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Fluorescence probes used for detection of reactive oxygen species

A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases

Superoxide dismutases (SOD) are essential enzymes that eliminate superoxide radical (O2-) and thus protect cells from damage induced by free radicals. The active O2- production and low SOD activity in cancer cells may render the malignant cells highly dependent on SOD for survival and sensitive to inhibition of SOD. Here we report that certain oestrogen derivatives selectively kill human leukaemia cells but not normal lymphocytes. Using complementary DNA microarray and biochemical approaches, we identify SOD as a target of this drug action and show that chemical modifications at the 2-carbon (2-OH, 2-OCH3) of the derivatives are essential for SOD inhibition and for apoptosis induction. Inhibition of SOD causes accumulation of cellular O2- and leads to free-radical-mediated damage to mitochondrial membranes, the release of cytochrome c from mitochondria and apoptosis of the cancer cells. Our results indicate that targeting SOD may be a promising approach to the selective killing of cancer cells, and that mechanism-based combinations of SOD inhibitors with free-radical-producing agents may have clinical applications.

Superoxide dismutase as a target for the selective killing of cancer cells

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species This notion has been supported by many groups but has been challenged recently Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2 We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality

https://www.pnas.org/content/pnas/111/20/E2100.full.pdf

Antibiotics induce redox-related physiological alterations as part of their lethality

Mitochondria are widely believed to be the source of reactive oxygen species (ROS) in a number of neurodegenerative disease states. However, conditions associated with neuronal injury are accompanied by other alterations in mitochondrial physiology, including profound changes in the mitochondrial membrane potential DeltaPsi(m). In this study we have investigated the effects of DeltaPsi(m) on ROS production by rat brain mitochondria using the fluorescent peroxidase substrates scopoletin and Amplex red. The highest rates of mitochondrial ROS generation were observed while mitochondria were respiring on the complex II substrate succinate. Under this condition, the majority of the ROS signal was derived from reverse electron transport to complex I, because it was inhibited by rotenone. This mode of ROS generation is very sensitive to depolarization of DeltaPsi(m), and even the depolarization associated with ATP generation was sufficient to inhibit ROS production. Mitochondria respiring on the complex I substrates, glutamate and malate, produce very little ROS until complex I is inhibited with rotenone, which is also consistent with complex I being the major site of ROS generation. This mode of oxidant production is insensitive to changes in DeltaPsi(m). With both substrates, ubiquinone-derived ROS can be detected, but they represent a more minor component of the overall oxidant signal. These studies demonstrate that rat brain mitochondria can be effective producers of ROS. However, the optimal conditions for ROS generation require either a hyperpolarized membrane potential or a substantial level of complex I inhibition.

Edward S Schulman

Papers

A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative.