Killing by Bactericidal Antibiotics Does Not Depend on Reactive Oxygen Species

Recent observations have suggested that classic antibiotics kill bacteria by stimulating the formation of reactive oxygen species (ROS). If true, this notion might guide new strategies to improve antibiotic efficacy. In this study, the model was directly tested. Contrary to the hypothesis, antibiotic treatment did not accelerate the formation of hydrogen peroxide in Escherichia coli and did not elevate intracellular free iron, an essential reactant for the production of lethal damage. Lethality persisted in the absence of oxygen, and DNA repair mutants were not hypersensitive, undermining the idea that toxicity arose from oxidative DNA lesions. We conclude that these antibiotic exposures did not produce ROS and that lethality more likely resulted from the direct inhibition of cell-wall assembly, protein synthesis, and DNA replication.

Albumin-bound fatty acids induce mitochondrial oxidant stress and impair antioxidant responses in proximal tubular cells

Specific activation of human neutrophils by scorpion venom: A flow cytometry assessment

Temperature (T) reduction increases lifespan, but the mechanisms are not understood. Because reactive oxygen species (ROS) contribute to aging, we hypothesized that lowering T might decrease mitochondrial ROS production. We measured respiratory response and ROS production in isolated mitochondria at 32, 35, and 37 °C. Lowering T decreased the rates of resting (state 4) and phosphorylating (state 3) respiration phases. Surprisingly, this respiratory slowdown was associated with an increase of ROS production and hydrogen peroxide release and with elevation of the mitochondrial membrane potential, ΔΨ(m). We also found that at lower T mitochondria produced more carbon-centered lipid radicals, a species known to activate uncoupling proteins. These data indicate that reduced mitochondrial ROS production is not one of the mechanisms mediating lifespan extension at lower T. They suggest instead that increased ROS leakage may mediate mitochondrial responses to hypothermia.

In the present study, the effects of C18 fatty acids with different numbers of double bonds, SA (stearic acid; C18:0), OA (oleic acid; C18:1), LA (linoleic acid; C18:2) and gamma-LNA (gamma-linolenic acid; C18:3), on ROS (reactive oxygen species) production by Jurkat (a human T-lymphocyte-derived cell line) and Raji (a human B-lymphocyte-derived cell line) cells were investigated. ROS production was determined by NBT (Nitro Blue Tetrazolium) reduction (intracellular and extracellular ROS production) and by dihydroethidium oxidation using flow cytometry (intracellular ROS production). The effectiveness on ROS production was gamma-LNA<SA<OA<LA in Jurkat cells and SA<gamma-LNA<OA<LA in Raji cells. LA (found in corn, soya bean and sunflower oils) was more potent than OA (found in olive oil) in stimulating ROS production in both Raji and Jurkat cells. The lower ROS production by OA compared with LA may be one of the benefits of olive oil consumption. As SA and gamma-LNA acids had little or no effect, further studies on the site of ROS production in these cells were carried out with OA and LA only. Activation of NADPH oxidase via PKC (protein kinase C) was found to be the major mechanism of ROS production induced by OA and LA in Jurkat and Raji cells.

Increased production of reactive oxygen species (ROS) in the vascular wall is a characteristic feature of disease states, including atherosclerosis, diabetes and hypertension. ROS, such as superoxide, reduce nitric oxide bioactivity by scavenging and cause oxidation of lipids and target proteins. In addition, recent work has revealed that ROS mediate a wide range of pathological processes in the endothelium, smooth muscle cells, and inflammatory cells.1 ROS are generated by enzyme systems present in cells in the vascular wall, including NAD(P)H oxidase, xanthine oxidase, and nitric oxide synthase. The activities and levels of these enzyme systems are increased in association with vascular disease risk factors2 and in vascular disease states in which oxidative stress is prominent, for example, in diabetes3 and atherosclerosis. See page 1838 The NAD(P)H oxidases appear to be particularly important sources of ROS production in blood vessels,4 where they are constitutively active, producing relatively low levels of ROS under basal conditions, but generating higher levels of oxidants in response to stimuli such as growth factors and cytokines. These factors are consistent with a role for nonphagocytic NAD(P)H oxidases in cellular signaling rather than the high-level burst activity characteristic of the phagocyte NAD(P)H oxidase. The NAD(P)H oxidases are multimeric enzymes composed of plasma membrane associated–proteins as well as cytosolic factors. In the phagocytic-type NAD(P)H oxidase, the plasma membrane–associated proteins gp91phox and p22phox compose the flavocytochrome b558 complex, which forms the catalytic subunit of the oxidase. The cytosolic subunits, including p47phox, p67phox, and the G-protein Rac, provide regulatory function. Azumi and colleagues,5 in this issue of Arteriosclerosis, Thrombosis and Vascular Biology …

This series of debates was initiated for the Journal of Applied Physiology because we believe an important means of searching for truth is through debate where contradictory viewpoints are put forward. This dialectic process whereby a thesis is advanced, then opposed by an antithesis, with a

Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice

Polyunsaturated fatty acids (PUFAs) can affect various functions of the immune system including inflammatory responses. An oxidative burst of phagocytes accompanied by reactive oxygen species (ROS) and reactive nitrogen species (RNS) formation is one of the phagocyte functions that could be modulated by PUFAs. To investigate the effects of omega-3 (alpha-linolenic, docosahexaenoic, eicosapentaenoic) and omega-6 (arachidonic, linoleic) PUFAs on lipopolysaccharide (LPS)-stimulated ROS and RNS production by the murine macrophage cell line RAW 264.7. Murine peritoneal macrophages RAW 264.7 were stimulated with LPS (0.1 microg/ml) and treated with 0.1-100 microM omega-3 or omega-6 PUFAs for either 8 (ROS production) or 20 h (RNS production). The cytotoxicity of PUFAs was evaluated by an ATP (adenosine triphosphate) test after both 8 and 20 h of treatment with PUFAs. Changes in ROS production by LPS-treated macrophages subsequently activated with phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP) were determined by luminol-enhanced chemiluminescence, whilst the production of RNS was determined as the concentration of nitrites in cell supernatants (Griess reaction). Changes in inducible nitric oxide synthase (iNOS) expression were evaluated by Western blot analysis. The antioxidant properties of PUFAs were tested by TRAP (total peroxyl radical-trapping antioxidant parameter) assay. All PUFAs in 100 microM concentration except eicosapentaenoic acid decreased ROS production. The effect was most significant when docosahexaenoic acid was used. Arachidonic acid decreased PMA-activated ROS production even in 1 and 10 microM concentrations. On the other hand, 10 and 100 microM eicosapentaenoic acid potentiated ROS production. As concerns RNS production, all the fatty acids that were tested in a concentration of 100 microM decreased iNOS expression and nitrite accumulation. Fatty acids had no significant effect on the viability and proliferation of RAW 264.7 cells. The TRAP assay confirmed that none of the tested PUFAs exerted any significant antioxidant properties. High concentrations of PUFAs of both omega-3 and omega-6 groups can inhibit ROS and RNS formation by stimulated macrophages. The expression of iNOS can also be inhibited. This effect, together with the absence of antioxidant activity and cytotoxic properties, indicates that PUFAs can participate in the regulation of enzymes responsible for reactive species production.

Non-ablative pulsed focused ultrasound (pFUS) has shown promise as an immunomodulatory anti-cancer therapeutic. While largely nondestructive to tissues compared ablative ultrasound, pFUS can generate anti-tumor molecular microenvironments and enhance migration of anti-tumor immune cells into B16 and 4T1 flank tumors in immune-competent mice. pFUS also increases the number of dsDNA breaks in nuclear DNA of tumor cells by TUNEL reactions. DNA damage can elicit immune responses by causing cells to express ligands for immune cells and thus may be a critical component of generating observed immune responses in flank tumors following pFUS treatment. Here we investigate potential phyisological mechanisms underlying pFUS-associated DNA damage. B16 melanoma, 4T1 or MBA-MD-231 breast tumors, or C6 glioma cells were cultured and treated with pFUS in vitro (1 MHz, 6 MPa peak negative pressure, 10% duty cycle, 100 pulses). Cells were analyzed for DNA damage by TUNEL, reactive oxygen species (ROS) production by DCFDA, and intracellular Ca2+ by fluo-4. pFUS increased TUNEL reactivity and ROS concentrations in all cell types 2-6 hr after pFUS. Increased TUNEL reactivity was not observed when cells were incubated with the ROS scavenger Trolox (need concentration) following pFUS. pFUS generated Ca2+ transients in all cell types during pFUS treatment. Ca2+ dysregulation can increase cellular ROS production and when cytosolic Ca2+ transients were blocked by BAPTA-AM, both ROS production and TUNEL reactivity was reduced in all cell types. Further investigating mechanisms of Ca2+ transients, application of verapamil (10 uM) during pFUS to inhibit voltage-gated Ca2+ channels (VGCC) also suppressed Ca2+ transients, ROS production, and TUNEL reactivity. Lastly, the magnitudes of Ca2+ transients, ROS production, and TUNEL reactivity induced by pFUS were nonetheless different for different cell types (C6 glioma exhibited the smallest changes after pFUS, while MBA-MD-231 exhibited the largest). This suggested different sensitivities for each cell type to pFUS-induced DNA damage. As Ca2+ homeostasis can be disrupted in tumor cells, we measured resting cytosolic Ca2+ concentrations in each cell type following plasma membrane disruption with digitonin (20 uM). Resting cytosolic Ca2+ concentrations inversely correlated with increased magnitudes of ROS production or TUNEL reactivity following pFUS treatment. In conclusion, pFUS causes DNA damage in tumor cells by initiating cytosolic Ca2+ transients, in part, through VGCC that increase cellular ROS production and lead to dsDNA breaks. The effectiveness of pFUS to stimulate this physiological pathway is related to resting cytosolic Ca2+ concentrations, which different tumor cells could display varying levels of disrupted homeostasis and, thus differing magnitudes of response to pFUS. Future directions will assess potential involvement of mitochondrial dynamics in Ca2+-induced ROS production and in vivo experiments to further understand these dynamics in solid tumor models. Citation Format: Robert B. Rosenblatt, Joseph A. Frank, Scott R. Burks. Pulsed focused ultrasound induces cytosolic calcium transients that increase free radical production and dsDNA breaks in tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3577.

UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

reactive oxygen species (ROS), such as superoxide or hydrogen peroxide, are usually thought of as damaging agents that nonspecifically disrupt proteins, lipids, and nucleic acids ([1][1]). However, there is now considerable evidence that ROS also act as signaling agents in a range of biological

Voltage gated proton channels modulate mitochondrial reactive oxygen species production by complex I in renal medullary thick ascending limb

Cancer treatments in general share various detrimental effects in common, especially upregulation of cardiovascular risk factors. Therefore, the science of onco-cardiology should not be restricted in scope to the side effects of each specific cancer drug. In particular, premature aging induced by cancer treatment may contribute to the chronic health problems of cancer survivors. About 1 660 000 people, including more than 12 000 children below the age of 18 years, are newly diagnosed with a malignancy in the United States every year.1 The American Cancer Society reported that in 2016 there are 15.5 million cancer survivors in the United States (http://www.cancer.org/cancer/news/news/report-number-of-cancer-survivors-continues-to-grow). At present, the 5-year survival rate of patients treated for cancer is 67%. Seventy-five percent of children in whom cancer is diagnosed today will live for at least 10 years; 20% will survive for longer than 35 years.1 Although these numbers are impressive compared with those from decades ago, further improvement of cancer survivors’ life span as well as quality of life and functional status is still necessary. Approximately 75% of cancer survivors have some form of chronic health problem. Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality in this population, particularly after recurrent or second malignancy. The risk of CVD in cancer survivors is 8× higher than that of the general population. The relative risks of coronary artery disease and heart failure in cancer survivors are 10× and 15× higher, respectively, than their siblings without cancer.1 Cancer treatments, including chemotherapy and radiation, can lead to both short- and long-term cardiovascular complications. Evidence of subclinical cardiac and vascular damage was observed in more than 50% of survivors 5 to 10 years after chemotherapy.1 Onco-cardiology is a medical subspecialty concerned with the diagnosis and treatment of CVDs and organ failure mediated by microcirculatory or macrocirculatory …

Circulating levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) play an important role in the development and progression of atherosclerosis. We hypothesized that the inflammatory marker C-reactive protein (CRP) might stimulate sLOX-1 release by activating tumor necrosis factor-α converting enzyme (TACE). Macrophages differentiated from THP-1 cells were stimulated with TNF-α and further treated with CRP in the absence or presence of specific inhibitors or small interfering RNA (siRNA). Our results showed that CRP increased sLOX-1 release from activated macrophages in a dose-dependent manner and that these effects were regulated by Fc γ receptor II (FcγRII)-mediated p47(phox) phosphorylation, reactive oxygen species (ROS) production, and TACE activation. CRP also enhanced sLOX-1 release from macrophages derived from peripheral blood mononuclear cells (PBMC) of patients with acute coronary syndrome (ACS). Pretreatment with antibody against FcγRII or with CD32 siRNA, p47(phox) siRNA, apocynin, N-acetylcysteine, tumor necrosis factor-α protease inhibitor 1 (TAPI-1) or TACE siRNA attenuated sLOX-1 release induced by CRP. CRP also elevated serum sLOX-1 levels in a rabbit model of atherosclerosis. Thus, CRP might stimulate sLOX-1 release, and the underlying mechanisms possibly involved FcγRII-mediated p47(phox) phosphorylation, ROS production, and TACE activation.