Evaluation of the effect of tetraethylammonium bromide and chloride on the growth and development of terrestrial plants.

Quaternary ammonium salts (QAS), which also include ionic liquids, constitute a vast group of chemical compounds that are increasingly common in the commercial use. This situation may lead to the contamination of the natural environment and may constitute a potential threat to all its elements, including terrestrial higher plants. This paper presents the effect of tetraethylammonium chloride [TEA][Cl] and tetraethylammonium bromide [TEA][Br] on the growth and development of spring barley and common radish. The applied QAS were characterized with phytotoxicity dependent on the concentration of compound and characteristics of the study plants. Spring barley turned out to be highly susceptible plant to the analyzed compounds, which was confirmed by % inhibition of length of plants, root length and fresh weight of plants and by calculated values for EC50, NOEC as well as LOEC. On the contrary, a common radish revealed the resistance to QAS used in the study; although, phytotoxic symptoms were still observed when high concentrations of dry weight of soil were applied (1000, 3000 and 5000 mg/kg). The applied QAS caused oxidative stress symptoms, mainly in spring barley seedlings, which were manifested by decreased assimilation of pigments content, increased hydrogen peroxide (H2O2) and malondialdehyde (MDA) content in plant cells and with a changed activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD).

A novel approach for in vivo screening of toxins using the Drosophila Giant Fiber circuit.

Finding compounds that affect neuronal or muscular function is of great interest as potential therapeutic agents for a variety of neurological disorders. Alternative applications for these compounds include their use as molecular probes as well as insecticides. We have developed a bioassay that requires small amounts of compounds and allows for unbiased screening of biological activity in vivo. For this, we paired administering compounds in a non-invasive manner with simultaneous electrophysiological recordings from a well-characterized neuronal circuit, the Giant Fiber System of Drosophila melanogaster, which mediates the escape response of the fly. The circuit encompasses a variety of neurons with cholinergic, glutamatergic, and electrical synapses as well as neuromuscular junctions. Electrophysiological recordings from this system allow for the detection of compound-related effects against any molecular target on these components. Here, we provide evidence that this novel bioassay works with small molecules such as the cholinergic receptor blocker mecamylamine hydrochloride and the potassium channel blocker tetraethylammonium hydroxide, as well as with venom from Conus brunneus and isolated conopeptides. Conopeptides have been developed into powerful drugs, such as the painkillers Prialt™ and Xen2174. However, most conopeptides have yet to be characterized, revealing the need for a rapid and straightforward screening method. Our findings show that mecamylamine hydrochloride, as well as the α-conotoxin ImI, which is known to be an antagonist of the human α7 nicotinic acetylcholine receptor, efficiently disrupted the synaptic transmission of a Drosophila α7 nicotinic acetylcholine receptor-dependent pathway in our circuit but did not affect the function of neurons with other types of synapses. This demonstrates that our bioassay is a valid tool for screening for compounds relevant to human health.

Dose-response regressions for algal growth and similar continuous endpoints: calculation of effective concentrations.

We derive equations for the effective concentration giving 10% inhibition (EC10) with 95% confidence limits for probit (log-normal), Weibull, and logistic dose-response models on the basis of experimentally derived median effective concentrations (EC50s) and the curve slope at the central point (50% inhibition). For illustration, data from closed, freshwater algal assays are analyzed using the green alga Pseudokirchneriella subcapitata with growth rate as the response parameter. Dose-response regressions for four test chemicals (tetraethylammonium bromide, musculamine, benzonitrile, and 4-4-(trifluoromethyl)phenoxy-phenol) with ranges of representative slopes at 50% response (0.54-2.62) and EC50s (2.20-357 mg/L) were selected. Reference EC50s and EC10s with 95% confidence limits using probit or Weibull models are calculated by nonlinear regression on the whole dataset using a dose-response regression program with variance weighting and proper inverse estimation. The Weibull model provides the best fit to the data for all four chemicals. Predicted EC10s (95% confidence limits) from our derived equations are quite accurate; for example, with 4-4-(trifluoromethyl)phenoxy-phenol and the probit model, we obtain 1.40 (1.22-1.61) mg/L versus 1.40 (1.20- 1.64) mg/L obtained from the nonlinear regression program. The main advantage of the approach is that EC10 or ECx (where x = 1-99) can be predicted from well-determined responses around EC20 to EC80 without experimental data in the low- or high-response range. Problems with the estimation of confidence interval for EClow,x (concentration predicted to cause x% inhibition) from algal growth inhibition also are addressed. Large confidence intervals may be the result of experimental error and lack of a well-defined reference response value.

Interaction of mitochondrial respiratory inhibitors and excitotoxins potentiates cell death in hippocampal slice cultures.

The broad-spectrum insecticide rotenone, an inhibitor of complex I of the mitochondrial electron transport chain (ETC), gives rise to oxidative stress and bioenergetic failure. Pesticides including rotenone have been implicated in human neurodegenerative diseases, including Parkinson's disease. Another intensively investigated hypothesis of neurodegenerative disease involves the toxic action of the excitatory neurotransmitter glutamate. In the present study, we determined whether concomitant exposure of rotenone plus tetraethylammonium chloride (TEA) or the specific glutamate receptor agonists N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) would cause greater cell death in organotypic hippocampal slice cultures than when given separately. Low, sublethal rotenone (100 nM), TEA (0.5-2.0 mM), NMDA (1.0-10 microM), and AMPA (1.0-10 microM) alone resulted in little cell death as determined by propidium iodide fluorescence. However, cell death was significantly to dramatically potentiated when the hippocampal slices were coincubated with comparable concentrations of rotenone plus TEA, NMDA, or AMPA. Similarly, in the presence of 10 microM NMDA, ETC inhibitors blocking other mitochondrial complexes also potentiated cell death. Immunohistochemical analysis using glial fibrillary acidic protein antibody determined that the cell death was preferentially neuronal. These results demonstrate that two different classes of toxicants can interact, resulting in potentiation of neurotoxicity, and further suggest that a combinatorial therapeutic approach may be required to ameliorate the potentiated cell death.

Glutamate decarboxylase protects neurons against excitotoxic injury.

Although the majority of agents with antiexcitotoxic action act as glutamate receptor antagonists, enzymatic degradation of glutamate can also be neuroprotective. The very low specific activity of the mammalian form of glutamate decarboxylase (GAD), the enzyme that catalyzes the formation of gamma-aminobutyric acid (GABA) from glutamate in neurons, is likely to limit its utility as an antiglutamate neuroprotectant. In contrast, the bacterial form of GAD can be isolated with relatively high specific activity and is most active in acidic environments. We have expressed and purified GAD from Escherichia coli (bGAD) and tested the ability of the enzyme to protect against glutamate excitotoxicity. Incubation of rat hipppocampal slices with the potassium channel antagonist tetraethyl ammonium (TEA) resulted in widespread excitotoxic death of pyramidal and granule cell neurons. bGAD alone showed no significant neurotoxicity and significantly reduced excitotoxicity induced by TEA. We hypothesize that bGAD may be internalized into the synaptic vesicle compartment by nonspecific endocytosis, where both the appropriate pH and high glutamate concentrations are present. Targeting of this enzyme to the interior of synaptic vesicles may enhance its potency as a neuroprotectant against excitotoxicity.

Adjuvant effect of quaternary ammonium compounds in a murine model.

It has been suggested that occupational exposure to quaternary ammonium compounds (QACs) may promote the development of allergic airway diseases. In this study, hazard identifications of the adjuvant effect of cetylpyridinium chloride (CPC), dimethyldioctadecylammonium bromide (DDA), hexadecyltrimethylammonium bromide (HTA), and tetraethylammonium chloride (TEA) were performed in a screening bioassay. Female BALB/c mice were injected subcutaneously with the model allergen ovalbumin (OVA) alone or together with different quantities of one of the QAC test compounds. After one or two boosters, levels of OVA-specific IgE, IgG1 and IgG2a antibodies were measured in sera. CPC and DDA increased IgE and IgG1 antibody production, respectively, compared to the OVA control group, whereas HTA and TEA showed no adjuvant effect. Nevertheless, when TEA was given in combination with DDA, the adjuvant effect was up to six-fold higher than the adjuvant effect of DDA alone. Only DDA had a statistically significant adjuvant effect on IgG2a antibody levels.