Correction: Reactions of nitroxides 16. First nitroxides containing tellurium atom.

[This corrects the article DOI: 10.1039/C6RA15880C.].

An In Vitro Comparative Study of the Effects of Tetrabromobisphenol A and Tetrabromobisphenol S on Human Erythrocyte Membranes-Changes in ATP Level, Perturbations in Membrane Fluidity, Alterations in Conformational State and Damage to Proteins.

Brominated flame retardants (BFRs) are substances used to reduce the flammability of plastics. Among this group, tetrabormobisphenol A (TBBPA) is currently produced and used on the greatest scale, but due to the emerging reports on its potential toxicity, tetrabromobisphenol S (TBBPS)-a compound with a very similar structure-is used as an alternative. Due to the fact that the compounds in question are found in the environment and in biological samples from living organisms, including humans, and due to the insufficient toxicological knowledge about them, it is necessary to assess their impacts on living organisms and verify the validity of TBBPA replacement by TBBPS. The RBC membrane was chosen as the research model. This is a widely accepted research model for assessing the toxicity of xenobiotics, and it is the first barrier to compounds entering circulation. It was found that TBBPA and TBBPS caused increases in the fluidity of the erythrocyte membrane in their hydrophilic layer, and conformational changes to membrane proteins. They also caused thiol group elevation, an increase in lipid peroxidation (TBBPS only) and decreases in the level of ATP in cells. They also caused changes in the size and shape of RBCs. TBBPA caused changes in the erythrocyte membrane at lower concentrations compared to TBBPS at an occupational exposure level.

Glyphosate and AMPA Induce Alterations in Expression of Genes Involved in Chromatin Architecture in Human Peripheral Blood Mononuclear Cells (In Vitro).

We have determined the effect of glyphosate and aminomethylphosphonic acid (AMPA) on expression of genes involved in chromatin architecture in human peripheral blood mononuclear cells (PBMCs). The cells were incubated with glyphosate and AMPA in the concentrations ranging from 0.5 to 100 µM and from 0.5, to 250 µM, respectively. The expression profile of the following genes by quantitative Real-Time PCR was evaluated: Genes involved in the DNA methylation (DNMT1, DNMT3A) and DNA demethylation process (TET3) and those involved in chromatin remodeling: genes involved in the modification of histone methylation (EHMT1, EHMT2) and genes involved in the modification of histone deacetylation (HDAC3, HDAC5). Gene profiling showed that glyphosate changed the expression of DNMT1, DMNT3A, and HDAC3, while AMPA changed the expression of DNMT1 and HDAC3. The results also revealed that glyphosate at lower concentrations than AMPA upregulated the expression of the tested genes. Both compounds studied altered expression of genes, which are characteristic for the regulation of transcriptionally inactive chromatin. However, the unknown activity of many other proteins involved in chromatin structure regulation prevents to carry out an unambiguous evaluation of the effect of tested xenobiotics on the studied process. Undoubtedly, we have observed that glyphosate and AMPA affect epigenetic processes that regulate chromatin architecture.

The selected epigenetic effects of aminomethylphosphonic acid, a primary metabolite of glyphosate on human peripheral blood mononuclear cells (in vitro).

Aminomethylphosphonic acid (AMPA) is a primary metabolite of glyphosate and amino-polyphosphonate. We have determined the effect of AMPA on selected epigenetic parameters and major cell cycle drivers in human peripheral blood mononuclear cells (PBMCs). The cells were incubated with AMPA at 0.5, 10 and 250 µM for 24 h. The performed analysis included: global DNA methylation by colorimetric measurement of 5-methylcytosine in DNA, methylation in the promoter regions of selected tumor suppressor genes (P16, P21, TP53) and proto-oncogenes (BCL2, CCND1) as well as the expression profile of the indicated genes by Real-Time PCR assays. The obtained results have revealed significant reduction of global DNA methylation level in PBMCs exposed to AMPA. Investigated xenobiotic changed methylation pattern of the P21 and TP53 suppressor gene promoters, but in case of other analyzed genes: P16, BCL2 and CCND1 no statistically significant changes have been noted. Gene profiling have shown that AMPA only changed the expression of CCND1. Summing up, our results have revealed a small potential disturbance in methylation processes and the absence of changes in expression of tested tumor suppressor genes (P16, P21, TP53) and protooncogenes (BCL2) in human PBMCs exposed to AMPA.

Evaluation of apoptotic potential of glyphosate metabolites and impurities in human peripheral blood mononuclear cells (in vitro study).

Glyphosate is used for cereal, vegetable and fruit crops for reducing or inhibiting the growth of weeds as well as a desiccant for various grain crops. That is why, glyphosate has been shown to be accumulated in humans and animals through ingestion of food of both plant and animal origin. The study aimed to assessed the effect of glyphosate, its metabolites: aminomethylphosphonic acid (AMPA), methylphosphonic acid and its impurities: PMIDA, N-methylglyphosate, hydroxymethylphosphonic acid and bis(phosphonomethyl)amine on apoptosis induction in human peripheral blood mononuclear cells (PBMCs). PBMCs were exposed to the compounds studied at the concentrations ranging from 0.01 to 5 mM for 4 h. We have observed an increase in reactive oxygen species (including hydroxyl radical) and cytosolic calcium ions levels as well as reduction of transmembrane mitochondrial potential (ΔΨm) in PBMCs exposed to the compounds examined. All substances studied changed PBMCs membrane permeability, activated caspase-8, -9, -3 and caused chromatin condensation, which showed that they were capable of inducing apoptosis both via extrinsic and particularly intrinsic pathway. Generally the study demonstrated that there were no differences between apoptotic changes induced by glyphosate, its metabolites or impurities, and observed changes were provoked by high concentrations of investigated compounds.

Reactions of Nitroxides, Part 17. Synthesis, Fungistatic and Bacteriostatic Activity of Novel Five- and Six-Membered Nitroxyl Selenoureas and Selenocarbamates.

The reactions of 3-isoselenocyanato-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 3-isoselenocyanatomethyl-2,2,5,5-tetramethyl-3-pyrrolidine-1-oxyl, and 4-isoselenocyanato-2,2,6,6-tetramethylpiperidine-1-oxyl with selected amines and alcohols give the corresponding novel nitroxyl selenoureas and selenocarbamates, all bearing a nitroxyl moiety. Synthesized selenoureas and selenocarbamates show significant activity against pathogenic fungi and bacteria. In contrast to piperidine nitroxides, pyrrolidine, five-membered nitroxyl selenoureas and selenocarbamates show excellent antifungal and antibacterial activity against pathogenic fungi and bacteria, respectively.

The mechanism of DNA damage induced by Roundup 360 PLUS, glyphosate and AMPA in human peripheral blood mononuclear cells - genotoxic risk assessement.

Glyphosate is the most heavily applied among pesticides in the world, and thus human exposure to this substance continues to increase. WHO changed classification of glyphosate to probably cancerogenic to humans, thus there is urgent need to assess in detail genotoxic mechanism of its action. We have assessed the effect of glyphosate, its formulation (Roundup 360 PLUS) and its main metabolite (aminomethylphosphonic acid, AMPA) in the concentration range from 1 to 1000 µM on DNA damage in human peripheral blood mononuclear cells (PBMCs). The cells were incubated for 24 h. The compounds studied and formulation induced DNA single and double strand-breaks and caused purines and pyrimidines oxidation. None of compounds examined was capable of creating adducts with DNA, while those substances increased ROS (including •OH) level in PBMCs. Roundup 360 PLUS caused damage to DNA even at 5 µM, while glyphosate and particularly AMPA induced DNA lesions from the concentration of 250 µM and 500 µM, respectively. DNA damage induced by glyphosate and its derivatives increased in order: AMPA, glyphosate, Roundup 360 PLUS. We may conclude that observed changes were not associated with direct interaction of xenobiotics studied with DNA, but the most probably they occurred through ROS-mediated effects.

The effect of two bromfenvinphos impurities: BDCEE and ß-ketophosphonate on oxidative stress induction, acetylcholinesterase activity, and viability of human red blood cells.

Numerous research works have shown that synthesis of pesticides leads to the formation of impurities that may substantially enhance pesticide toxicity. In this study, the effect of manufacturing impurities of pesticide bromfenvinphos (BFVF) such as 1-bromo-2-(2,4-dichlorophenyl)-2-ethoxy ethene (BDCEE) and diethyl [2-(2,4-dichlorophenyl)-2-oxo-ethyl] phosphonate (ß-ketophosphonate) on human erythrocytes, being significantly exposed to xenobiotics has been studied. The cells were treated with the compounds studied in the concentrations ranging from 0.1 µM to 250 µM for 4 h. In order to assess the effect of BDCEE and ß-ketophosphonate on red blood cells hemolytic changes, changes in cell size (FSC parameter) and oxidation of hemoglobin were studied. Moreover, alterations in reactive oxygen species (ROS) formation, reduced glutathione (GSH) level and acetylcholinesterase (AChE) activity were determined. BDCEE induced an increase in ROS level and caused strong oxidation of hemoglobin as well as a slight change in erythrocytes size and hemolysis, while it did not change GSH level and AChE activity. ß-ketophosphonate has not been shown to affect most parameters studied, but it strongly reduced AChE activity. Because changes in the parameters examined were noted at low concentrations of BFVF impurities (5-250 µM), those substances should not negatively affect on red blood cells of humans environmentally exposed to this pesticide.

The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells.

The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide-glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01-10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances.

Reactions of nitroxides 15. Cinnamates bearing a nitroxyl moiety synthesized using a Mizoroki-Heck cross-coupling reaction.

Cinnamic acid derivatives bearing a nitroxyl moiety (2,2,6,6-tetramethyl-1-oxyl-4-piperidyl 3-E-aryl acrylates) were synthesized in 30-100% yield using a Mizoroki-Heck cross-coupling reaction between 4-acryloyloxy-2,2,6,6-tetramethylpiperidine-1-oxyl and iodobenzene derivatives in the presence of palladium(II) acetate coordinated with a tri(o-tolyl)phosphine ligand immobilized in a polyurea matrix.

Oxidative stress in human erythrocytes treated with bromfenvinphos and its impurities.

Bromfenvinphos (BFVF) is an organophosphorus (OP) pesticide which was widely used in agriculture and veterinary practice. During synthesis of this insecticide five main impurities are formed: dihydro-bromfenvinphos, dibromo-bromfenvinphos, 2,4-dichlorophenacyl bromide, 2,4-dichlorophenacylidene bromide and 2,4-dichlorophenacylidyne bromide, which can be present in technical grade bromfenvinphos in amounts from 0.1 to 4%. The aim of this study was to examine the influence of bromfenvinphos and its manufacturing impurities on parameters of oxidative stress, the activity of antioxidative enzymes and the level of reduced glutathione. Human erythrocytes were incubated with bromfenvinphos and its impurities in the concentrations range from 0.5 to 500 µM for 1 h. This study indicated that 2,4-dichlorophenacyl derivatives more strongly oxidized analyzed parameters in human erythrocytes than bromfenvinphos. Investigated compounds caused an increase in lipid peroxidation and oxidation of fluorescent probe DCFH2 - the strongest pro-oxidative changes were provoked by 2,4-dichlorophenacyl bromide. None of the compounds studied in the concentrations from 0.5 to 500 µM changed the activity of SOD and only 2,4-dichlorophenacyl decreased activity of CAT. The level of GSH was only altered by 2,4-dichlorophenacyl derivatives. It was observed that increasing number of bromine atoms in the side chain of those derivatives was associated with decreased GSH level.

The effect of metabolites and impurities of glyphosate on human erythrocytes (in vitro).

The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study was undertaken to evaluate toxic potential of widely used pesticide - glyphosate, its metabolites: aminomethylphosphonic acid (AMPA); methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine. We evaluated the effect of those compounds on hemolysis, hemoglobin oxidation, reactive oxygen species (ROS) formation and changes in morphology of human erythrocytes. The erythrocytes were exposed to different concentrations of glyphosate and its metabolites and impurities (0.01-5mM) for 1, 4 and 24h. Glyphosate, its metabolites and impurities induced a little hemolysis and hemoglobin oxidation. All changes were very low, even after 24h incubation. Most of the investigated compounds induced reactive oxygen species formation from 0.25mM, except the N-methylglyphosate which caused an increase in ROS formation from 0.5mM. Moreover, the investigated xenobiotics did not change the size and shape (except bis-(phosphonomethyl)amine) of the human erythrocytes. Changes in human erythrocytes were observed only when high concentrations of the compounds were applied. Some investigated metabolites and impurities caused a slight stronger damage to human erythrocytes than a glyphosate. The results clearly show that the changes induced in the erythrocytes can occur only as a result of poisoning with these compounds.

The effect of bromfenvinphos, its impurities and chlorfenvinphos on acetylcholinesterase activity.

The aim of this work was to examine the effect of two organophosphorous compounds i.e. bromfenvinphos (BFVF) and chlorfenvinphos (CFVF) possessing acaricidal and insecticidal properties, on the activity of human erythrocytes acetylcholinesterase (AChE, EC 3.1.1.7). Moreover, the effect of five bromfenvinphos production impurities on AChE activity was studied. The erythrocytes were incubated with the compounds studied in the concentrations range from 0.05 to 250 µM for 1h. The organophosphorous compounds studied in low concentrations increased Km value but they did not change Vmax value (competitive inhibition). Higher concentrations of the compounds studied decreased Vmax value and increased Km value, what revealed a mixed type of AChE inhibition by these xenobiotics. Basic significance in AChE activity inhibition has the type of halogen in vinyl group. Chlorfenvinphos (insecticide) exhibited stronger enzyme inhibition than bromfenvinphos. CFVF and dibromo-bromfenvinphos possessed the lowest Ki and Ki' values among all the compounds studied. The presence of Cl atom (chlorfenvinphos) instead of Br atom (bromfenvinphos) considerably increases antiesterase activity of the individual compound. Three impurities like 2,4-dichlorophenacyl bromide, 2,4-dichlorophenacylidene bromide and 2,4-dichlorophenacylidyne bromide did not induce any statistically changes in AChE activity. Two impurities of bromfenvinphos such as: dihydro-bromfenvinphos and dibromo-bromfenvinphos revealed significant effect on the AChE activity, which may be connected with the presence a phosphate group in these substances. It was proven that an increase in antiesterase activitiy of the compounds studied corresponded with the increase in the number of Br atoms at carbon of their vinyl group: dibromo-bromfenvinphos>bromfenvinphos>dihydro-bromfenvinphos.

The effect of bromfenvinphos and its impurities on human erythrocyte.

Bromfenvinphos - (E,Z)-O,O-diethyl-O-[1-(2,4-dichlorophenyl)-2-bromovinyl] phosphate (BFVF) is the insecticide elaborated in Poland, which has been used against Varroa destructor causing honey bees disease called as varroosis. The substances that are formed as a result of bromfenvinphos synthesis are dihydro-bromfenvinphos (O,O-diethyl O-[1-(2,4-dichlorophenyl)vinyl] phosphate); dibromo-bromfenvinphos (O,O-diethyl O-[1-(2,4-dichlorophenyl)-2,2-dibromovinyl] phosphate); 2,4-dichlorophenacyl bromide; 2,4-dichlorophenacylidene bromide and 2,4-dichlorophenacylidyne bromide. In this work, we evaluated the effect of these compounds on hemolysis and hemoglobin oxidation (met-Hb formation) in human erythrocytes. Moreover, the changes in the size (FSC-A) and the shape (SSC-A) of red blood cells were assessed using flow cytometry and phase contrast microscopy. It was proven that bromfenvinphos at concentrations ranging from 0.5 to 250 µM during 1h incubation did not change the parameters examined in human erythrocytes. Similarly, most of bromfenvinphos impurities did not increase hemolysis and methemoglobin level nor changed the size and shape of the erythrocytes. The exception was dibromo-bromfenvinphos, which changed the FSC-A and SSC-A parameters, as well as 2,4-dichlorophenacyl bromide which induced hemolysis, increased the level of met-Hb and changed erythrocytes morphology.