy of current acaricides for Sarcoptes scabiei var. hominis.

Resistance of Sarcoptes scabiei to various topical therapies has been described, but clinical assessment of treatment failure is problematic and in-vitro assays are generally not available. We describe a simple in-vitro analysis used to evaluate the relative efficacy of a range of topical, oral, and herbal treatments available in Australia for the treatment of scabies. S. scabiei var. hominis mites were collected from skin scrapings obtained from 7 crusted scabies patients over a period of 2 years (1997 and 1998). Larvae, nymphal instars, and adult mites were tested within 3 h of collection and continuously exposed to selected commercially available treatment products until death, with the elapsed time recorded. Neem was the only product to show little acaricidal activity. Survival curves indicated that, of the other agents, 5% permethrin (Lyclear) had the slowest killing time, with 35% of mites still alive after 3 h, and 4% still alive after 18-22 h of constant exposure. In contrast, no mites were alive after 3 h exposure to 25% benzyl benzoate (Ascabiol), 1% lindane (Quellada), 5% tea tree oil and 100-8000 ng/g of ivermectin (Equimec). Despite the slower killing time with 5% permethrin, there was no evidence of any mite tolerance in vivo or treatment failure in any patients or contact cases.

Concentration of putrescine in plasma, frontal cortex and hippocampus of rats after systemic administration of the convulsants N-methyl-D-aspartate, pentylentetrazol, picrotoxinine, lindane and 4-aminopyridine.

The motor responses (such as stereotypic behavior or convulsions induced in rats by N-methyl-D-aspartate (NMDA) administered systemically were followed by a rapid, moderate increase in the putrescine concentration in plasma which preceded an increase in this amine in the brain. This effect was not observed following the convulsions evoked by pentylentetrazol, picrotoxinine, lindane or 4-aminopyridine. However, all the convulsants assayed induced a mild increase in the concentration of putrescine in the frontal cortex and hippocampus. A differential activation of the ornithine decarboxylase (ODC)/polyamine system in both cerebral and peripheral tissues could account for these results.

Role of calcium in biphasic immunomodulation by gamma-HCH (lindane) in mice.

gamma-HCH (Lindane) is reported to cause a biphasic immunomodulation-stimulation followed by suppression-after oral administration in mice. Role of calcium in this biphasic immunomodulation was assessed after 4, 12 and 24 wks of gamma-HCH administration. 45Ca-uptake was enhanced during the initial immunostimulation followed by decrease concomitant with immunosuppression. Lymphocyte proliferation was inhibited during both the phases of immune response by verapamil, a calcium channel blocker, and by trifluoperazine, a calmodulin inhibitor. These findings show an impairment of calcium homeostasis in lymphocytes culminating into the biphasic immunomodulatory effects of gamma-HCH.

Effects of midazolam, DMCM and lindane on potentiated startle in the rat.

Forty-eight male Wistar rats were exposed to contingent light-shock combinations and 48 rats received light and shock stimuli in a random order. One day after fear conditioning the animals were tested for startle potentiation after injection of midazolam (0, 0.5, 1.0, 2.0 mg/kg, IP) or DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate; 0, 0.1, 0.2, 0.4 mg/kg IP) or lindane (0, 7.5, 15.0, 30.0 mg/kg PO). Midazolam attenuated potentiated startle dose dependently and the inverse benzodiazepine agonist DMCM had the opposite effect. The effects of lindane on startle amplitudes were identical to those of DMCM, indicating that lindane has anxiogenic effects on behavior. It is suggested that the anxiogenic effects of lindane are mediated by an effect at the GABA-ionophore complex.

[Continuous interpretation system for the cytophotometer "Amplival photometry"=-studies with pesticide combinations]. / Kontinuierliches Auswertesystem zum Cytophotometer "Amplival photometrie"--Untersuchungen mit Pestizidkombinationen.

By means of complementary technique the cytophotometer "Amplival photonetrie" is converted in a continuous interpretation system. Thereby it is possible to move the slides by controlling step motors in alpha- and/or gamma-axis. The number of stepts and the speed of them can be predetermined. For the quantitative histochemical examination the sum of the extinctions of single steps is determined. therefore the arythmetical average can be calculated per arbitrary unit tissue. In examinations with combinations of pesticides the usefulness of the interpretation system is demonstrated.

[Biogenic amine metabolism in homeotherms exposed to pesticides of different chemical nature]. / Osobennosti obmena biogennykh aminov u teplokrovnykh pri vozdeistvii pestitsidami razlichnoi khimicheskoi prirody.

Some aspects of the biogenic amines -- serotonin and histamine metabolism in rats and guinea pigs following introduction to them of pesticides of different chemical nature, viz. gamma-isomer of hexachlorcyclohexane (lindane) and dimethyldithiocarbamate zinc (zyram) were studied. These pesticides were found to dissimilarly affect the passage with urine of the principal serotonin metabolite -- 5-oxyindol-acetic acid. A short-term (2 days) introduction to rats of large doses (34 mg/kg) of lindane and a protracted (90 days) action of low doses (1.7 mg/kg) produce changes in the content of serotonin and of 5-oxyindol-acetic acid in the brain, liver and kidneys. In the blood of guinea pigs receiving for a lenthy time (120 days) small amounts (4 mg/kg) of zyram shifts in the histamine-histaminase-histaminopexy system were revealed, this pointing to the ability of this pesticide to provoke the state of sensitization.