Intracellular Reactive Species Production Research Articles

Accelerated apoptosis, oxidative stress, and cholinergic inflammation in blood of metalworkers

Metalworkers are exposed to numerous chemicals in their workplace environment, such as solvents, heavy metals, and metalworking fluids, that have a negative impact on their health. Furthermore, there is an increase in the prevalence of chronic diseases among metalworkers; however, the molecular mechanisms involved in this increased predisposition to chronic diseases are unclear. Considering that occupational exposure represents a potential risk for metalworkers, the aim of this study was to measure biomarkers of oxidative stress, inflammation, and cytotoxicity in the peripheral blood of metalworkers from Southern Brazil. The study included 40 metalworkers and 20 individuals who did not perform activities with any recognized exposure to chemical substances, such as those working in administration, commerce, and education, as controls. Cellular and molecular biomarkers as leukocyte viability, intracellular production of reactive species, mitochondrial mass and membrane potential and plasma lipid peroxidation, sulfhydryl groups, total antioxidant capacity, and butyrylcholinesterase activity were evaluated in the blood of metalworkers and controls. Metalworkers were found to have higher rates of apoptosis, increased production of reactive species, and increased mitochondrial potential and mass in leukocytes associated with decreased antioxidant defenses and increased activity of the butyrylcholinesterase enzyme in their plasma. It can be concluded that cytotoxicity, oxidative stress, and inflammation are involved in the multiplicity of health outcomes related to chemical exposure in the metalworking industry.

The synthetic tubulysin derivative, tubugi-1, improves the innate immune response by macrophage polarization in addition to its direct cytotoxic effects in a murine melanoma model

Synthetic tubugis are equally potent but more stable than their natural forms. Their anticancer potential was estimated on a solid melanoma in vitro and in vivo. Tubugi-1 induced the apoptosis in B16 cells accompanied with strong intracellular production of reactive species, subsequently imposing glutathione and thiol group depletion. Paradoxically, membrane lipids were excluded from the cascade of intracellular oxidation, according to malondialdehyde decrease. Although morphologically apoptosis was typical, externalization of phosphatidylserine (PS) as an early apoptotic event was not detected. Even their exposition is pivotal for apoptotic cell eradication, primary macrophages successfully eliminated PS-deficient tubugi-1 induced apoptotic cells. The tumor volume in animals exposed to the drug in therapeutic mode was reduced in comparison to control as well as to paclitaxel-treated animals. Importantly, macrophages isolated from tubugi-1 treated animals possessed conserved phagocytic activity and were functionally and phenotypically recognized as M1. The cytotoxic effect of tubugi-1 is accomplished through its ability to polarize the macrophages toward M1, probably by PS independent apoptotic cell engulfment. The unique potential of tubugi-1 to prime the innate immune response through the induction of a specific pattern of tumor cell apoptosis can be of extraordinary importance from fundamental and applicable aspects.

Acute Consumption of Bordo Grape Juice and Wine Improves Serum Antioxidant Status in Healthy Individuals and Inhibits Reactive Oxygen Species Production in Human Neuron-Like Cells.

Few studies investigated the biological effects of American grape cultivars. We investigated the metabolic response after acute consumption of grape juice or wine from Bordo grapes (Vitis labrusca) in a placebo-controlled crossover study with fifteen healthy volunteers. Blood samples were collected 1 hour after the intake of 100 mL of water, juice, or wine to measure TBARS, ABTS, FRAP, glucose, and uric acid levels. To evaluate differences in cellular response, intracellular reactive species production (DCFH-DA) and metabolic mitochondrial viability (MTT) were assessed after exposure of human neuron-like cells (SH-SY5Y) to juice or wine. Glycemia was reduced after juice or wine consumption, whereas blood levels of uric acid were reduced after juice consumption but increased after wine consumption. Juice and wine consumption reduced plasma lipid peroxidation and increased plasma antioxidant capacity (ABTS and FRAP assays). Furthermore, juice inhibited H2O2-induced intracellular production of reactive species (RS) and increased the viability of SH-SY5Y cells. In contrast, wine (dealcoholized) exhibited a per se effect by inducing the production of RS and reducing cell viability. These results indicate a positive impact of acute consumption of Bordo juice and wine on human oxidative status, whereas only juice had protective effects against oxidative stress-induced cytotoxicity.

Effects of 47C allele (rs4880) of the SOD2 gene in the production of intracellular reactive species in peripheral blood mononuclear cells with and without lipopolysaccharides induction

Challenging of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharides (LPS) has been shown to activate monocytes and macrophages, leading to the production of pro-inflammatory cytokines and reactive oxygen species (ROS). Manganese superoxide dismutase (MnSOD) is an important enzyme that may play a central role in the response to oxidative stress. 47C> T SNP of the SOD2 gene, the -9Val MnSOD is less efficient than the -9Ala version. We have previously characterized the cellular redox status of human PBMCs expressing either -9Ala (CC) or -9Val (TT) SOD2 and analyzed the responses of these cells to oxidative stress induced by LPS. Due to the observed alterations in the activities of these antioxidant enzymes, we decided to investigate their immunocontent and analyze the production of intracellular oxidants, as well as any resulting DNA damage. PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers per allele). We then analyzed levels of nitrite, DNA damage by comet assay, TNF-α, carboxymethyl lysine and nitrotyrosine and assessed production of intracellular reactive species by the DCFH-DA-based assay and western blots were used to analyze protein levels. Our results show that there occurs an increase in nitric oxide production in both allele groups after challenge with LPS. A significant increase in DNA damage was observed in PBMCs after an 8-h LPS challenge. Cells expressing the SOD2 47C allele quickly adapt to a more intense metabolism by upregulating cellular detoxification mechanisms. However, when these cells are stressed over a long period, they accumulate a large quantity of toxic metabolic byproducts.

Modulation of Inflammatory Markers by miR-146a during Replicative Senescence in Trabecular Meshwork Cells

To investigate the alterations in microRNA (miRNA) expression during replicative senescence (RS) in human trabecular meshwork (HTM) cells. Two HTM cell lines were serially passaged until they reached RS. Changes in expression of 30 miRNAs were assessed by real-time quantitative (q)-PCR. The effects of miR-146a on gene expression were analyzed with gene arrays and the results confirmed by real-time q-PCR. Protein levels of IRAK1 and PAI-1 were analyzed by Western blot and those of IL6 and IL8 by ELISA. Senescence-associated markers were monitored by flow cytometry and cell proliferation by BrdU incorporation. RS of HTM cells was associated with significant changes in expression of 18 miRNAs, including the upregulation of miR-146a. miR-146a downregulated multiple genes associated with inflammation, including IRAK1, IL6, IL8, and PAI-1, inhibited senescence-associated beta-galactosidase (SA-beta-gal) activity and production of intracellular reactive species (iROS), and increased cell proliferation. Overexpression of either IRAK1 or PAI-1 inhibited the effects of miR-146a on cell proliferation and iROS production in senescent cells. RS in HTM cells was associated with changes in miRNA expression that could influence the senescent phenotype. Upregulation of the anti-inflammatory miR-146a may serve to restrain excessive production of inflammatory mediators in senescent cells and limit their deleterious effects on the surrounding tissue. Among the different proteins repressed by miR-146a, the inhibition of PAI-1 may act to minimize the effects of senescence on the generation of iROS and growth arrest and prevent alterations of the extracellular proteolytic activity of the TM.

Retinol increases catalase activity and protein content by a reactive species-dependent mechanism in Sertoli cells

Vitamin A (retinol) is widely used as an antioxidant in therapeutic interventions and dietary supplementations. However, the redox properties of retinoids have been the subject of intense debate in the last few years, as recent works observed deleterious effects caused by retinol supplementation in clinical trials. In the present work, we show that retinol treatment (7 μM, 24 h) led to catalase (EC 1.11.1.6; CAT) activation in cultured Sertoli cells by increasing its protein content in a reactive species-dependent manner. Retinol treatment also increased cell lipoperoxidation, assessed by determination of thiobarbituric acid-reactive substances (TBARS), and intracellular reactive species production, determined by the real-time dihydrochlorofluorescein (DCFH-DA) assay. However, no alterations on CAT mRNA expression (assessed by RT-PCR) were observed, indicating an effect independent of CAT gene-transcription regulation. Importantly, all the effects induced by retinol were inhibited by the antioxidant Trolox, a hydrophilic analogue of alpha-tocopherol. These results show for the first time that retinol increases CAT activity by a redox-dependent modulation of its protein content in a cell culture model. CAT activity or expression are widely used as indexes of oxidative stress in biological systems; since no changes in CAT mRNA expression were detected in these conditions, the use of CAT gene-transcription activation when assessing oxidative stress should be re-evaluated.