Relative hypoxia and oxidative stress in spleen lymphocytes of immunized Balb/c mice as indicated by HIF-1α, HIF-2α, Nrf2 expression, and glutathione peroxidase activity

Background: Lymphocytes activated by immunization must increase their metabolism to meet the energy requirements for mitosis, differentiation, and protein synthesis, which may subject the cell to conditions of relative hypoxia and oxidative stress. This study was conducted to investigate the increase in the levels of transcription factors involved in both conditions. Methods: Male Balb/c mice were divided into the following four groups, each consisting of six animals: the control and three experimental groups. The experimental groups were immunized by injection of 0.2 ml of 2% sheep red blood cells (SRBC) suspended in phosphate-buffered saline (PBS). Lymphocytes were harvested from the spleens of each group at time intervals of 24-, 48-, and 72-h post-immunization. The buffy coat from splenocytes was separated using Ficoll Histopaque as the medium. The lymphocytes were separated from adherent cells by incubating the purified splenocytes in microtubes for 2-h. Cells were lysed by three freeze–thaw cycles (−80°C and 37°C) and used to analyze the levels of HIF-1α and HIF-2α (mRNA and protein), Nrf2 (protein), and glutathione peroxidase (GPx) activity. Results: The treatment caused an increase in GPx activity and HIF-1α protein concentration 24-h post-immunization, whereas the HIF-1α mRNA levels remained static. Elevated Nrf2 protein levels were detected within 48-h after treatment. Meanwhile, the HIF-2α mRNA and protein levels increased within72-h after immunization. Conclusion: Immunization with SRBC suspension induced relative hypoxia, elevated reactive oxygen species (ROS), and oxidative stress in the lymphocytes as indicated by the increase in both HIF-1α and HIF-2α protein and mRNA levels, GPx activity, and Nrf2 protein levels

Ascorbate Uptake and Retention by Breast Cancer Cell Lines and the Intracellular Distribution of Sodium-Dependent Vitamin C Transporter 2

Ascorbate plays a vital role as a co-factor for a superfamily of enzymes, the 2-oxoglutarate dependent dioxygenases (2-OGDDs), which govern numerous pathways in cancer progression, including the hypoxic response and the epigenetic regulation of gene transcription. Ascorbate uptake into most cells is through active transport by the sodium-dependent vitamin C transporter 2 (SVCT2). The aims of this study were to determine the kinetics of ascorbate uptake and retention by breast cancer cell lines under various oxygen conditions, and to investigate the role of SVCT2 in mediating ascorbate uptake and intracellular trafficking. Human MDA-MB231 cells accumulated up to 5.1 nmol ascorbate/106 cells, human MCF7 cells 4.5 nmol/106 cells, and murine EO771 cells 26.7 nmol/106 cells. Intracellular ascorbate concentrations decreased rapidly after reaching maximum levels unless further ascorbate was supplied to the medium, and there was no difference in the rate of ascorbate loss under normoxia or hypoxia. SVCT2 was localised mainly to subcellular compartments, with the nucleus apparently containing the most SVCT2 protein, followed by the mitochondria. Much less SVCT2 staining was observed on the plasma membrane. Our data showed that careful management of the doses and incubation times with ascorbate in vitro allows for an approximation of in vivo conditions. The localisation of SVCT2 suggests that the distribution of ascorbate to intracellular compartments is closely aligned to the known function of ascorbate in supporting 2-OGDD enzymatic functions in the organelles and with supporting antioxidant protection in the mitochondria