High Oxygen Stress Research Articles

Abstract 3694: Metabolic response in human cervical cancer cells to modifications in bioavailable ATP

Abstract Introduction: Cancer cells utilize a markedly reduced oxidative phosphorylation (OXPHOS) when experiencing conditions of high oxygen stress (Warburg Effect/aerobic glycolysis), a phenomenon first studied by Otto Warburg. While there have been many discoveries associated with metabolic differences across cancer cells, one of the most common alterations is the primary means of generating ATP. The importance of deciphering these cellular decisions will enable an understanding of the multifaceted role ATP has in cancer proliferation, senescence, and chemotherapeutic resistance. The use of synthetic protein DX, a man-made ATP chelator with high specificity and affinity, is a proposed method of investigating the metabolic response in cancer cells. Hypothesis: Following DX expression, HeLa cells respond to energy stress (reduced intracellular ATP) via metabolic adaptations, specifically inducing the spare respiratory capacity. Methods: The impact of DX on cell viability was determined using a tetrazolium-based colorimetric cell viability assay and a caspase 3/7 assay. To correlate phenotypic/viability change with DX activity, bioavailable ATP levels were measured at specific time points following DX expression. Additionally, the relative contribution of glycolysis and OXPHOS to the total ATP production rate was measured using the label-free XF Real Time ATP Rate Assay and XF Cell Mito Stress Test (XFe96 Seahorse, Agilent Technologies) over time post DX expression. To determine the impact of ATP stress on mitochondrial and nuclear content, both were quantified following DX-expression. Results: In a time- and dose- dependent manner, DX negatively impacted cell growth and induced cell death via apoptosis, at a time concomitant with a decrease in bioavailable ATP. In response to DX over time, the total ATP production rates in HeLa cells significantly decreased. Importantly, this reduction in the rate of ATP production was associated with a significant downregulation in both OXPHOS and glycolysis. Conclusion: Advances in synthetic biology have allowed for the intentional design of artificial proteins that maintain function in vivo. These proteins can be developed into powerful investigative tools to study biological questions, such as cellular decisions involved in cancer cell metabolism. In response to DX-potentiated ATP stress, the significant reduction in both glycolysis and oxidative phosphorylation suggests metabolic downregulation is required to maintain HeLa cell viability. Citation Format: Parth K. Jayaswal, Taha Muhammad, Ashley Brown, Jeffrey Norris, Shaleen B. Korch. Metabolic response in human cervical cancer cells to modifications in bioavailable ATP. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3694.

Nannochloropsis oceanica (Eustigmatophyceae) mutants resistant to rose bengal demonstrate high tolerance to oxygen

ABSTRACT The genus Nannochloropsis (Eustigmatophyceae), including N. oceanica, has attracted considerable interest, due to its ability to accumulate high levels of lipids. Under large-scale cultivation outdoors, cells are exposed to a combination of high oxygen and light irradiance conditions that may promote photoinhibition, so selecting strains able to tolerate oxidative stress is desirable. Rose bengal (RB) reacts with oxygen to form singlet oxygen in the presence of light. Therefore, RB can be a very useful tool for testing the sensitivity of algae to oxidative stress and thus identify strains capable of photosynthesis under oxidative stress. In this study, two mutants of N. oceanica, RB2 and RB113, which are resistant to high concentrations of rose bengal, were phenotypically characterized for their sensitivity to high oxygen concentrations. Both strains exhibited levels of catalase and ascorbate peroxidase 1.5- and 3-fold higher, respectively, than the wild type. In addition, the synthesis of carotenoids was lower than in the wild type, indicating lower oxidative stress. These results were confirmed by chlorophyll fluorescence measurements, which indicated a lower sensitivity of the photosynthetic apparatus of the mutants, especially of RB2, even in the presence of H2O2. The results support the hypothesis that resistance to rose bengal may also induce resistance to high dissolved oxygen stress. The ability shown by the mutants to perform photosynthesis more efficiently under high oxygen stress than the wild type makes them promising candidates for outdoor cultures.

Exploring the oxidative, antimicrobial and genomic properties of Campylobacter jejuni strains isolated from poultry

Campylobacter jejuni is the leading cause of food-borne bacterial enteritis in humans, with contaminated poultry products considered the main source of infection. To survive the food chain, C. jejuni utilizes multiple defense mechanisms that counter oxidative and aerobic stresses. In this study, we phenotypically characterised 63 C. jejuni strains with oxidative stress survival and antimicrobial susceptibility testing to investigate correlations between these two phenotypes against the source of the strains and the presence of the MarR regulators RrpA and RrpB which have a role in regulating the response to oxidative and aerobic stress. C. jejuni strains isolated from meat and neck skin displayed the highest resistance to oxidative stress. In addition, C. jejuni strains that have an rrpA+rrpB− profile exhibit increased resistance to oxidative stress and to antimicrobials. Here we establish a preliminary link between the distribution of RrpA and RrpB and the increased resistance to antimicrobials. This study provides insight into how the genotypic make up of C. jejuni can influence the ability of the bacterium to survive within areas of high oxygen stress, such as the food chain, and subsequently can have a potential negative impact on human health.

A systematic approach to expound the variations in taxane production under different dissolved oxygen conditions in Taxus chinensis cells.

Our results provide an evidence that the changes in taxane production caused by dissolved oxygen shifts could be associated with the global variations in the cell central carbon metabolism. Taxol is an important taxane synthesized by the Taxus plant. A two-stage culture of Taxus in vitro has been considered as an attractive alternative approach to produce Taxol and its precursors. To investigate the consequences of dissolved oxygen (DO) shifts for cell primary and secondary metabolism, we conducted metabolomic and transcriptomic profiling analyses under low dissolved oxygen (LDO), medium dissolved oxygen (MDO), and high dissolved oxygen (HDO) conditions in a suspension culture of Taxus chinensis cells. Under LDO, the results indicate a significant increase in the production of Taxol and its main precursors by 3.4- to 1.4-fold compared with those under MDO and HDO on 9th day. Multiple acyl taxanes (MAT) are abundant taxanes in the cells, and exhibited only a slight increase under the same conditions. Metabolomic analysis based on 209 primary metabolites indicated that several pathways in central carbon metabolism were involved, including the enhancement of the glycolysis pathway of glucose-6-phosphate to fructose-6-phosphate and pyruvate and the mevalonate pathway of terpene biosynthesis, and decline in the tricarboxylic acid pathway under LDO. These results indicate the mechanism by which related taxanes accumulate through enhancing the supplies of substrates and expression levels of hydroxylases. Excess acetyl-CoA supply induced by high oxygen stress was found to be correlated with high productivity of MAT. Our results provide an evidence that the changes in taxane production caused by DO shifts could be associated with the global variations in the cell central carbon metabolism.

Response of three krill species to hypoxia and warming: an experimental approach to oxygen minimum zones expansion in coastal ecosystems

AbstractTo understand the adaptation of euphausiid (krill) species to oxygen minimum zones (OMZs), respiratory response and stress experiments combining hypoxia‐reoxygenation exposure with warming were conducted. Experimental krill species were obtained from the Antarctic (South Georgia area), the Humboldt Current System (HCS, Chilean coast) and the Northern California Current System (NCCS, Oregon).Euphausia mucronatafrom theHCSshowed oxyconformingpO2‐dependent respiration below 80% air saturation (18 kPa). Normoxic subsurface oxygenation in winter posed a ‘high oxygen stress’ for this species. TheNCCSkrill,Euphausia pacifica, and the Antarctic krill,Euphausia superba, maintained respiration rates constant down to low criticalpO2values of 6 kPa (30% air saturation) and 11 kPa (55% air saturation), respectively. Antarctic krill had low antioxidant enzyme activities, but high concentrations of the molecular antioxidant glutathione (GSH) and was not lethally affected by 6 h exposure to moderate hypoxia. The temperate krill species (E. pacifica) had higher superoxide dismutase (SOD) values in winter than in summer, which relates to a higher winter metabolic rate. In all species, antioxidant enzyme activities remained constant during hypoxic exposure at the typical temperature for their habitat. Warming by 7 °C above habitat temperature in summer increasedSODactivities andGSHlevels inE. mucronata(HCS), but no oxidative damage occurred. In winter, when theNCCSis well mixed and theOMZis deeper, +4 °C of warming combined with hypoxia represents a lethal condition forE. pacifica. In summer, when theOMZexpands upwards (100 m subsurface), antioxidant defences counteracted hypoxia and reoxygenation effects inE. pacifica, but only at mildly elevated temperature (+2 °C). In this season, experimental warming by +4 °C reduced antioxidant activities and the combination of warming with hypoxia again caused mortality of exposed specimens. We conclude that a climate change scenario combining warming and hypoxia represents a serious threat toE. pacificaand, as a consequence,NCCSfood webs.

Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration.

Campylobacter jejuni is an important pathogen of foodborne illness. Transmigration across the intestinal epithelial barrier and invasion are considered as primary reasons for tissue damage triggered by C. jejuni. Using knockout mutants, it was shown that the serine protease HtrA may be important for stress tolerance and physiology of C. jejuni. HtrA is also secreted in the extra-cellular environment, where it can cleave junctional host cell proteins such as E-cadherin. Aim of the present study was to establish a genetic complementation system in two C. jejuni strains in order to introduce the wild-type htrA gene in trans, test known htrA phenotypes, and provide the basis to perform further mutagenesis. We confirm that reexpression of the htrA wild-type gene in ΔhtrA mutants restored the following phenotypes: 1) C. jejuni growth at high temperature (44 °C), 2) growth under high oxygen stress conditions, 3) expression of proteolytically active HtrA oligomers, 4) secretion of HtrA into the supernatant, 5) cell attachment and invasion, and 6) transmigration across polarized epithelial cells. These results establish a genetic complementation system for htrA in C. jejuni, exclude polar effects in the ΔhtrA mutants, confirm important HtrA properties, and permit the discovery and dissection of new functions.

Identification and characterization of the chromium(VI) responding protein from a newly isolated Ochrobactrum anthropi CTS-325

A Gram-negative, chromium(VI) tolerant and reductive strain CTS-325, isolated from a Chinese chromate plant, was identified as Ochrobactrum anthropi based on its biochemical properties and 16S rDNA sequence analysis. It was able to tolerate up to 10 mmol/L Cr(VI) and completely reduce 1 mmol/L Cr(VI) to Cr(III) within 48 h. When the strain CTS-325 was induced with Cr(VI), a protein increased significantly in the whole cell proteins. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed that this protein was a superoxide dismutase (SOD) homology. The measured superoxide dismutase activity was 2694 U/mg after three steps of purification. The SOD catalyzes the dismutation of the superoxide anion (O2*-) into hydrogen peroxide and molecular oxygen. This protein is considered to be one of the most important anti-oxidative enzymes for O. anthropi as it allows the bacterium to survive high oxygen stress environments, such as the environment produced during the reduction process of Cr(VI).

AML1-ETO Inhibits DNA Repair Pathways and Promotes the Accumulation of DNA Damage and the Activation of p53.

Malignant transformation is a multistep process that is promoted by the accumulation of genetic changes over time. The contribution of individual genetic changes to the development of leukemia and the processes that induce these changes are not yet fully understood. The generation of the fusion gene AML1-ETO is thought to be an early event in the development of AML with t(8;21). Cells expressing AML1-ETO have been detected in samples from healthy newborns, as well as in patient samples analyzed long before the manifestation of AML. For these reasons it is assumed that a pre-leukemic clone expressing AML1-ETO requires additional genetic changes to complete the malignant transformation. However, the mechanism whereby these changes are acquired and the role that AML1-ETO plays in this process remain unclear. We used a model of human CD34+ cells expressing the AML1-ETO fusion protein to elucidate the role of AML1-ETO in leukemogenesis. AML1-ETO expressing (AE) cells displayed characteristics of a pre-leukemia, with enhanced self-renewal and NOD/SCID engraftment but no ability to induce malignancy in vivo. Analysis of gene expression using Affymetrix HG U133 Plus2.0 gene chips revealed that multiple genes important in various DNA repair pathways were suppressed in AE cells: OGG1, UNG, TDG, MBD4, POLB, POLE and FEN1 (Base Excision Repair), MRE11, RAD50, ATM, CHEK1 and CDC25A (ATM pathway), and FANCA, FANCL and BRCA2 (Fanconi anemia proteins). Treatment with DNA interstrand crosslinking agents mitomycin C or melphalan revealed a block in the S-phase of the cell cycle in AE cells at doses not affecting control cells. AE cells were also more sensitive to gamma irradiation compared to vector-transduced cells. In AE cultures, a gradual accumulation of cells with DNA damage was detected by immunofluorescence microscopy, using phosphorylated histone H2A.X as a marker. AE cells had a hyperactive p53 pathway with increased p53 protein levels, upregulation of its target genes (p21, TP53I3 and DAPK1), and increased apoptosis compared to control cultures, as might be expected in cells with chronic DNA damage. Incubation under low oxygen conditions prevented the accumulation of DNA damage and resulted in decreased expression of p53 target genes (TP53I3 and DAPK1), indicating that a sub-optimal response to the high oxygen stress of culture could be responsible for the accumulation of DNA damage foci and activation of the p53 pathway in AE cultures. Inhibition of p53 by shRNA led to increased resistance of AE cells to gamma irradiation but with continued high levels of DNA damage as shown by phosphorylated H2A.X. Interestingly, the POU4f1 transcription factor, a modifier of the p53 response pathway, has been found to be overexpressed in t(8;21) patient samples. In our system, AE cells that ectopically overexpressed POU4f1 following retroviral transduction showed decreased levels of p53 compared to vector-transduced AE cells. Inhibition of the p53 pathway in pre-leukemic clones expressing AML1-ETO could lead to a proliferative or survival advantage during periods of genotoxic stress. The impairment of DNA repair pathways and the accumulation of DNA damage in AML1-ETO-expressing cells may promote an elevated mutation rate and increase the chances of acquiring subsequent genetic alterations.

The expanding network of redox signaling: new observations, complexities, and perspectives.

Over thirty years ago, the observations that eventually led to the discovery of the NADPH oxidase were made by Baehner, Karnovsky, and colleagues (1–3). These have served as a focal point of interest in placing reactive oxygen species (ROS) in the conceptual forefront of the biomedical community. Over the last decade, the examination of the roles of oxygen and redox tone in regulating cell function has turned inward to the intracellular environment. Because oxidative metabolism is central to the biology and health of all humans, how we respond to conditions of low and high oxygen stress has become a critical consideration in biology and medicine. Humans live in a world where we continually balance the use of oxygen as a source of energy, and as a source of cellular injury. The generation of oxygen radicals secondary to mitochondrial disruption, the activation of cellular NADPH oxidases, the metabolism of xenobiotics, or other forms of oxidative stress can lead to mutations in DNA, lipid peroxidation, and protein damage. We have therefore evolved a marvelously complex system of both defense mechanisms and sensing mechanisms for changes in cellular redox tone. These include the enzymes superoxide dismutase, catalase, and glutathione peroxidase that detoxify ROS. We have also developed signaling mechanisms that utilize ROS to initiate processes that allow cells to survive exposure to oxidative stress within certain tolerances, but also, when stress and damage become too great, to ensure cell death. How these pathways are initiated and controlled on a molecular basis by ROS and also molecular oxygen is at the heart of what is generally considered redox signaling and the response to oxidative stress. The molecular species that fall under the term ROS include superoxide, hydrogen peroxide (H2O2), hydroxyl radical, and singlet oxygen. Each of these can play a role in a variety of intracellular processes. Finally, we have adapted these molecular species, particularly H2O2 and (NO), as signaling molecules in multiple biological processes.

ON‐LINE MONITORING OF CHLOROPHYLL FLUORESCENCE TO ASSESS THE EXTENT OF PHOTOINHIBITION OF PHOTOSYNTHESIS INDUCED BY HIGH OXYGEN CONCENTRATION AND LOW TEMPERATURE AND ITS EFFECT ON THE PRODUCTIVITY OF OUTDOOR CULTURES OF SPIRULINA PLATENSIS (CYANOBACTERIA)

The saturating pulse fluorescence technique was applied to study photoinhibition of photosynthesis in outdoor cultures of the cyanobacterium Spirulina platensis (Nordstedt) Geitler strain M2 grown under high oxygen and low temperature stress. Diurnal changes in maximum photochemical yield (Fv/Fm), photon yield of PSII (ΔF/F′m), and nonphotochemical quenching (qN) were measured using a portable, pulse‐amplitude–modulated fluorometer. When solar irradiance reached the maximum value, the Fv/Fm and ΔF/F′m ratios of theSpirulina cultures grown under high oxygen stress decreased by 35% and 60%, respectively, as compared with morning values. The depression of the Fv/Fm and ΔF/F′m ratios reached 55% and 84%, respectively, when high oxygen stress was combined with low temperature (i.e. 10° C below the optimal value for growth). Photoinhibition reduced the daily productivity of the culture grown under high oxygen stress by 33% and that of the culture grown under high oxygen–low temperature stress by 60%. Changes in the biomass yield of the cultures correlated well with changes in the daily integrated value of the estimated electron transport rate through the PSII (ΔF/F′m × photon flux density). The results indicate that on‐line chlorophyll fluorescence measurement is a powerful tool for assessing the photosynthetic performance of outdoorSpirulina cultures.

Hyperoxia (145 mmHg pO2) and tissue normoxia (20-40 mmHg pO2) modulate human vascular cell functions.

Between aggression and defence, the endothelial cell, as the pluripotent control cell of the vessel wall, fulfills its functions in regulated interactions between the blood, the smooth muscle cells (SMC) and the fibroblasts of the vessel wall [1–5]. Oxygen influences various cellular functions like the cell proliferation, the angiogenesis, the lipid metabolism and others [2–11]. The oxygen partial pressure (pO2) in the arterial blood is between 70–100 mmHg, in the venous blood around 40 mmHg, in the aortic media between 30–40 mmHg and in the bovine pulmonary artery between 9–22 mmHg pO2 [7, 11]. Therefore, many years ago, we postulated that 145 mmHg pO2 (21 vol% O2) applied in vessel wall cell cultivation is hyperoxic, does not resemble in vivo tissue normoxia and induces an unphysiologically high oxygen stress to the vascular cells in vitro, thereby, artificially modulate their functional behavior [2, 4, 5, 12].KeywordsBovine Aortic Endothelial CellPulmonary Arterial Smooth Muscle CellPreventive CardiologyHuman Smooth Muscle CellProliferation IncreaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Anoxic/Oxic Activated Sludge Treatment Kinetics of Cyanides and Phenols

The anoxic/oxic activated sludge treatment kinetics of cyanides (i.e., cyanide and thiocyanate) and phenols (i.e., phenol, cresols, and catechol) are studied. The Michaelis‐Menten expression or its simplified forms are used to model the following microbial reactions associated with the process: (1) Anoxic/oxic TOC utilization; (2) anoxic denitrification; (3) nitrification; and (4) cyanide and thiocyanate utilization. The steady‐state experimentation is performed to obtain experimental data for the validation of kinetic models and evaluation of kinetic coefficients. The results obtained indicate that all oxic microbial reactions occurring in an anoxic/oxic activated sludge process are not affected kinetically by the presence of the anoxic environment with high oxygen stress. As a result, the results collected from both conventional and anoxic/oxic activated sludge units exhibit identical kinetic patterns. Anoxic TOC utilization, anoxic denitrification, and nitrification reactions are describable by zero‐or...

Effects of Low-Temperature Acclimation and Oxygen Stress on Tocopheron Production in Euglena gracilis Z

The effects of low-temperature acclimation and oxygen stress on tocopheron production were examined in the unicellular phytoflagellate Euglena gracilis Z. Cells were cultured photoheterotrophically at 27.5 +/- 1 degrees C with 5% carbon dioxide-95% air and 740 microeinsteins m s (photosynthetically active radiation) and served as controls. Low-temperature acclimation (12.5 +/- 1 degrees C) and high-oxygen stress (5% carbon dioxide-95% oxygen) were individually examined in the mass culturing of the algae. Chromatographic analyses demonstrated a six-to sevenfold enhancement of alpha-tocopherol production in temperature-stressed cells, along with a concomitant decline in the levels of alpha-tocotrienol and the absence of other tocopherol homologs. Oxygen-stressed cultures demonstrated the presence of high levels of alpha-tocopherylquinone; alpha-tocopheron and its homologs and precursors were absent or declined markedly. These findings are discussed in terms of the feasibility of microbial production of natural tocopherols. In addition, these results lend themselves to speculation regarding the biological role(s) of tocopherols as antioxidants and free radical scavengers in reducing photo-induced oxidative damage or lipid peroxidation toxicities or both in photosynthetically active E. gracilis Z.