Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

The National Health Commission of the People’s Republic of China has reported that as of February 24, 2020, a total of 3387 of 77,262 patients with Covid-19 (4.4%) in China were health care workers or others who worked in medical facilities.^1,2 According to the Chinese Red Cross Foundation, the National Health Commission of the People’s Republic of China, and public media, as of April 3, a total of 23 of the health care professionals among these 3387 persons had died from Covid-19 after they became infected during the practice of medicine in Wuhan and elsewhere in China.^3,4

Iron is a critical component of many cellular functions including DNA replication and repair, and it is essential for cell vitality. As an essential element, iron is critical for maintaining human health. However, excess iron can be highly toxic, resulting in oxidative DNA damage. Many studies have observed significant associations between iron and cancer, and the association appears to be more than just coincidental. The chief characteristic of cancers, hyper-proliferation, makes them even more dependent on iron than normal cells. Cancer therapeutics are becoming as diverse as the disease itself. Targeting iron metabolism in cancer cells is an emerging, formidable field of therapeutics. It is a strategy that is highly diverse with regard to specific targets and the various ways to reach them. This review will discuss the importance of iron metabolism in cancer and highlight the ways in which it is being explored as the medicine of tomorrow.

Background & Aims Hypoxic inflammation (decreased oxygen tension at sites of inflammation) is a feature of inflammatory bowel disease (IBD). The hypoxia response is mediated by the transcription factors hypoxia-inducible factor (HIF) 1α and endothelial PAS domain protein 1 (EPAS1 or HIF2α), which are induced in intestinal tissues of patients with IBD. HIF1α limits intestinal barrier dysfunction, but the role of EPAS1 has not been assessed under conditions of hypoxic inflammation or in models of IBD. Methods Acute colitis was induced by administration of Citrobacter rodentium or dextran sulfate sodium (DSS) to transgenic hypoxia reporter mice (oxygen-dependent degradation−luciferase), mice with conditional overexpression of Epas1 (Epas1^LSL/LSL), mice with intestinal epithelium-specific deletion of Epas1 (Epas1^ΔIE ), or wild-type littermates (controls). Colon tissues from these mice and from patients with ulcerative colitis or Crohn's disease were assessed by histologic and immunoblot analyses, immunohistochemistry, and quantitative polymerase chain reaction. Results Levels of hypoxia and EPAS1 were increased in colon tissues of mice after induction of colitis and patients with ulcerative colitis or Crohn's disease compared with controls. Epas1^ΔIE mice had attenuated colonic inflammation and were protected from DSS-induced colitis. Intestine-specific overexpression of EPAS1, but not HIF-1α, led to spontaneous colitis, increased susceptibility to induction of colitis by C rodentium or DSS, and reduced survival times compared with controls. Disruption of intestinal epithelial EPAS1 attenuated the inflammatory response after administration of DSS or C rodentium, and intestine-specific overexpression of EPAS1 increased this response. We found EPAS1 to be a positive regulator of tumor necrosis factor−α production by the intestinal epithelium. Blocking tumor necrosis factor−α completely reduced hypoxia-induced intestinal inflammation. Conclusions EPAS1 is a transcription factor that activates mediators of inflammation, such as tumor necrosis factor−α, in the intestinal epithelium and promotes development of colitis in mice.

Oxygen dynamics in the liver is a central signaling mediator controlling hepatic homeostasis, and dysregulation of cellular oxygen is associated with liver injury. Moreover, the transcription factor relaying changes in cellular oxygen levels, hypoxia-inducible factor (HIF), is critical in liver metabolism, and sustained increase in HIF signaling can lead to spontaneous steatosis, inflammation, and liver tumorigenesis. However, the direct responses and genetic networks regulated by HIFs in the liver are unclear. To help define the HIF signal-transduction pathway, an animal model of HIF overexpression was generated and characterized. In this model, overexpression was achieved by Von Hippel-Lindau ( Vhl ) disruption in a liver-specific temporal fashion. Acute disruption of Vhl induced hepatic lipid accumulation in an HIF-2α–dependent manner. In addition, HIF-2α activation rapidly increased liver inflammation and fibrosis, demonstrating that steatosis and inflammation are primary responses of the liver to hypoxia. To identify downstream effectors, a global microarray expression analysis was performed using livers lacking Vhl for 24 hours and 2 weeks, revealing a time-dependent effect of HIF on gene expression. Increase in genes involved in fatty acid synthesis were followed by an increase in fatty acid uptake-associated genes, and an inhibition of fatty acid β-oxidation. A rapid increase in proinflammatory cytokines and fibrogenic gene expression was also observed. In vivo chromatin immunoprecipitation assays revealed novel direct targets of HIF signaling that may contribute to hypoxia-mediated steatosis and inflammation. Conclusion: These data suggest that HIF-2α is a critical mediator in the progression from clinically manageable steatosis to more severe steatohepatitis and liver cancer, and may be a potential therapeutic target. (Hepatology 2011;)