Proteomics Study of Benzene Metabolite Hydroquinone Induced Hematotoxicity in K562 Cells.

Objective: Hydroquinone (HQ), one of the phenolic metabolites of benzene, is widely recognized as an important participant in benzene-induced hematotoxicity. However, there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn't been fully understood yet. Methods: In this study, we treated K562 cells with 40 µmol/L HQ for 72 h, examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring (PRM), and performed bioinformatics analysis to identify interaction networks. Results: One hundred and eighty-seven upregulated differentially expressed proteins (DEPs) and 279 downregulated DEPs were identified in HQ-exposed K562 cells, which were involved in neutrophil-mediated immunity, blood microparticle, and other GO terms, as well as the lysosome, metabolic, cell cycle, and cellular senescence-related pathways. Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways, we constructed the network of protein interactions and determined 6 DEPs (STAT1, STAT3, CASP3, KIT, STAT5B, and VEGFA) as main hub proteins with the most interactions, among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity. Conclusion: Our work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification.

Facile green approach towards the synthesis of some phenyl piperazine based dithiocarbamates as potent hemolytic and thrombolytic agents.

The facile and efficient protocol for the synthesis of N-phenyl piperazine based di-thio-carbamates has been reported under neat conditions. A library of novel piperazine based di-thio-carbamates (3a-h) in excellent yields has been prepared. Solvent free, catalyst free and easy work up conditions make this protocol an attractive synthetic protocol to achieve novel biologically active di-thio-carbamates. The synthesized molecules have been characterized by FT-IR, 1HNMR and 13CNMR spectroscopic techniques. The pharmacological aspects of these derivatives have been evaluated via hemolysis and thrombolysis. All the target molecules (3a-h) exhibit mild to medium potential as hemolytic and thrombolytic agents. Among the synthesized derivatives, compound 3c showed least cytotoxicity and better thrombolytic potential.

Haematotoxicity during peptide receptor radionuclide therapy: Baseline parameters differences and effect on patient's therapy course.

BACKGROUND: Mainly severe (CTCAE grade 3-4) haematotoxicity during peptide receptor radionuclide therapy (PRRT) is reported in literature due to major clinical impact, however moderate (CTCAE grade 2) haematotoxicity is common and could affect therapy management. The aim of this study was to evaluate the haematotoxicity course during PRRT and to compare baseline parameters between haematotoxicity grades. METHODS: In this retrospective study, 100 patients with a neuroendocrine tumour treated with PRRT were included. Patients were treated with an aimed number of four cycles with 7.4 GBq [177Lu]Lu-DOTA-TATE administered every 10 weeks. Haematological assessment was performed at baseline and frequently up to 10 weeks after the fourth cycle. The lowest haematological value was graded according to CTCAE v5.0, and patients were classified using the highest observed grade. Differences in baseline parameters, including [68Ga]Ga-DOTA-TATE positive tumour volume, were evaluated between CTCAE grades. RESULTS: Four cycles were completed by 86/100 of patients, 4/100 patients discontinued due to haematotoxicity, and 10/100 patients due to progressive disease. The treatment course was adjusted due to haematotoxicity in 24/100 patients, including postponed next cycle (n = 17), reduced administered activity (n = 13), and both adjustments (n = 10). The most observed haematotoxicity grade was grade 0-1 in 54/100 patients, grade 2 in 38/100 and grade 3-4 in 8/100. Significant differences in baseline leucocyte, neutrophil and platelet counts were observed between grade 0-1 and grade 2. However, the correlation between baseline and lowest observed values was poor to moderate. No differences between haematotoxicity grades and baseline parameters or somatostatin receptor positive tumour volume was observed. CONCLUSIONS: The incidence of severe haematotoxicity was low with extensive screening and monitoring. The vast majority of patients (96/100) was not restricted in treatment continuation by haematotoxicity; therefore, our selection criteria appeared appropriate for safe PRRT treatment. Baseline parameters showed limited correlation with the degree of decline in haematological values.

Global Identification of HIF-1α Target Genes in Benzene Poisoning Mouse Bone Marrow Cells.

Benzene is a hematopoietic toxicant, and hematopoietic cells in bone marrow (BM) are one of the main targets for its action, especially hematopoietic stem cells (HSCs). Hypoxia-inducible factor-1α (HIF-1α) is associated with the metabolism and physiological functions of HSCs. We previously found that the mechanism of regulation of HIF-1α is involved in benzene-induced hematopoietic toxicity. In this study, chromatin immunoprecipitation sequencing (ChIP-Seq) technologies were used to analyze the genome-wide binding spectrum of HIF-1α in mouse BM cells, and specific HIF-1α target genes and pathways associated with benzene toxicity were screened and validated. By application of the ChIP-Seq technique, we identified target genes HIF-1α directly binds to and regulates. Forty-two differentially down-regulated genes containing the HIF-1α specific binding site hypoxia response element (HRE) were found, of which 25 genes were with biological function. Moreover, the enrichment analysis of signal pathways indicated that these genes were significantly enriched in the Jak-STAT signaling pathway, Natural killer cell mediated cytotoxicity, the Fc epsilon RI signaling pathway, Pyrimidine metabolism, the T cell receptor signaling pathway, and Transcriptional misregulation in cancer. After verification, 11 genes involved in HSC self-renewal, cell cycle, differentiation, and apoptosis pathways were found to be significantly reduced, and may participate in benzene-induced hematotoxicity. Our study provides a new academic clue for the mechanism of benzene hematotoxicity.

pH-Dependent exhibition of hemolytic activity by an extract of Hypsizygus marmoreus fruiting bodies.

The current study found that an extract from the fruiting bodies of the edible mushroom Hypsizygus marmoreus exhibited hemolytic activity against sheep red blood cells when its pH was lowered. Although hemolytic activity was not detected when an extract had a neutral pH, an extract with a low pH exhibited potent hemolytic activity. The maximal hemolytic activity was exhibited by an extract with a pH of 5.5. A heat-treated extract did not exhibit hemolytic activity before its pH was lowered, and that activity was inhibited in the presence of PMSF and EDTA. The turbidity of the extract increased during lowering of its pH, and the precipitate fraction exhibited hemolytic activity. Fractionation by a modified Bligh and Dyer method and TLC analyses suggested that a hemolytic compound in the extract might be a type of lipid. These results suggest that a hemolytic lipid-like compound in an extract of H. marmoreus fruiting bodies may be released by a non-active precursor substance(s) through metalloenzyme(s) while the extract has a low pH.

HemoPred: a web server for predicting the hemolytic activity of peptides.

AIM: Toxicity arising from hemolytic activity of peptides hinders its further progress as drug candidates. MATERIALS & METHODS: This study describes a sequence-based predictor based on a random forest classifier using amino acid composition, dipeptide composition and physicochemical descriptors (named HemoPred). RESULTS: This approach could outperform previously reported method and typical classification methods (e.g., support vector machine and decision tree) verified by fivefold cross-validation and external validation with accuracy and Matthews correlation coefficient in excess of 95% and 0.91, respectively. Results revealed the importance of hydrophobic and Cys residues on α-helix and ß-sheet, respectively, on the hemolytic activity. CONCLUSION: A sequence-based predictor which is publicly available as the web service of HemoPred, is proposed to predict and analyze the hemolytic activity of peptides.

Efficient and non-toxic gene delivery by anionic lipoplexes based on polyprenyl ammonium salts and their effects on cell physiology.

BACKGROUND: One of the major challenges limiting the development of gene therapy is an absence of efficient and safe gene carriers. Among the nonviral gene delivery methods, lipofection is considered as one of the most promising. In the present study, a set of cationic polyprenyl derivatives [trimethylpolyprenylammonium iodides (PTAI)] with different lengths of polyprenyl chains (from 7, 8 and 11 to 15 isoprene units) was suggested as a component of efficient DNA vehicles. METHODS: Optimization studies were conducted for PTAI in combination with co-lipid dioleoylphosphatidylethanolamine on DU145 human prostate cancer cells using: size and zeta potential measurements, confocal microscopy, the fluorescein diacetate/ethidium bromide test, cell counting, time-lapse monitoring of cell movement, gap junctional intercellular coupling analysis, antimicrobial activity assay and a red blood cell hemolysis test. RESULTS: The results obtained show that the lipofecting activity of PTAI allows effective transfection of plasmid DNA complexed in negatively-charged lipoplexes of 200-500 nm size into cells without significant side effects on cell physiology (viability, proliferation, morphology, migration and gap junctional intercellular coupling). Moreover, PTAI-based vehicles exhibit a potent bactericidal activity against Staphylococcus aureus and Escherichia coli. The developed anionic lipoplexes are safe towards human red blood cell membranes, which are not disrupted in their presence. CONCLUSIONS: The developed carriers constitute a group of promising lipofecting agents of a new type that can be utilized as effective lipofecting agents in vitro and they are also an encouraging basis for in vivo applications.

Intracellular haemolytic agents of Heterocapsa circularisquama exhibit toxic effects on H. circularisquama cells themselves and suppress both cell-mediated haemolytic activity and toxicity to rotifers (Brachionus plicatilis).

A harmful dinoflagellate, Heterocapsa circularisquama, is highly toxic to shellfish and the zooplankton rotifer Brachionus plicatilis. A previous study found that H. circularisquama has both light-dependent and -independent haemolytic agents, which might be responsible for its toxicity. Detailed analysis of the haemolytic activity of H. circularisquama suggested that light-independent haemolytic activity was mediated mainly through intact cells, whereas light-dependent haemolytic activity was mediated by intracellular agents which can be discharged from ruptured cells. Because H. circularisquama showed similar toxicity to rotifers regardless of the light conditions, and because ultrasonic ruptured H. circularisquama cells showed no significant toxicity to rotifers, it was suggested that live cell-mediated light-independent haemolytic activity is a major factor responsible for the observed toxicity to rotifers. Interestingly, the ultrasonic-ruptured cells of H. circularisquama suppressed their own lethal effect on the rotifers. Analysis of samples of the cell contents (supernatant) and cell fragments (precipitate) prepared from the ruptured H. circularisquama cells indicated that the cell contents contain inhibitors for the light-independent cell-mediated haemolytic activity, toxins affecting H. circularisquama cells themselves, as well as light-dependent haemolytic agents. Ethanol extract prepared from H. circularisquama, which is supposed to contain a porphyrin derivative that displays photosensitising haemolytic activity, showed potent toxicity to Chattonella marina, Chattonella antiqua, and Karenia mikimotoi, as well as to H. circularisquama at the concentration range at which no significant toxicity to rotifers was observed. Analysis on a column of Sephadex LH-20 revealed that light-dependent haemolytic activity and inhibitory activity on cell-mediated light-independent haemolytic activity existed in two separate fractions (f-2 and f-3), suggesting that both activities might be derived from common compounds. Our results suggest that the photosensitising haemolytic toxin discharged from ruptured H. circularisquama cells has a relatively broad spectrum of phytoplankton toxicity, and that physical collapse of H. circularisquama cells can lead not only to the disappearance of its own toxicity, but also to mitigation of the effects of other HABs.

Comparison of hematological alterations and markers of B-cell activation in workers exposed to benzene, formaldehyde and trichloroethylene.

Benzene, formaldehyde (FA) and trichloroethylene (TCE) are ubiquitous chemicals in workplaces and the general environment. Benzene is an established myeloid leukemogen and probable lymphomagen. FA is classified as a myeloid leukemogen but has not been associated with non-Hodgkin lymphoma (NHL), whereas TCE has been associated with NHL but not myeloid leukemia. Epidemiologic associations between FA and myeloid leukemia, and between benzene, TCE and NHL are, however, still debated. Previously, we showed that these chemicals are associated with hematotoxicity in cross-sectional studies of factory workers in China, which included extensive personal monitoring and biological sample collection. Here, we compare and contrast patterns of hematotoxicity, monosomy 7 in myeloid progenitor cells (MPCs), and B-cell activation biomarkers across these studies to further evaluate possible mechanisms of action and consistency of effects with observed hematologic cancer risks. Workers exposed to benzene or FA, but not TCE, showed declines in cell types derived from MPCs, including granulocytes and platelets. Alterations in lymphoid cell types, including B cells and CD4+ T cells, and B-cell activation markers were apparent in workers exposed to benzene or TCE. Given that alterations in myeloid and lymphoid cell types are associated with hematological malignancies, our data provide biologic insight into the epidemiological evidence linking benzene and FA exposure with myeloid leukemia risk, and TCE and benzene exposure with NHL risk.

Synthesis of 2-{(5-phenyl-1,3,4-Oxadiazol-2-yl)sulfanyl}-N-substituted acetamides as potential antimicrobial and hemolytic agents.

A new series of N-substituted derivatives of 2-{(5-phenyl-1,3,4-Oxadiazol-2-yl)sulfanyl}acetamides was synthesized. The synthesis was carried out by converting benzoic acid (1) into ethyl benzoate (2), benzohydrazide (3) and then 5-pheny-1,3,4-Oxadiazol-2-thiol (4) step by st0ep. The target compounds 6a-p were synthesized by reaction of compound 4 with equimolar ratios of different N-alkyl/aryl substituted 2-bromoacetamide (5a-p) in the presence of DMF and sodium hydride (NaH). The spectral (EI-MS, IR, (1)H-NMR) characterization of all the synthesized compounds reveal their successful synthesis. The compounds were also screened for antimicrobial & hemolytic activity and most of them were found to be active against the selected microbial species at variable extent relative to reference standards. But 6h was the most active against the selected panel of microbes. This series showed less toxicity and may be considered for further biological screening and application trial except 6m, possessing higher cytotoxicity.

The multigene families of actinoporins (part II): Strategies for heterologous production in Escherichia coli.

The sea anemone venom contains pore-forming proteins (PFP) named actinoporins, due to their purification from organisms belonging to Actiniaria order and its ability to form pores in sphingomyelin-containing membranes. Actinoporins are generally basic, monomeric and single-domain small proteins (∼20 kDa) that are classified as α-type PFP since the pore formation in membranes occur through α-helical elements. Different actinoporin isoforms have been isolated from most of the anemones species, as was analyzed in the first part of this review. Several actinoporin full-length genes have been identified from genomic-DNA libraries or messenger RNA. Since the actinoporins lack carbohydrates and disulfide bridges, their expression in bacterial systems is suitable. The actinoporins heterologous expression in Escherichia coli simplifies their production, replaces the natural source reducing the ecological damage in anemone populations, and allows the production of site-specific mutants for the study of the structure-function relationship. In this second part of the review, the strategies for heterologous production of actinoporins in Escherichia coli are analyzed, as well as the different approaches used for their purification. The activity of the recombinant proteins with respect to the wild-type is also reviewed.

Genotypic and phenotypic analysis of clinical isolates of Staphylococcus aureus revealed production patterns and hemolytic potentials unlinked to gene profiles and source.

BACKGROUND: Nosocomial infections caused by the bacterial pathogen Staphylococcus aureus can lead to serious complications due to the varying presence of secreted toxins. Comparative studies of genomic information and production rates are needed to assess the pathogenic potential of isolated strains. Genotypic and phenotypic profiling of clinical and colonising isolates of S. aureus was used to characterise the release of exotoxins. Blood isolates were compared with colonisation strains to determine similarities and differences of single strains and clusters. RESULTS: Fifty-one fresh isolates obtained from colonised individuals (n = 29) and S. aureus bacteremia (SAB) patients (n = 22) were investigated. The prevalence of genes encoding for three cytolysins (alpha/beta/gamma toxin) and twenty-four superantigens (SEA-SElX) was determined. Isolates exhibited eighteen distinct combinations of superantigens. Sequence analysis identified mutated open reading frames in hla in 13.7% of all strains, in selw (92.2%) and in selx (15.7%). All corrupted genes were associated with specific clonal complexes. Functional assessment of alpha toxin activity by a rabbit erythrocyte lysis assay revealed that supernatants lacking alpha toxin still displayed hemolysis. This was due to the presence of gamma toxin, as proven by inhibition experiments using antisera raised against the respective recombinant proteins. Alpha toxin, SEC, and TSST1 production was quantified by enzyme-linked immunosorbent assays on supernatants of all hla, sec, and tst positive isolates. Blood isolates and colonising strains showed comparable amounts of secreted proteins within a wide range. Agr types I to IV were identified, but did not allow a prediction of high or low production rates. In contrast, alpha toxin production rates between distinct clonal complexes clearly differed. Spa typing was performed and revealed thirty-two unique spa gene patterns and eight small clusters comprising nineteen isolates. Recognised spa-typing clusters displayed highly similar production rates. CONCLUSION: Production rates of the three most prevalent exotoxins varied within both groups of blood isolates and colonising strains. By comparing genotypes and secretion, we found that identical complex gene patterns did not allow predictions of toxin production and function. However, identification of spa typing clusters was suitable to predict similar quantities of released exotoxins.

Venom from Opisthacanthus elatus scorpion of Colombia, could be more hemolytic and less neurotoxic than thought.

We report the first biochemical, biological, pharmacological and partial proteomic characterization studies of the Opisthancanthus elatus venom (Gervais, 1844) from Colombia. The Reverse Phase High-Performance Liquid Chromatography venom profile showed 28 main well-defined peaks, most eluting between 20 and 45min (18-30% of acetonitrile, respectively). High-resolution mass analysis indicates the presence of 106 components ranging from 806.59742Da to 16849.4139Da. O. elatus venom showed hemolytic activity and hydrolyzed the specific substrate BapNa suggesting the presence of proteins with serine-protease activity. Collected RP-HPLC fractions eluting at 52.6, 55.5, 55.8, 56.2, and 63.9min (PLA2 region between 33 and 40% of acetonitrile), showed hemolytic activity and hydrolyzed the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid, indicating the presence of compounds with phospholipases A2 activity. These RP-HPLC fractions, showed molecular masses values up to 13978.19546Da, corroborating the possible presence of the mentioned enzymes. Tryptic digestion and MS/MS analysis showed the presence of a phospholipase like fragment, similar to on described in other Opisthacanthus genus studies. No coagulant activity was observed. No larvicidal or antimicrobial activity was observed at concentrations evaluated. Lethal and toxic activity is expected at doses above 100mg/kg, no neurotoxic effects were detected at lower doses. In conclusion, O. elatus exhibits a venom with a predominant phospholipase A2 activity than thought; mammal's neurotoxic activity is expected above the 100mg/kg, which is very high compared to the venom from other neurotoxic scorpions.

Contribution to the food products' analysis: A research and evaluation on the hemolytic effect of some pesticides used in Lebanon.

Pesticides are a real concern for the society as their use has become critical, leading sometimes to their accumulation as residues in fruits and vegetables. After examining the pesticides sold in Northern Lebanon, this study is focused on the analysis and identification of pesticides residues in fruits and vegetables that are harvested in this region and treated with the locally sold pesticides. Results show: first, (i) a use of Zineb by the name of another pesticide Micronized Sulfur to avoid prosecution; (ii) a significant presence of Metalaxyl in lemons and oranges; (iii) a significant presence of Trifluralin in strawberries; and (iv) a significant presence of Zineb in lemons and tomatoes. Second, with the use of hemolytic tests on human blood results show: (i) a critical concentration and a significant hemolytic effect of some pesticides used in Lebanon; and (ii) an absence of hemolytic effect in the collected fractions of the different analyzed fruit extracts containing pesticides. Finally, this work is the first step for pesticides' analysis in vegetables and fruits in Lebanon, initiating a wider analytical study in order to control and examine the use of pesticides which, according to our results, could have an adverse effect on human health over a long term.

Protective effects of bacterial osmoprotectant ectoine on bovine erythrocytes subjected to staphylococcal alpha-haemolysin.

Ectoine (ECT) is a bacterial compatible solute with documented protective action however no data are available on its effects on various cells against bacterial toxins. Therefore, we determined the in vitro influence of ECT on bovine erythrocytes subjected to staphylococcal α-haemolysin (HlyA). The cells exposed to HlyA alone showed a distinct haemolysis and reduced glutathione (GSH)/oxidised glutathione (GSSG) level, however the toxic effects were attenuated in the combinations of HlyA + ECT suggesting ECT-induced protection of erythrocytes from HlyA.

The effect of cholesterol on the long-range network of interactions established among sea anemone Sticholysin II residues at the water-membrane interface.

Actinoporins are α-pore forming proteins with therapeutic potential, produced by sea anemones. Sticholysin II (StnII) from Stichodactyla helianthus is one of its most extensively characterized members. These proteins remain stably folded in water, but upon interaction with lipid bilayers, they oligomerize to form a pore. This event is triggered by the presence of sphingomyelin (SM), but cholesterol (Chol) facilitates pore formation. Membrane attachment and pore formation require changes involving long-distance rearrangements of residues located at the protein-membrane interface. The influence of Chol on membrane recognition, oligomerization, and/or pore formation is now studied using StnII variants, which are characterized in terms of their ability to interact with model membranes in the presence or absence of Chol. The results obtained frame Chol not only as an important partner for SM for functional membrane recognition but also as a molecule which significantly reduces the structural requirements for the mentioned conformational rearrangements to occur. However, given that the DOPC:SM:Chol vesicles employed display phase coexistence and have domain boundaries, the observed effects could be also due to the presence of these different phases on the membrane. In addition, it is also shown that the Arg51 guanidinium group is strictly required for membrane recognition, independently of the presence of Chol.

Compensatory erythropoiesis has no impact on the outcome of the in vivo Pig-a mutation assay in rats following treatment with the haemolytic agent 2-butoxyethanol.

The Pig-a assay has rapidly gained international interest as a useful tool for assessing the mutagenic potential of compounds in vivo. Although a large number of compounds, including both mutagens and non-mutagens, have been tested in the rat Pig-a assay in haematopoietic cells, there is limited understanding of how perturbations in haematopoiesis affect assay performance. Of particular concern is the possibility that regenerative haematopoiesis alone, without exposure to a genotoxic agent, could result in elevated Pig-a mutant cell frequencies. To address this concern, Wistar-Han rats were dosed by oral gavage with a non-genotoxic haemolytic agent, 2-butoxyethanol (2-BE). Dose levels ranging from 0 to 450 mg/kg were tested using both single administration and 28-day treatment regimens. Haematology parameters were assessed at minimum within the first 24h of treatment and 8 days after the final administration. Pig-a mutant frequencies were assessed on Days 15 and ~30 for both treatment protocols and also on Days 43 and 57 for the 28-day protocol. Even at doses of 2-BE that induced marked intravascular lysis and strong compensatory erythropoiesis, the average Pig-a mutant phenotype red blood cell and reticulocyte frequencies were within the historical vehicle control distribution. 2-BE therefore showed no evidence of in vivo mutagenicity in these studies. The data suggest that perturbations in haematopoiesis alone do not lead to an observation of increased mutant frequency in the Pig-a assay.

Structural basis for self-assembly of a cytolytic pore lined by protein and lipid.

Pore-forming toxins (PFT) are water-soluble proteins that possess the remarkable ability to self-assemble on the membrane of target cells, where they form pores causing cell damage. Here, we elucidate the mechanism of action of the haemolytic protein fragaceatoxin C (FraC), a α-barrel PFT, by determining the crystal structures of FraC at four different stages of the lytic mechanism, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore. The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore also exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of a haemolytic toxin.

Hyparrhenia hirta: A potential protective agent against hematotoxicity and genotoxicity of sodium nitrate in adult rats.

The present study was carried out to examine the adverse hematotoxic and genotoxic effects of water nitrate pollution on male adult rats and the use of hyparrhenia hirta methanolic extract in alleviating these effects. Sodium nitrate (NaNO3 ) was administered to adult rats by oral gavage at a dose of 400 mg kg(-1) bw daily for 50 days, while hyparrhenia hirta methanolic extract was given by drinking water at a dose of 1.5 mg mL(-1) (200 mg kg(-1) bw). The NaNO3 -treated group showed a significant decrease in red blood cell count, hemoglobin and hematocrit and a significant increase in total white blood cell, in neutrophil and eosinophil counts. Platelet count, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration remained unchanged in treated groups compared to those of controls. Meanwhile, the results showed a marked reduction in the antioxidant enzyme activities, such as superoxide dismutase, catalase, and glutathione peroxidase, along with an elevation in the level of lipid peroxidation and a reduction in the total glutathione content, indicating the induction of oxidative stress in the erythrocytes of NaNO3 -treated group. Interestingly, NaNO3 treatment showed a significant increase in the frequencies of total chromosomal aberrations, aberrant metaphases and micronucleus in bone-marrow cells. The oxidative stress induced by nitrate treatment might be the major cause for chromosomal rearrangements as free radicals leading to DNA damage. Hyparrhenia hirta methanolic extract appeared to be effective against hematotoxic and genotoxic changes induced by nitrate, as evidenced by the improvement of the markers cited above.

Why does the hemolytic activity of silica predict its pro-inflammatory activity?

BACKGROUND: The hemolytic activity of inhaled particles such as silica has been widely investigated in the past and represents a usual toxicological endpoint to characterize particle reactivity despite the fact that red blood cells (RBCs) are not involved in the pathogenesis of pulmonary inflammation or fibrosis caused by some inhaled particles. The inflammatory process induced by silica starts with the activation of the inflammasome, which leads to the release of mature IL-1ß. One of the upstream mechanisms causing activation of the inflammasome is the labilization of the phagolysosomal membrane after particle phagocytosis. Considering RBC lysis as a model of membrane damage, we evaluated the relationship between hemolytic activity and inflammasome-dependent release of IL-1ß for a panel of selected silica particles, in search of the toxicological significance of the hemolytic activity of an inhaled particle. METHODS: Well-characterized silica particles, including four quartz samples and a vitreous silica, with different surface properties and hemolytic potential were tested for their capacity to induce inflammasome-dependent release of IL-1ß in LPS-primed primary murine peritoneal macrophages by ELISA and Western blot analysis. The mechanisms of IL-1ß maturation and release were clarified by using ASC-deficient cells and inhibitors of phagocytosis and cathepsin B. RESULTS: The silica samples induced dose-dependent hemolysis and IL-1ß release of different amplitudes. A significant correlation between IL-1ß release and hemolytic activity was evidenced (r = 0.827) by linear regression analysis. IL-1ß release was completely abolished in ASC-deficient cells and reduced by inhibitors, confirming the involvement of the inflammasome and the requirement of phagocytosis and cathepsin B for activation. CONCLUSIONS: The same physico-chemical properties of silica particles which are relevant for the lysis of the RBC membrane also appear implicated in the labilization of the phagolysosome, leading to inflammasome activation and release of the pro-inflammatory cytokine IL-1ß. These findings strengthen the relevance of the hemolysis assay to predict the pro-inflammatory activity of silica dusts.