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Previously, we reported that 3H-1,2-dithiole-3-thione (D3T), an Nrf2 activator, acted as a potential chemoprotectant against lipopolysaccharide (LPS)-induced mortality in mice. In view of the critical involvement of macrophages in the pathogenesis of LPS-induced endotoxemia, in the present study, we investigated the protective effects of D3T on LPS-induced proinflammatory responses in cultured murine RAW 264.7 macrophage cell line and primary peritoneal macrophages and the potential involvement of antioxidant induction, NF-κB, and Nrf2. We showed that treatment with D3T resulted in increased levels of a series of antioxidants in RAW 264.7 cells in a concentration-dependent manner. These included the reduced form of glutathione, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and NADPH:quinone oxidoreductase 1. Catalase was also potently induced by D3T which, however, did not show a concentration dependency. Concurrent with the ability to induce the above cellular antioxidants, D3T pretreatment of RAW 264.7 cells also led to a concentration-dependent suppression of LPS-induced interleukin-1beta (IL-1β) production and nitric oxide release. LPS-stimulated tumor necrosis factor-alpha (TNF-α) production was also suppressed by D3T, but to a much lesser extent. Using NF-κB reporter gene-expressing RAW 264.7 cells, we further showed that D3T pretreatment also suppressed LPS-induced NF-κB activation. To investigate the potential involvement of Nrf2, a chief regulator of cellular antioxidant genes, we used peritoneal macrophages isolated from Nrf2

Cerium oxide nanoparticles, also known as nanoceria, possess antioxidative and anti-inflammatory activities in animal models of inflammatory disorders, such as sepsis. However, it remains unclear how nanoceria affect cellular superoxide fluxes in macrophages, a critical type of cells involved in inflammatory disorders. Using human ML-1 cell-derived macrophages, we showed that nanoceria at 1–100 μg/ml potently reduced superoxide flux from the mitochondrial electron transport chain (METC) in a concentration-dependent manner. The inhibitory effects of nanoceria were also shown in succinate-driven mitochondria isolated from the macrophages. Furthermore, nanoceria markedly mitigated the total intracellular superoxide flux in the macrophages. These data suggest that nanoceria could readily cross the plasma membrane and enter the mitochondrial compartment, reducing intracellular superoxide fluxes in unstimulated macrophages. In macrophages undergoing respiratory burst, nanoceria also strongly reduced superoxide flux from the activated macrophage plasma membrane NADPH oxidase (NOX) in a concentration-dependent manner. Token together, the results of the present study demonstrate that nanoceria can effectively diminish superoxide fluxes from both METC and NOX in human macrophages, which may have important implications for nanoceria-mediated protection against inflammatory disease processes.

Vitamin C, also known as ascorbic acid or ascorbate, is a water-soluble vitamin synthesized in plants as well as in animals except humans and several other animal species. Humans obtain vitamin C from dietary sources and via vitamin supplementation. Vitamin C possesses important biological functions, including serving as a cofactor for many enzymes, acting as an antioxidant and anti-inflammatory compound, and participating in regulating stem cell biology and epigenetics. The multifunctional nature of vitamin C contributes to its essentialness in maintaining and safeguarding physiological homeostasis, especially regulation of immunity and inflammatory responses. In this context, vitamin C has been investigated for its efficacy in treating diverse inflammatory disorders, including sepsis, one of the major causes of death globally and for which currently there is no cure. Accordingly, this Mini-Review surveys recent major research findings on the effectiveness of vitamin C and the underling molecular mechanisms in sepsis intervention in both experimental animal models and randomized controlled trials. To set a stage for discussing the effects and mechanisms of vitamin C in sepsis intervention, this Mini-Review begins with an overview of vitamin C redox biochemistry and its multifunctional properties.

Epidemiological studies have exhibited a strong correlation between exposure to air pollution and deaths due to vascular diseases such as atherosclerosis. Benzo-a-pyrene-1,6-quinone (BP-1,6-Q) is one of the components of air pollution. This study was to examine the role of GSH in BP-1,6-Q mediated cytotoxicity in human EA.hy96 endothelial cells and demonstrated that induction of cellular glutathione by a potent triterpenoid, CDDO-Im (1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole), protects cells against BP-1,6-Q induced protein and lipid damage. Incubation of EA.hy926 endothelial cells with BP-1,6-Q caused a significant increase in dose-dependent cytotoxicity as measured by LDH release assay and both apoptotic and necrotic cell deaths as measured by flow cytometric analysis. Incubation of EA.hy926 endothelial cells with BP-1,6-Q also caused a significant decrease in cellular GSH levels. The diminishment of cellular GSH by buthionine sulfoximine (BSO) potentiated BP-1,6-Q-induced toxicity significantly suggesting a critical involvement of GSH in BP-1,6-Q induced cellular toxicity. GSH-induction by CDDO-Im significantly protects cells against BP-1,6-Q induced protein and lipid damage as measured by protein carbonyl (PC) assay and thiobarbituric acid reactive substances (TBARS) assay, respectively. However, the co-treatment of cells with CDDO-Im and BSO reversed the cytoprotective effect of CDDO-Im on BP-1,6-Q-mediated lipid peroxidation and protein oxidation. These results suggest that induction of GSH by CDDO-Im might be the important cellular defense against BP-1,6-Q induced protein and lipid damage. These findings would contribute to better understand the action of BP-1,6-Q and may help to develop novel therapies to protect against BP-1,6-Q-induced atherogenesis.

Peroxiredoxin (Prx) refers to a family of thiol-dependent peroxidases that decompose hydrogen peroxide, lipid hydroperoxides, as well as peroxynitrite, and protect against oxidative and inflammatory stress. There are six mammalian Prx isozymes (Prx1-6), classified as typical 2-Cys, atypical 2-Cys, or 1-Cys Prxs based on the mechanism and the number of cysteine residues involved during catalysis. In addition to their well-established peroxide-scavenging activity, some Prxs also participate in the regulation of various cell signaling pathways. Extensive animal studies employing primarily gene knockout models provide substantial evidence supporting a critical protective role of Prxs in various disease processes involving oxidative and inflammatory stress. This review surveys recent research findings, published primarily in influential journals, on the involvement of various Prx isozymes in protecting against cardiovascular injury and related disorders, including diabetes, metabolic syndromes, and sepsis, whose pathophysiology all intimately involves oxidative stress and inflammation.

Strong epidemiological evidence supports the association between increased air pollution and the risk of developing atherosclerotic cardiovascular diseases (CVDs). However, the mechanism remains unclear. As an environmental air pollutant and benzo-a-pyrene (BP) metabolite, BP-1,6-quinone (BP-1,6-Q) is present in the particulate phase of air pollution. This study was undertaken to examine the redox activity of BP-1,6-Q and mechanisms associated with it using EA.hy926 endothelial cells. BP-1,6-Q at 0.01–1 μM significantly stimulated the production of reactive oxygen species (ROS)·in intact cells and isolated mitochondria. Furthermore, BP-1,6-Q-induced ROS was altered by mitochondrial electron transport chain (METC) inhibitors of complex I (rotenone) and complex III (antimycin A), denoting the involvement of mitochondrial electron transport chain (METC) in BP-1,6-Q mediated ROS production. In METC deficient cells, interestingly, BP-1,6-Q-mediated ROS production was enhanced, suggesting that overproduction of ROS by BP-1,6-Q is not only produced from mitochondria but can also be from the cell outside of mitochondria (extramitochondrial). BP-1,6-Q also triggered endothelial-monocyte interaction and stimulated expression of vascular adhesion molecule-1 (VCAM-1). In conclusion, these results demonstrate that BP-1,6-Q can generate ROS within both mitochondria and outside of mitochondria, resulting in stimulation of adhesion of monocytes to endothelial cells, a key event in the pathogenesis of atherosclerosis.

Previously, we reported that 3H-1,2-dithiole-3-thione (D3T), an Nrf2 activator, acted as a potential chemoprotectant against lipopolysaccharide (LPS)-induced mortality in mice. In view of the critical involvement of macrophages in the pathogenesis of LPS-induced endotoxemia, in the present study, we investigated the protective effects of D3T on LPS-induced proinflammatory responses in cultured murine RAW 264.7 macrophage cell line and primary peritoneal macrophages and the potential involvement of antioxidant induction, NF-κB, and Nrf2. We showed that treatment with D3T resulted in increased levels of a series of antioxidants in RAW 264.7 cells in a concentration-dependent manner. These included the reduced form of glutathione (GSH), glutathione peroxidase, glutathione reductase, glutathione S-transferase, and NADPH:quinone oxidoreductase 1. Catalase was also potently induced by D3T which, however, did not show a concentration dependency. Concurrent with the ability to induce the above cellular antioxidants, D3T pretreatment of RAW 264.7 cells also led to a concentration-dependent suppression of LPS-induced interleukin-1beta (IL-1β) production and nitric oxide release. LPS-stimulated tumor necrosis factor-alpha (TNF-α) production was also suppressed by D3T, but to a much lesser extent. Using NF-κB reporter gene-expressing RAW 264.7 cells, we further showed that D3T pretreatment also suppressed LPS-induced NF-κB activation. To investigate the potential involvement of Nrf2, a chief regulator of cellular antioxidant genes, we used peritoneal macrophages isolated from Nrf2+/+ and Nrf2−/− mice. Our results showed that D3T pretreatment suppressed LPS-induced proinflammatory responses in Nrf2+/+ macrophages, and this inhibitory effect of D3T was completely lost in Nrf2−/− macrophages. Collectively, the results of the present study demonstrated that D3T acted as a potent suppressor of LPS-induced proinflammatory responses in macrophages. Antioxidant induction, NF-κB suppression, and Nrf2 activation appeared to contribute to the anti-proinflammatory activity of D3T in macrophages.

Numerous studies conducted in the past have reported deaths in the human population due to cardiovascular diseases (CVD) on exposure to air particulate matter (APM). BP-1,6-quinone (BP-1,6-Q) is one of the significant components of APM. However, the mechanism(s) by which it can exert its toxicity in endothelial cells is not yet completely understood. NAD(P)H: quinone oxidoreductase-1 (NQO1) is expressed highly in myocardium and vasculature tissues of the heart and plays a vital role in maintaining vascular homeostasis. This study, demonstrated that BP-1,6-Q diminishes NQO1 enzyme activity in a dose-dependent manner in human EA.hy926 endothelial cells. The decrease in the NQO1 enzyme causes potentiation in BP-1,6-Q-mediated toxicity in EA.hy926 endothelial cells. The enhancement of NQO1 in endothelial cells showed cytoprotection against BP-1,6-Q-induced cellular toxicity, lipid, and protein damage suggesting an essential role of NQO1 in cytoprotection against BP-1,6-Q toxicity. Using various biochemical assays and genetic approaches, results from this study further demonstrated that NQO1 also plays a crucial role in BP-1,6-Q-induced production of reactive oxygen species (ROS). These findings will contribute to elucidating BP-1,6-Q mediated toxicity and its role in the development of atherosclerosis.

Regulation of antioxidant gene expression is essential for controlling oxidative stress and maintaining physiological homeostasis. In this context, the nuclear factor E2-related factor 2 (Nrf2) has been identified as the chief regulator of the transcription of diverse antioxidant genes as well as many other cytoprotective genes. Nrf2 activity is subjected to the regulation at various levels including protein stability, transcription, and post-transcription. Among the various regulatory pathways, the Keap1-Cul3-Rbx1 axis is the most prominent regulator of Nrf2 activity. Being a tightly controlled transcriptional activator of antioxidant genes, Nrf2 signaling is intimately involved in health and disease. While Nrf2 is a protector against oxidative and electrophilic tissue injury, persistent activation of Nrf2 signaling may also contribute to disease pathophysiology, such as cancer progression.

While it is well known that bacterial infection is the predominant cause of sepsis, the molecular pathophysiology of this clinical syndrome remains ill-defined. In this Research Highlights article, we discuss the recent research findings regarding a protective role for glutathione peroxidase-4 (GPx4) in bacterial infection and polymicrobial sepsis via modulating ferroptosis and pyroptosis, two novel modes of regulated cell death. It is suggested that GPx4, being a requisite gateway to both ferroptosis and pyroptosis, may serve as a critical molecular target for developing effective drugs for controlling infection and sepsis.

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.

In vivo imaging of cancer cell growth and invasion is instrumental in studying cancer cell behavior and in developing effective anticancer agents. In this ROS Protocols article, we report the experimental protocol and steps involving the implantation of luciferase-expressing Lewis lung carcinoma (LLC) cells in normal syngeneic C57BL/6 mice. Using the Berthold NightOwl LB981 in vivo imaging system, we observe the time-dependent growth and invasion of the lung cancer cells following subcutaneous injection of luciferase-expressing LLC cells. The three-dimensional image and counts of photon emission of the tumor mass are obtained to estimate the relative size of the tumor. Ex vivo imaging of the isolated lungs supplemented with D-luciferin and adenosine triphosphate (ATP) is obtained to determine lung metastasis of the LLC cells. The LLC cell load in entire mouse lungs is further determined by quantitative bioluminometry with a concurrently run standard curve of the number of LLC cells versus bioluminescence intensity. This in vivo imaging system in live mice, in combination with ex vivo imaging of isolated lungs as well as quantitative bioluminometry of target tissues, may provide important information on the in vivo cancer cell dynamics in immunocompetent syngeneic C57BL/6 mice and offer a valuable tool for studying experimental anticancer agents, including redox-modulating compounds, which are promising anticancer modalities.

Early work in the 1970s by Linus Pauling, a twice-honored Nobel laureate, led to his proposal of using high-dose vitamin C to treat cancer patients. Over the past several decades, a number of studies in animal models as well as several small-scale clinical studies have provided substantial support of Linus Pauling's early proposal. Production of reactive oxygen species (ROS) via oxidation of vitamin C appears to be a major underlying event, leading to the selective killing of cancer cells. However, it remains unclear how vitamin C selectively kills cancer cells while sparing normal cells and what the molecular targets of high-dose vitamin C are. In a recent article published in Science (2015 December 11; 350(6266):1391-6. doi: 10.1126/science.aaa5004), Yun et al. reported that vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) through an ROS-dependent mechanism. This work by Yun et al. along with other findings advances our current understanding of the molecular basis of high-dose vitamin C-mediated cancer cell killing, which will likely give an impetus to the continued research efforts aiming to further decipher the novel biochemistry of vitamin C and its unique role in cancer therapy.

Doxorubicin (also called Adriamycin) is effective in treating a wide range of human cancers and currently considered as one of the most important drugs in cancer chemotherapeutics. The clinical use of doxorubicin is, however, associated with dosage-dependent cardiotoxicity and development of heart failure, which diminish the therapeutic index of this widely used anticancer drug. This article first surveys key research findings on doxorubicin redox biology that may impact its cardiotoxicity as well as anticancer activity. It then discusses emerging concepts, especially the topoisomerase IIb-p53-mitochondrion axis that may lead to the development of mechanistically based novel strategies to protect against cardiotoxicity and enhance the effectiveness of doxorubicin therapy.

Molecular dioxygen (O2) is an essential element of aerobic life, yet incomplete reduction or excitation of O2 during aerobic metabolisms generates diverse oxygen-containing reactive species, commonly known as reactive oxygen species (ROS). On the one hand, ROS pose a serious threat to aerobic organisms via inducing oxidative damage to cellular constituents. On the other hand, these reactive species, when their generation is under homeostatic control, also play important physiological roles (e.g., constituting an important component of immunity and participating in redox signaling). This article defines oxygen and the key facts about oxygen, and discusses the relationship between oxygen and the emergence of early animals on Earth. The article then describes the discovery of oxygen by three historical figures and examines the birth of the concepts of oxygen toxicity and the underlying free radical mechanisms. The article ends with a brief introduction to the emerging field of ROS-mediated redox signaling and physiological responses.

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione- S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity.

Objective To investigate the potential compensatory effects of hOGG1 and hMTH1 in the repair of oxidative DNA damage. Methods The hOGG1 and hMTH1 gene knockdown human embryonic pulmonary fibroblast cell lines were established by lentivirus-mediated RNA interference. The messenger RNA (mRNA) levels of hOGG1 and hM1TH1 were analyzed by the real-time polymerase chain reaction, and 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) formation was analyzed in a high-performance liquid chromatography-electrochemical detection system. Results The hOGG1 and hMTH1 knockdown cells were obtained through blasticidin selection. After transfection of hOGG1 and hMTH1 small interfering RNA, the expression levels of the mRNA of hOGG1 and hMTH1 genes were decreased by 97.2% and 96.2%, respectively. The cells then were exposed to 100 μmol/L of hydrogen peroxide (H2O2) for 12 h to induce oxidative DNA damage. After H2O2 exposure, hMTH1 mRNA levels were increased by 25% in hOGG1 gene knockdown cells, whereas hOGG1 mRNA levels were increased by 52% in hMTH1 gene knockdown cells. Following the treatment with H2O2, the 8-oxo-dG levels in the DNA of hOGG1 gene knockdown cells were 3.1-fold higher than those in untreated HFL cells, and 1.67-fold higher than those in H2O2-treated wild-type cells. The 8-oxo-dG levels in hMTH1 gene knockdown cells were 2.3-fold higher than those in untreated human embryonic pulmonary fibroblast cells, but did not differ significantly from those in H2O2-treated wild-type cells. Conclusion Our data suggested that hOGG1 could compensate for hMTH1 during oxidative DNA damage caused by H2O2, whereas hMTH1 could not compensate sufficiently for hOGG1 during the process.

To evaluate how exposure to deep-frying oils, repeated frying oil (RFO) and restaurant waste oil (RWO) affects emission of polycyclic aromatic hydrocarbons (PAHs) and oxidative stress in male restaurant workers.The study participants included 236 male restaurant workers in 12 restaurants in Shenzhen. Airborne particulate PAHs were measured over 12h on each of two consecutive work days. Urinary 1-hydroxypyrene (1-OHP) measurements were used to indicate cooking oil fumes (COF) exposure, and urinary malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were adopted as oxidative stress markers.The production and emission rates of ultrafine particles (UFPs) and PM2.5 were higher in the exposed groups than in the control group. The concentrations of summed PAHs were in the order of RFO-frying groupRWO-frying groupdeep-frying groupunexposed control group. Urinary 1-OHP was found to be a significant predictor of elevated urinary MDA and 8-OHdG concentrations (all, P0.05). UFPs were a significant predictor of elevated urinary 8-OHdG for restaurant workers (P0.05). The RFO- and RWO-frying groups had higher mean urinary concentrations of 1-OHP, MDA and 8-OHdG than the control group (P0.05). RFO exposure was found to be a significant risk factor for elevated urinary 8-OHdG and RWO exposure was found to be a significant risk factor for elevated urinary MDA (both, P0.001).Concentrations of urinary 1-OHP, MDA and 8-OHdG reflect occupational exposure to PAHs from COFs and oxidative stress in restaurants workers. Exposure to RFO may cause increased oxidative DNA damage, and exposure to RWO might cause increased lipid peroxidation.

Inflammatory bowel disease (IBD) comprises primarily the chronic relapsing inflammatory disorders, Crohn's disease and ulcerative colitis, with the former affecting any part of the gastrointestinal tract and the latter mainly afflicting the colon. The precise etiology of IBD remains unclear, and it is thought that interactions among various factors, including genetic factors, the host immune system and environmental factors, cause disruption of intestinal homeostasis, leading to dysregulated inflammatory responses of the gut. As inflammation is intimately related to formation of reactive intermediates, including reactive oxygen and nitrogen species (ROS/RNS), oxidative stress has been proposed as a mechanism underlying the pathophysiology of IBD. This review is intended to summarize succinctly recent new experimental and clinical evidence supporting oxidative stress as a pathophysiological component of IBD and point to the potential of using antioxidant compounds as promising therapeutic modalities of human IBD. The sources of ROS/RNS and the redox signaling mechanism underlying oxidative stress and inflammation in IBD are discussed to provide insight into the molecular basis of oxidative stress as a pathophysiological factor in IBD.

Alcoholic liver disease (ALD) is a major cause of morbidity and mortality worldwide. The spectrum of ALD ranges from fatty liver to alcoholic hepatitis and cirrhosis, which may eventually lead to the development of hepatocellular carcinoma. In developed countries as well as many developing nations, ALD is a major cause of end-stage liver disease that requires liver transplantation. The most effective therapy for ALD is alcohol abstinence; however, for individuals with severe forms of ALD and those in whom alcohol abstinence is not achievable, targeted therapies are absolutely necessary. In this context, advances of our understanding of the pathophysiology of ALD over the past two decades have contributed to the development of therapeutic modalities (e.g., pentoxifylline and corticosteroids) for the disease though the efficacy of the available treatments remains limited. This article is intended to succinctly review the recent experimental and clinical findings regarding the involvement of oxidative stress and redox signaling in the pathophysiology of ALD and the development of mechanistically-based antioxidant modalities to target the oxidative stress and redox signaling mechanisms of ALD. The biochemical and cellular sources of reactive oxygen and nitrogen species (ROS/RNS) and the dysregulated redox signaling pathways associated with alcohol consumption are particularly discussed to provide insight into the molecular basis of hepatic cell dysfunction and destruction as well as tissue remodeling underlying ALD.

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.

The nuclear factor E2-related factor 2 (Nrf2) is known as the chief regulator of cellular antioxidant defenses as well as a suppressor of inflammation. Macrophages act as major players in inflammatory responses. Because oxidative stress and inflammation are two intertwined processes, the anti-inflammatory activity of Nrf2 signaling is believed to result from its upregulation of cellular antioxidant defenses via the antioxidant response element-driven transcription. In a recent article published in Nature Communications (May 23, 2016; doi: 10.1038/ncomms11624), Kobayashi et al. reported that Nrf2 suppresses transcriptional upregulation of pro-inflammatory cytokines independent of its role in regulating cellular antioxidants and redox status. This study by Kobayashi et al. provides novel insights into the molecular basis of Nrf2 acting as a suppressor of inflammation.

Over the past 40 years, there has been a tremendous amount of research on the dual role of free radicals as both toxic and beneficial species. Free radicals are produced as by-products of normal cellular metabolism or generated by chemicals in our external environment (e.g., cigarette smoke, air and water pollution, exposure to sunlight, gamma-irradiation, and certain chemotherapeutic drugs). At low to intermediate concentrations, free radicals exert their effects through regulation of cell signaling cascades. At high concentrations, they damage all macromolecules, inducing DNA damage, lipid peroxidation, protein modification, and eventually cell death. Free radicals have been implicated in the pathogenesis of a number of conditions, such as aging, atherosclerosis, ischemic heart disease, cancer, and Alzheimer’s disease. Aerobic organisms have evolved sophisticated antioxidant systems to protect themselves from cellular damage and death caused by free radicals. The ability to estimate chemical biomarkers of free radical damage in body fluids and tissues is an important step in understanding the mechanisms contributing to disease processes. This review of a large amount of research studies discusses formation of free radicals in normal cells, their basic properties, toxic effects on cellular processes, potential beneficial role in signaling and phagocytosis, and the part the oxidative stress plays in some major disease processes.

Acrolein is a ubiquitous unsaturated aldehyde has been implicated in the pathogenesis of various neurological disorders. However, limited study has been conducted into potential therapeutic protection and underlying mechanism against acrolein-induced cytotoxicity via upregulation of cellular aldehyde-detoxification defenses. In this study we have utilized RA-differentiated human SH-SY5Y cells and primary human astrocytes to investigate the induction of glutathione (GSH) by the synthetic triterpenoid 2-cyano-3,12-dixooleana-1,9-dien-28-imidazolide (CDDO-Im) and the protective effects CDDO-Im-mediated antioxidant defenses on acrolein toxicity. Acrolein exposure to RA-differentiated SH-SY5Y cells resulted in a significant time dependent depletion of cellular GSH preceding a reduction in cell viability and LDH release. Further, we demonstrated the predominance of cellular GSH in protection against acrolein-induced cytotoxicity. Buthionine sulfoximine (BSO) at 25μM dramatically depleted GSH and significantly potentiated acrolein-induced cytotoxicity. Pretreatment of the cells with 100nM CDDO-Im afforded a dramatic protection against acrolein-induced cytotoxicity. Pretreatment of BSO and CDDO was found to prevent the CDDO-Im-mediated GSH induction and partially reversed the cytoprotective effects of CDDO-Im against acrolein cytotoxicity. Overall, this study represents for the first time the CDDO-Im mediated upregulation of GSH is a predominant mechanism against acrolein-induced neurotoxicity.

Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ~ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells.

Paraoxonase (PON) refers to a family of three enzymes, namely PON1, PON2, and PON3. PON1 and PON3 are found in circulation bound to high-density lipoprotein, whereas PON2 is an intracellular protein. PON1 was first discovered as an enzyme to hydrolyze the organophosphate pesticide paraoxon, an activity that both PON2 and PON3 lack. All three PON enzymes are able to degrade oxidized lipids and protect against oxidative stress. PON enzymes also act to suppress inflammation. Animal studies show a critical role for PON enzymes, especially PON1 in protecting against cardiovascular diseases and related disorders, including diabetes and metabolic syndrome. In line with the findings in experimental animals, accumulating evidence from clinical research also indicates that PON enzymes function as potential protectors in human cardiovascular diseases and related disorders. Identification of PON enzymes as important players in cardiovascular health will facilitate the development of novel preventive and therapeutic modalities targeting PON enzymes to combat cardiovascular diseases and related disorders, which collectively constitute the chief contributors to the global burden of disease. This review describes the biochemical properties and molecular regulation of PON and summarizes the major recent findings on the functions of PON in protecting against cardiovascular diseases and related disorders.

Case reports and studies may be defined as the nonexperimental description of an individual or a few of cases in terms of new or unusual presentation of the diseases, an unexpected disease course or pathophysiology, and new effects (either beneficial or detrimental) of existing medications or procedures. Although they suffer from the non-experimental nature and other potential bias and errors, case reports and studies have played and will continue to play an important part in the advancement of medicine. They often serve as “primers” leading to discoveries of new diseases/disease pathophysiology as well as development of new preventive and therapeutic measures. Case reports and case studies are also employed as a platform for the training of medical students and/or resident doctors in scientific writing and critical thinking. Although the significance of case reports and studies in medicine has being recognized since the early stage of development of clinical medicine, their value needs to be appreciated in the context of modern clinical research design and the hierarchy of strength of evidence for guiding patient care. This paper discusses case reports and studies within the big picture of clinical research, research design, and evidence-based practice. Understanding the Value of Case Reports and Studies in the Context of Clinical Research, Research Design, and Evidence-Based Practice Y. Robert Li*1,2,3,4, Zhenquan Jia2 and Hong Zhu1