How to Use Respiratory Chain Inhibitors in Toxicology Studies-Whole-Cell Measurements.

Mitochondrial electron transport chain (ETC) inhibition is a phenomenon interesting in itself and serves as a tool for studying various cellular processes. Despite the fact that searching the term "rotenone" in PubMed returns more than 6900 results, there are many discrepancies regarding the directions of changes reported to be caused by this RTC inhibitor in the delicate redox balance of the cell. Here, we performed a multifaceted study of the popular ETC inhibitors rotenone and antimycin A, involving assessment of mitochondrial membrane potential and the production of hydrogen peroxide and superoxide anions at cellular and mitochondrial levels over a wide range of inhibitor concentrations (1 nmol/dm3-100 µmol/dm3). All measurements were performed with whole cells, with accompanying control of ATP levels. Antimycin A was more potent in hindering HepG2 cells' abilities to produce ATP, decreasing ATP levels even at a 1 nmol/dm3 concentration, while in the case of rotenone, a 10,000-times greater concentration was needed to produce a statistically significant decrease. The amount of hydrogen peroxide produced in the course of antimycin A biological activity increased rapidly at low concentrations and decreased below control level at a high concentration of 100 µmol/dm3. While both inhibitors influenced cellular superoxide anion production in a comparable manner, rotenone caused a greater increase in mitochondrial superoxide anions compared to a modest impact for antimycin A. IC50 values for rotenone and antimycin A with respect to HepG2 cell survival were of the same order of magnitude, but the survival curve of cells treated with rotenone was clearly biphasic, suggesting a concentration-dependent mode of biological action. We propose a clear experimental setup allowing for complete and credible analysis of the redox state of cells under stress conditions which allows for better understanding of the effects of ETC inhibition.

A Vanadium-Catalyzed Synthesis of Fully Substituted Pyrroles.

We present a straightforward, fast, inexpensive, and environmentally friendly synthesis of 1,2,3,4,5-pentasubstituted derivatives of pyrrole, which were produced in one-pot reactions of 3-oxoanilides with hydrazides of carboxylic acids, catalyzed by 10 mol % VOSO4·H2O. The reactions were carried out in ethanol in contact with air as the oxidant. The 19 pyrroles obtained were usually crystalline and did not require purification. The reaction tolerates various substituents in both substrates. All products were characterized by infrared, nuclear magnetic resonance, and ultraviolet-visible spectroscopy and elemental analysis. The molecular structures of the products and the intermediates were unambiguously determined by X-ray single-crystal analysis.

Products of Lipid Peroxidation as a Factor in the Toxic Effect of Silver Nanoparticles.

In our previous study we have shown that nanoparticles have different effects depending on the energy metabolism of the cell, which is an important factor in the context of oncology and diabetes. Here we assess the influence of AgNPs on cellular lipid components in varying glucose concentrations. To assess the effect of silver nanoparticles on cell lipids, we measured cell viability, the fluidity of the cell membranes, the content of amino groups in proteins, the level of lipid peroxidation products, the concentration of 4-hydroxynonenal (4-HNE), and the concentration of lipid peroxides. The obtained results show differences in the formation of lipid peroxidation products in cells exposed to oxidative stress induced by nanoparticles. In addition, we have shown that the metabolic state of the cell is a factor significantly affecting this process.

Silver nanoparticles can attenuate nitrative stress.

We have reported previously that glucose availability can modify toxicity of silver nanoparticles (AgNPs) via elevation of antioxidant defence triggered by increased mitochondrial generation of reactive oxygen species. In this study, we examined the effect of glucose availability on the production of reactive nitrogen species in HepG2 cells and modification of nitrative stress by AgNPs. We found that lowering the glucose concentration increased expression of genes coding for inducible nitric oxide syntheas, NOS2 and NOS2A resulting in enhanced production of nitric oxide. Surprisingly, AgNPs decreased the level of nitric oxide accelerated denitration of proteins nitrated by exogenous peroxynitrite in cells grown in the presence of lowered glucose concentration, apparently due to further induction of protective proteins.

PIN2-like proteins may contribute to the regulation of morphogenetic processes during spermatogenesis in Chara vulgaris.

KEY MESSAGE: PIN2-like auxin transporters are expressed, preferentially in a polarized manner, in antheridial cells of freshwater green alga Chara vulgaris , considered to be the closest relative of the present-day land plants. Chara vulgaris represents a group of advanced multicellular green algae that are considered as the closest relatives of the present-day land plants. A highly specialized structure of its male sex organs (antheridia) includes filaments consisting of generative cells, which after a series of synchronous divisions transform into mature sperm, and non-generative cells comprising outer shield cells, cylindrical manubria, and central complex of capitular cells from which antheridial filaments arise. Immunofluorescence observations indicate that PIN2-like proteins (PIN2-LPs), recognized by antibodies against PIN-FORMED2 (PIN2) auxin transporter in Arabidopsis thaliana, are expressed in both types of antheridial cells and, in most of them, preferentially accumulate in a polarized manner. The appearance of PIN2-LPs in germ-line cells is strictly confined to the proliferative period of spermatogenesis and their quantities increase steadily till antheridial filaments reach the 16-celled stage. An enhanced level of PIN2-LPs observed in the central cell walls separating two asynchronously developing parts of antheridial filaments (characterized by the plugged plasmodesmata) is correlated with an enhanced deposition of callose. Intense PIN2-LPs immunofluorescence maintained in the capitular cells and its altering polarity in manubria suggest a pivotal role of these cells in the regulation of auxin transport directionality during the whole time of antheridial ontogenesis. Immunohistochemical staining of IAA revealed a clear-cut correspondence between localization sites of auxins and PIN2-LPs. It seems probable then that a supplementary developmental mechanism has evolved in Chara, by which all antheridial elements may be integrated at the supra-cellular level via plasma membrane-targeted PIN2-LPs and auxin-mediated processes.

A simple and safe method for the preparation of bis[2-(2H-tetrazol-5-yl)pyridinium] tetrachloridozincate(II).

In the title complex salt, (C6H6N5)2[ZnCl4], the Zn(II) cation is coordinated by four chloride ligands in a distorted tetrahedral geometry. The organic cations and complex anions are connected by N-H···Cl hydrogen bonds, leading to the formation of a three-dimensional network. The title complex salt was synthesized by the reaction of sodium azide, pyridine-2-carbonitrile and ZnCl2 in aqueous solution. The salt was characterized by elemental analysis and IR and UV-Vis spectroscopy.