Search

Hydrogen peroxide (H2O2) is a major reactive oxygen species (ROS) produced by various cellular sources, especially mitochondria. At high levels, H2O2 causes oxidative stress, leading to cell injury, whereas at low concentrations, this ROS acts as an important second messenger to participate in cellular redox signaling. Detection and measurement of the levels or rates of production of cellular H2O2 are instrumental in studying the biological effects of this major ROS. While a number of assays have been developed over the past decades for detecting and/or quantifying biological H2O2 formation, none has been shown to be perfect. Perhaps there is no perfect assay for sensitively and accurately quantifying H2O2 as well as other ROS in cells, wherein numerous potential reactants are present to interfere with the reliable measurement of the specific ROS. In this context, each assay has its own advantages and intrinsic limitations. This article describes a highly sensitive assay for real-time detection of H2O2 formation in cultured cells and isolated mitochondria. This assay is based on the luminol/horseradish peroxidase-dependent chemiluminescence that is inhibitable by catalase. The article discusses the usefulness and shortcomings of this chemiluminometric assay in detecting biological H2O2 formation induced by beta-lapachone redox cycling with both cells and isolated mitochondria.

Dietary supplementation with omega-3 fatty acids, also known as n-3 fatty acids, has been widely considered cardiovascular protective in the general human population. This widely acclaimed status of omega-3 fatty acids as cardiovascular protective molecules has, however, been questioned by findings from multiple rigorously designed randomized controlled trials, recently reported in the New England Journal of Medicine. Although the anti-inflammatory and other beneficial effects of omega-3 fatty acids are substantiated by research in experimental models as well as findings from observational epidemiological studies, dietary supplementation with omega-3 fatty acids at the typical dosage of 1 g daily does not appear to be an effective strategy for either primary or secondary prevention of cardiovascular disease in humans.

Through the history of modern medicine, bioactive components in natural products have been either employed directly as medicines or used as prototypes for synthetic drug development. This brief Research Highlights paper considers 3H-1,2-dithiole-3-thione (D3T), a member of the 1,2-dithiole-3-thiones—compounds which may naturally occur in cruciferous vegetables. Among 1,2-dithiole-3-thiones, D3T is the most potent member with regard to the capacity of inducing tissue defenses against oxidative and inflammatory stress. Oxidative and inflammatory stress is a major pathophysiological process involved in numerous human disorders, including cancer, cardiovascular diseases, neurodegeneration, and sepsis, to name just a few. This article surveys recent major research findings on D3T as an inducer of tissue antioxidative and anti-inflammatory defenses and as a potential therapeutic modality for sepsis intervention.

MitoSOX-based assays are widely used to detect mitochondrial reactive oxygen species (ROS), especially superoxide. To this end, 5 μM MitoSOX is commonly used. In this ROS Protocols article, we described the flow cytometric protocol involving the use of various concentrations of MitoSOX (1, 2.5, 5 μM) for detecting mitochondrial ROS in control and mitochondrial DNA-deficient (MD) melanoma B16-F10 cells. We also compared the MitoSOX-based flow cytometry with lucigenin-derived chemiluminometry for their ability to reliably detect the relative differences in mitochondrial ROS formation in the control and MD cells. Our results suggested that 1 μM, rather than the commonly used 5 μM, appeared to be the optimal concentration of MitoSOX for detecting mitochondrial ROS via flow cytometry.

Utilization of molecular oxygen by aerobic organisms inevitably results in the formation of a number of oxygen-containing reactive species that are collectively known as reactive oxygen species (ROS). ROS play important roles in both physiology and pathophysiology of aerobic life. The field of 'ROS biology and medicine' deals with the involvement of ROS and related species in contemporary biology and medicine. The purpose of this article is to survey common terms and concepts in ROS biology and medicine. It also introduces the 'ROS paradigm' so as to provide a conceptual framework for understanding the rapidly evolving field of ROS biology and medicine.

Animal models are essential for developing effective drugs for treating human cancer. Examination of the formation of lung surface foci of B16-F10 melanoma cells is a widely used animal model for studying cancer metastasis and drug intervention. This model, however, suffers from several drawbacks, including its non-quantitative nature and inability to yield information on cancer cell load inside the target organ. Here we report the development of a highly sensitive, bioluminescence-based method for quantifying melanoma cell load in mouse lungs following intravenous injection of luciferase-expressing B16-F10 melanoma cells. This method could readily detect as few as 1-10 cells in the samples and enable quantification of cancer cell load before the formation of surface foci in mouse lungs following metastasis of intravenously inoculated B16-F10 melanoma cells. This innovative bioluminometry-based method has important implications for studying anticancer drugs, including naturally occurring redox-active quinones that generate reactive oxygen species to kill cancer cells.

The role of Nrf2, a key regulator of antioxidant and cytoprotective genes, in tumorigenesis remains controversial. Here we showed that Nrf2 deficiency led to increased local tumor growth in mice following subcutaneous injection of B16-F10 melanoma cells, as indicated by increased proportion of animals with locally palpable tumor mass and time-dependent increases in tumor volume at the injection site. In vivo bioluminescence imaging also revealed increased growth of melanoma in Nrf2-null mice as compared with wild-type mice. By using a highly sensitive bioluminometric assay, we further found that Nrf2 deficiency resulted in a remarkable increase in lung metastasis of B16-F10 melanoma cells as compared with wild-type mice. Taken together, the results of this short communication for the first time demonstrated that Nrf2 deficiency promoted melanoma growth and lung metastasis following subcutaneous inoculation of B16-F10 cells in mice.

Detection and measurement of doxorubicin in biological systems, including body fluids, cells, and tissues, are instrumental in understanding the mechanisms of action of this widely used drug in treating cancer as well as in causing adverse effects. In this article, we, for the first time, characterized the use of fluorescence-based techniques, including fluorescence spectrometry, microscopy, and flow cytometry in measuring and/or detecting doxorubicin in biological systems, including cell lysates and cultured intact cells. We showed that doxorubicin has a maximum excitation and emission wavelength of 470 and 560 nm, respectively. The detection sensitivity by fluorescence spectrometry is less than 0.1 μM in buffers and cell lysates. Fluorescence microscopy demonstrated the readily detection of concentration-dependent accumulation of doxorubicin in cultured cells via either green or red fluorescence, but with green fluorescence showing a higher sensitivity of detection. Flow cytometry also revealed sensitive detection of doxorubicin accumulation in cell suspensions in a concentration-dependent manner. The readily and sensitive measurement and detection of doxorubicin by the above three fluorescence-based techniques has important implications in studying the cellular dynamics of doxorubicin in both cancer and normal cells under various experimental conditions.