Vitamin CVitamin C is one of the most extensively studied vitamins in modern medicine. Its diverse biological activities have made it a promising agent for human disease interventionIntervention. This chapter discusses recent advances in redox biologyRedox biology of vitamin C and its potential clinical implications in the management of common diseasesDisease, including cardiovascular disorders, cancerCancer, neurodegenerationNeurodegeneration, and others. The chapter focuses on the latest cutting-edge discoveries reported in influential journals rather than surveying all research findings in the literature. It is hoped that by emphasizing the novel findings from high-profile research studies, this chapter would stimulate critical thinking and future innovative research on vitamin C in health and diseaseDisease.

Doxorubicin (Dox) is among the most widely used cancer chemotherapeutic drugs. The clinical use of Dox is, however, limited due to its cardiotoxicity. Studies over the past several decades have suggested various mechanisms of Dox-induced cardiotoxicity (DIC). Among them are oxidative stress, topoisomerase inhibition, and mitochondrial damage. Several novel molecular targets and signaling pathways underlying DIC have emerged over the past few years. The most notable advances include discovery of ferroptosis as a major form of cell death in Dox cytotoxicity, and elucidation of the involvement of cardiogenetics and regulatory RNAs as well as multiple other targets in DIC. In this review, we discuss these advances, focusing on latest cutting-edge research discoveries from mechanistic studies reported in influential journals rather than surveying all research studies available in the literature.

Background: Metformin is among the most frequently prescribed antidiabetic drugs worldwide and remains the first-line therapy for type 2 diabetes due to its well-established glucose-lowering efficacy and favorable safety profile. Summary: Studies over the past decades show that metformin also exerts many other beneficial effects independent of its glucose-lowering effect both in experimental models and human subjects. Among them, the most notable is its cardiovascular protective effect. In this review, we discuss the latest cutting-edge research findings on metformin's cardiovascular protection from both preclinical studies and randomized clinical trials. We focus on describing novel basic research discoveries reported in influential journals and discussing their implications in the context of latest clinical trial findings related to common cardiovascular and metabolic disorders, including atherosclerosis and dyslipidemia, myocardial injury, and heart failure. Key Messages: While substantial preclinical and clinical evidence suggests metformin as a potential cardiovascular protectant, large-scale randomized controlled trials are warranted to establish its clinical efficacy in treating patients with atherosclerotic cardiovascular disease and heart failure.

Metformin is among the most widely used antidiabetic drugs. Studies over the past few years have identified multiple novel molecular targets and pathways that metformin acts on to exert its beneficial effects in treating type 2 diabetes as well as other disorders involving dysregulated inflammation and redox homeostasis. In this mini-review, we discuss the latest cutting-edge research discoveries on novel molecular targets of metformin in glycemic control, cardiovascular protection, cancer intervention, anti-inflammation, antiaging, and weight control. Identification of these novel targets and pathways not only deepens our understanding of the molecular mechanisms by which metformin exerts diverse beneficial biological effects, but also provides opportunities for developing new mechanistically based drugs for human diseases.

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+/+ 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.

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.

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.

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 mu M 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 endothelialmonocyte 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.

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.

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.

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.

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.

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.

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 antiinflammatory defenses and as a potential therapeutic modality for sepsis intervention.

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.

Our early work suggested that graphene quantum dots (GQDs) block Cu(II)/Cu(I) redox cycle in biological systems. Here we report that GQDs could also potently protect against copper redox-mediated oxidative DNA damage. Using Cu(II)/hydrogen peroxide, Cu(II)/hydroquinone, and Cu(II)/ascorbic acid as three biologically relevant systems for inducing oxidative DNA damage, we demonstrated that GQDs protected against the above system-induced DNA strand breaks in ϕx-174 plasmid DNA in a concentration-dependent manner. Notably, a significant protection was observed with GQDs at 1 μg/ml, and a nearly complete protection was shown with 10 and 100 μg/ml of GQDs. Using electron paramagnetic resonance (EPR) spectrometry in conjunction with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN)-spin trapping, we showed that the above three systems generated hydroxyl radicals, as evidenced by the formation of a POBN-CH3 radical adduct in the presence of 0.5 M dimethyl sulfoxide (DMSO). Consistent with the protective effects of GQDs on DNA damage, the hydroxyl radical formation was markedly reduced in the presence of GQDs in a concentration dependent manner. A nearly complete blockage of the hydroxyl radical generation was seen with GQDs at 10 and 100 μg/ml. Taken together, our results showed that GQDs potently protected against oxidative DNA damage. Considering the critical role of copper in cancer development, our findings might have important implications for cancer intervention with GQD-based nanotech modality.

In this work, we investigated the effects of graphene quantum dots (GQDs) on copper redox-mediated free radical generation and cell injury. Using electron paramagnetic resonance (EPR) spectrometry in conjunction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, we found that GQDs at a concentration as low as 1 μg/ml significantly inhibited Cu(II)/H2O2-mediated hydroxyl radical formation. GQDs also blocked Cu(II)-catalyzed nucleophilic addition of H2O to DMPO to form a DMPO-OH adduct in the absence of H2O2, suggesting a potential for GQDs to inhibit copper redox activity. Indeed, we observed that the presence of GQDs prevented H2O2-mediated reduction of Cu(II) to Cu(I) though GQDs themselves also caused the reduction of Cu(II) to Cu(I). To further investigate the effects of GQDs on copper redox activity, we employed the Cu(II)/hydroquinone system in which copper redox activity plays an essential role in the oxidation of hydroquinone to semiquinone radicals with consequent oxygen consumption. Using oxygen polarography as well as EPR spectrometry, we demonstrated that the presence of GQDs drastically blocked the oxygen consumption and semiquinone radical formation resulting from the reaction of Cu(II) and hydroquinone. These results suggested that GQDs suppressed free radical formation via inhibiting copper redox activity. Lastly, using cultured human cardiomyocytes, we demonstrated that the presence of GQDs also protected against Cu(II)/H2O2-mediated cardiac cell injury as indicated by morphological changes (e.g., cell shrinkage and degeneration). In conclusion, our work shows, for the first time, the potential for using GQDs to counteract copper redox-mediated biological damage.

This study investigated the hypothesis that acetaminophen at pharmacologically relevant concentrations may act as a protector against peroxynitrite toxicity.  Our results showed that acetaminophen inhibited SIN-1 (a peroxynitrite generator)-induced DNA cleavage in a concentration-dependent manner in an in vitro model. With bicarbonate-enhanced luminol-dependent chemiluminometry, we further showed a nearly complete blockage of peroxynitrite-derived chemiluminescence by acetaminophen at 25‒100 µM with minimal effects on SIN-1-mediated oxygen consumption, suggesting acetaminophen as a potent scavenger of peroxynitrite. Electron spin resonance spectrometry in combination with 5,5-dimethyl-1-pyrroline N -oxide (DMPO)-spin-trapping supported the ability of acetaminophen at high concentrations to diminish the production of a free radical species (likely hydroxyl radical) from SIN-1. Fenton chemistry-based DMPO-spin trapping further demonstrated the hydroxyl radical-scavenging capacity of acetaminophen. Collectively, the results of this study for the first time revealed the potential of acetaminophen to protect against peroxyitrite toxicity, which may have important implications in neuroprotection associated with the use of this popular analgesic and antipyretic drug.