The oxidation of p-phenylenediamine, an ingredient used for permanent hair dyeing purposes, leads to the formation of hydroxyl radicals: oxidative stress and DNA damage in human immortalized keratinocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Low-level laser therapy (LLLT) in human progressive-intensity running: effects on exercise performance, skeletal muscle status, and oxidative stress

The aim of this work was to evaluate the effects of low-level laser therapy (LLLT) on exercise performance, oxidative stress, and muscle status in humans. A randomized double-blind placebo-controlled crossover trial was performed with 22 untrained male volunteers. LLLT (810 nm, 200 mW, 30 J in each site, 30 s of irradiation in each site) using a multi-diode cluster (with five spots - 6 J from each spot) at 12 sites of each lower limb (six in quadriceps, four in hamstrings, and two in gastrocnemius) was performed 5 min before a standardized progressive-intensity running protocol on a motor-drive treadmill until exhaustion. We analyzed exercise performance (VO(2 max), time to exhaustion, aerobic threshold and anaerobic threshold), levels of oxidative damage to lipids and proteins, the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and the markers of muscle damage creatine kinase (CK) and lactate dehydrogenase (LDH). Compared to placebo, active LLLT significantly increased exercise performance (VO(2 max) p = 0.01; time to exhaustion, p = 0.04) without changing the aerobic and anaerobic thresholds. LLLT also decreased post-exercise lipid (p = 0.0001) and protein (p = 0.0230) damages, as well as the activities of SOD (p = 0.0034), CK (p = 0.0001) and LDH (p = 0.0001) enzymes. LLLT application was not able to modulate CAT activity. The use of LLLT before progressive-intensity running exercise increases exercise performance, decreases exercise-induced oxidative stress and muscle damage, suggesting that the modulation of the redox system by LLLT could be related to the delay in skeletal muscle fatigue observed after the use of LLLT.

Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.

Stress free configuration of the human eye

Numerical simulations of eye globes often rely on topographies that have been measured in vivo using devices such as the Pentacam or OCT. The topographies, which represent the form of the already stressed eye under the existing intraocular pressure, introduce approximations in the analysis. The accuracy of the simulations could be improved if either the stress state of the eye under the effect of intraocular pressure is determined, or the stress-free form of the eye estimated prior to conducting the analysis. This study reviews earlier attempts to address this problem and assesses the performance of an iterative technique proposed by Pandolfi and Holzapfel [1], which is both simple to implement and promises high accuracy in estimating the eye's stress-free form. A parametric study has been conducted and demonstrated reliance of the error level on the level of flexibility of the eye model, especially in the cornea region. However, in all cases considered 3-4 analysis iterations were sufficient to produce a stress-free form with average errors in node location <10(-6)mm and a maximal error <10(-4)mm. This error level, which is similar to what has been achieved with other methods and orders of magnitude lower than the accuracy of current clinical topography systems, justifies the use of the technique as a pre-processing step in ocular numerical simulations.

DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.

Acute Stress Reduces Wound-Induced Activation of Microbicidal Potential of Ex Vivo Isolated Human Monocyte-Derived Macrophages

Background Psychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a) acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM), and (b) that these reductions are modulated by stress hormone release. Methods Fourty-one healthy men (mean age 35±13 years) were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker) before assessing HMDM microbicidal potential. Results Compared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p’s <.05). Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14–44.72). Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001). This effect was blocked by prior incubation with phentolamine. Conclusions Our results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing.

Metamorphosis of human lumbar vertebrae induced by VEPTR growth modulation and stress shielding

INTRODUCTION Distraction-based spinal growth modulation by growing rods or vertical expandable prosthetic titanium ribs (VEPTRs) is the mainstay of instrumented operative strategies to correct early onset spinal deformities. In order to objectify the benefits, it has become common sense to measure the gain in spine height by assessing T1-S1 distance on anteroposterior (AP) radiographs. However, by ignoring growth changes on vertebral levels and by limiting measurement to one plane, valuable data is missed regarding the three-dimensional (3D) effects of growth modulation. This information might be interesting when it comes to final fusion or, even more so, when the protective growing implants are removed and the spine re-exposed to physiologic forces at the end of growth. METHODS The goal of this retrospective radiographic study was to assess the growth modulating impact of year-long, distraction-based VEPTR treatment on the morphology of single vertebral bodies. We digitally measured lumbar vertebral body height (VBH) and upper endplate depth (VBD) at the time of the index procedure and at follow-up in nine patients with rib-to-ileum constructs (G1) spanning an anatomically normal lumbar spine. Nine patients with congenital thoracic scoliosis and VEPTR rib-to-rib constructs, but uninstrumented lumbar spines, served as controls (G2). All had undergone more than eight half-yearly VEPTR expansions. A Wilcoxon signed-rank test was used for statistical comparison of initial and follow-up VBH, VBD and height/depth (H/D) ratio (significance level 0.05). RESULTS The average age was 7.1 years (G1) and 5.2 year (G2, p > 0.05) at initial surgery; the average overall follow-up time was 5.5 years (p = 1). In both groups, VBH increased significantly without a significant intergroup difference. Group 1 did not show significant growth in depth, whereas VBD increased significantly in the control group. As a consequence, the H/D ratio increased significantly in group 1 whereas it remained unchanged in group 2. The growth rate for height in mm/year was 1.4 (group 1) and 1.1 (group 2, p = 0.45), and for depth, it was -0.3 and 1.1 (p < 0.05), respectively. CONCLUSIONS VEPTR growth modulating treatment alters the geometry of vertebral bodies by increasing the H/D ratio. We hypothesize that the implant-related deprivation from axial loads (stress-shielding) impairs anteroposterior growth. The biomechanical consequence of such slender vertebrae when exposed to unprotected loads in case of definitive VEPTR removal at the end of growth is uncertain.

Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress

The yeast peptide-methionine sulfoxide reductase (MsrA) was overexpressed in a Saccharomyces cerevisiae null mutant of msrA by using a high-copy plasmid harboring the msrA gene and its promoter. The resulting strain had about 25-fold higher MsrA activity than its parent strain. When exposed to either hydrogen peroxide, paraquat, or 2,2′-azobis-(2-amidinopropane) dihydrochloride treatment, the MsrA overexpressed strain grew better, had lower free and protein-bound methionine sulfoxide and had a better survival rate under these conditions than did the msrA mutant and its parent strain. Substitution of methionine with methionine sulfoxide in a medium lacking hydrogen peroxide had little effect on the growth pattern, which suggests that the oxidation of free methionine in the growth medium was not the main cause of growth inhibition of the msrA mutant. Ultraviolet A radiation did not result in obvious differences in survival rates among the three strains. An enhanced resistance to hydrogen peroxide treatment was shown in human T lymphocyte cells (Molt-4) that were stably transfected with the bovine msrA and exposed to hydrogen peroxide. The survival rate of the transfected strain was much better than its parent strain when grown in the presence of hydrogen peroxide. These results support the proposition that the msrA gene is involved in the resistance of yeast and mammalian cells to oxidative stress.

Fluid shear stress inhibits TNF-α activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and IL-1 stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. TNF-α and IL-1 regulate gene expression in ECs, in part, by stimulating mitogen-activated protein kinases (MAPK), which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAPK in EC. To test this hypothesis, we determined the effects of flow (shear stress = 12 dynes/cm2) on TNF-α and IL-1-stimulated activity of three MAPK in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 and p38 activity but decreased JNK activity compared with static controls. TNF-α or IL-1 alone activated ERK1/2, p38, and JNK maximally at 15 min in HUVEC. Preexposing HUVEC for 10 min to flow inhibited TNF-α and IL-1 activation of JNK by 46% and 49%, respectively, but had no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, which inhibits flow-mediated ERK1/2 activation, prevented flow from inhibiting cytokine activation of JNK. Phorbol 12-myristate 13-acetate, which strongly activates ERK1/2, also inhibited TNF-α activation of JNK. These findings indicate that fluid shear stress inhibits TNF-α-mediated signaling events in HUVEC via the activation of the ERK1/2 signaling pathway. Inhibition of TNF-α signal transduction represents a mechanism by which steady laminar flow may exert atheroprotective effects on the endothelium.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

Dynamic contrast-enhanced magnetic resonance imaging reveals stress-induced angiogenesis in MCF7 human breast tumors.

The mechanism of contrast enhancement of tumors using magnetic resonance imaging was investigated in MCF7 human breast cancer implanted in nude mice. Dynamic contrast-enhanced images recorded at high spatial resolution were analyzed by an image analysis method based on a physiological model, which included the blood circulation, the tumor, the remaining tissues, and clearance via the kidneys. This analysis enabled us to map in rapidly enhancing regions within the tumor, the capillary permeability factor (capillary permeability times surface area per voxel volume) and the fraction of leakage space. Correlation of these maps with T2-weighted spin echo images, with histopathology, and with immunohistochemical staining of endothelial cells demonstrated the presence of dense permeable microcapillaries in the tumor periphery and in intratumoral regions that surrounded necrotic loci. The high leakage from the intratumoral permeable capillaries indicated an induction of a specific angiogenic process associated with stress conditions that cause necrosis. This induction was augmented in tumors responding to tamoxifen treatment. Determination of the distribution and extent of this stress-induced angiogenic activity by contrast-enhanced MRI might be of diagnostic and of prognostic value.

Transcription of liver X receptor is down-regulated by 15-deoxy-Δ12,14-prostaglandin J2 through oxidative stress in human neutrophils

Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. They play important roles in controlling cholesterol homeostasis and as regulators of inflammatory gene expression and innate immunity, by blunting the induction of classical pro-inflammatory genes. However, opposite data have also been reported on the consequences of LXR activation by oxysterols, resulting in the specific production of potent pro-inflammatory cytokines and reactive oxygen species (ROS). The effect of the inflammatory state on the expression of LXRs has not been studied in human cells, and constitutes the main aim of the present work. Our data show that when human neutrophils are triggered with synthetic ligands, the synthesis of LXRα mRNA became activated together with transcription of the LXR target genes ABCA1, ABCG1 and SREBP1c. An inflammatory mediator, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), hindered T0901317-promoted induction of LXRα mRNA expression together with transcription of its target genes in both neutrophils and human macrophages. This down-regulatory effect was dependent on the release of reactive oxygen species elicited by 15dPGJ2, since it was enhanced by pro-oxidant treatment and reversed by antioxidants, and was also mediated by ERK1/2 activation. Present data also support that the 15dPGJ2-induced serine phosphorylation of the LXRα molecule is mediated by ERK1/2. These results allow to postulate that down-regulation of LXR cellular levels by pro-inflammatory stimuli might be involved in the development of different vascular diseases, such as atherosclerosis.

Investigation of oxidative stress defenses of Neisseria gonorrhoeae by using a human polymorphonuclear leukocyte survival assay

Neisseria gonorrhoeae has well-characterized oxidative stress defense systems that protect against oxidative killing in in vitro assays. In contrast, mutant strains of N. gonorrhoeae lacking oxidative stress defenses are identical to the wild type when tested in an ex vivo survival assay using human polymorphonuclear leukocytes.