The effect of baseline O2 conditions on the response of prostate cancer cells to hypoxia.

The transcriptional response to hypoxia is largely regulated by the hypoxia-inducible factors (HIFs), which induce the expression of genes involved in glycolysis, angiogenesis, proliferation, and migration. Virtually all cell culture-based hypoxia experiments have used near-atmospheric (18% O2) oxygen levels as the baseline for comparison with hypoxia. However, this is hyperoxic compared with mammalian tissue microenvironments, where oxygen levels range from 2% to 9% O2 (physioxia). Thus, these experiments actually compare hyperoxia to hypoxia. To determine how the baseline O2 level affects the subsequent response to hypoxia, we cultured PC-3 prostate cancer cells in either 18% or 5% O2 for 2 wk before exposing them to hypoxia (∼1.1% pericellular O2) for 12-48 h. RNA-seq revealed that the transcriptional response to hypoxia was dependent on the baseline O2 level. Cells grown in 18% O2 before hypoxia exposure showed an enhanced induction of HIF targets, particularly genes involved in glucose metabolism, compared with cells grown in physioxia before hypoxia. Consistent with this, hypoxia significantly increased glucose consumption and metabolic activity only in cells previously cultured in 18% O2, but not in cells preadapted to 5% O2. Transcriptomic analyses also indicated effects on cell proliferation and motility, which were followed up by functional assays. Although unaffected by hypoxia, both proliferation and migration rates were greater in cells cultured in 5% O2 versus 18% O2. We conclude that an inappropriately hyperoxic starting condition affects the transcriptional and metabolic responses of PC-3 cells to hypoxia, which may compromise experiments on cancer metabolism in vitro.NEW & NOTEWORTHY Although human cell culture models have been instrumental to our understanding of the mechanisms involved in the cellular response to hypoxia, in virtually all experiments, cells are routinely cultured in near-atmospheric (∼18% O2) oxygen levels, which are hyperoxic relative to physiological conditions in vivo. Here, we show for the first time that cells cultured in physiological O2 levels (5% O2) respond differently to subsequent hypoxia than cells grown at 18%.

Revisiting reactive oxygen species production in hypoxia.

Cellular responses to hypoxia are crucial in various physiological and pathophysiological contexts and have thus been extensively studied. This has led to a comprehensive understanding of the transcriptional response to hypoxia, which is regulated by hypoxia-inducible factors (HIFs). However, the detailed molecular mechanisms of HIF regulation in hypoxia remain incompletely understood. In particular, there is controversy surrounding the production of mitochondrial reactive oxygen species (ROS) in hypoxia and how this affects the stabilization and activity of HIFs. This review examines this controversy and attempts to shed light on its origin. We discuss the role of physioxia versus normoxia as baseline conditions that can affect the subsequent cellular response to hypoxia and highlight the paucity of data on pericellular oxygen levels in most experiments, leading to variable levels of hypoxia that might progress to anoxia over time. We analyze the different outcomes reported in isolated mitochondria, versus intact cells or whole organisms, and evaluate the reliability of various ROS-detecting tools. Finally, we examine the cell-type and context specificity of oxygen's various effects. We conclude that while recent evidence suggests that the effect of hypoxia on ROS production is highly dependent on the cell type and the duration of exposure, efforts should be made to conduct experiments under carefully controlled, physiological microenvironmental conditions in order to rule out potential artifacts and improve reproducibility in research.

Oxygen toxicity: cellular mechanisms in normobaric hyperoxia.

In clinical settings, oxygen therapy is administered to preterm neonates and to adults with acute and chronic conditions such as COVID-19, pulmonary fibrosis, sepsis, cardiac arrest, carbon monoxide poisoning, and acute heart failure. In non-clinical settings, divers and astronauts may also receive supplemental oxygen. In addition, under current standard cell culture practices, cells are maintained in atmospheric oxygen, which is several times higher than what most cells experience in vivo. In all the above scenarios, the elevated oxygen levels (hyperoxia) can lead to increased production of reactive oxygen species from mitochondria, NADPH oxidases, and other sources. This can cause cell dysfunction or death. Acute hyperoxia injury impairs various cellular functions, manifesting ultimately as physiological deficits. Chronic hyperoxia, particularly in the neonate, can disrupt development, leading to permanent deficiencies. In this review, we discuss the cellular activities and pathways affected by hyperoxia, as well as strategies that have been developed to ameliorate injury. • Hyperoxia promotes overproduction of reactive oxygen species (ROS). • Hyperoxia dysregulates a variety of signaling pathways, such as the Nrf2, NF-κB and MAPK pathways. • Hyperoxia causes cell death by multiple pathways. • Antioxidants, particularly, mitochondria-targeted antioxidants, have shown promising results as therapeutic agents against oxygen toxicity in animal models.

Alteration in liver function tests among patients hospitalized for COVID-19: a multicentric study in Peru.

INTRODUCTION: COVID-19 affects the liver, causing alteration in liver biochemistry tests such as aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), total bilirubin and albumin. OBJECTIVE: To determine the prevalence of alteration in liver functions tests and associated factors for severity among Peruvian COVID-19 patients. MATERIALS AND METHODS: A descriptive, retrospective and cross-sectional study was performed in 4 public hospitals in Peru. Patients admitted to hospitalization wards and intensive care units with a diagnosis COVID-19 were enrolled. The evaluation of AST, ALT, ALP, totalbilirubin and albumin was performed. Associations with demographic and medical data were assessed. RESULTS: 1,100 patients were enrolled, of which 81.7% had altered liver function tests. Only 2.8% of the patients had cirrhosis and 2.1% hepatitis B/C virus. AST and ALT were altered at admission in 64.7% and 63.7%, of the patients respectively. Factors associated with liver injury were: being female OR=0.53 (95% CI: 0.39-0.73; p<0.01), dyslipidemia OR=1.72 (95% CI: 1.10-2.70; p=0.01), previous medication OR=1.56 (95% CI: 1.12 -2.16, p<0.01) and fever OR=1.43 (95% CI: 1.03-1.199, p=0.03). Disease severity was associated with levels of AST and ALT (p<0.01). Patients taking self-medication OR=1.56 (95% CI: 1.12-2.16; p<0.01) and paracetamol OR= 1.41 (95% CI:1.01-1.98; p=0.04) had higher risk of liver injury. Meanwhile, corticosteroids OR=0.55 (95% CI: 0.38-0.78; p<0.01) and enoxaparin OR=0.53 (95% CI: 0.35- 0.81; p<0.01) were protective factors. CONCLUSIONS: Peruvian patients with COVID-19 presented high prevalence of alteration in liver function tests, high levels of AST and ALT were related to disease severity.

Transmembrane Chemical Absorption Process for Recovering Ammonia as an Organic Fertilizer Using Citric Acid as the Trapping Solution.

Membrane contactors are among the available technologies that allow a reduction in the amount of ammoniacal nitrogen released into the environment through a process called transmembrane chemical absorption (TMCA). This process can be operated with different substances acting as trapping solutions; however, strong inorganic acids have been studied the most. The purpose of this study was to demonstrate, at laboratory scale, the performance of citric acid as a capturing solution in TMCA processes for recovering ammonia as an organic fertilizer from anaerobic digestor reject water using membrane contactors in a liquid-liquid configuration and to compare it with the most studied solution, sulfuric acid. The experiments were carried out at 22 °C and 40 °C and with a feed water pH of 10 and 10.5. When the system was operated at pH 10, the rates of recovered ammonia from the feed solution obtained with citric acid were 10.7-16.5 percentage points (pp) lower compared to sulfuric acid, and at pH 10.5, the difference decreased to 5-10 pp. Under all tested conditions, the water vapor transport in the system was lower when using citric acid as the trapping solution, and at pH 10 and 40 °C, it was 5.7 times lower. When estimating the operational costs for scaling up the system, citric acid appears to be a better option than sulfuric acid as a trapping solution, but in both cases, the process was not profitable under the studied conditions.

Culture of Cancer Cells at Physiological Oxygen Levels Affects Gene Expression in a Cell-Type Specific Manner.

Standard cell culture is routinely performed at supraphysiological oxygen levels (~18% O2). Conversely, O2 levels in most mammalian tissues range from 1-6% (physioxia). Such hyperoxic conditions in cell culture can alter reactive oxygen species (ROS) production, metabolism, mitochondrial networks, and response to drugs and hormones. The aim of this study was to investigate the transcriptional response to different O2 levels and determine whether it is similar across cell lines, or cell line-specific. Using RNA-seq, we performed differential gene expression and functional enrichment analyses in four human cancer cell lines, LNCaP, Huh-7, PC-3, and SH-SY5Y cultured at either 5% or 18% O2 for 14 days. We found that O2 levels affected transcript abundance of thousands of genes, with the affected genes having little overlap between cell lines. Functional enrichment analysis also revealed different processes and pathways being affected by O2 in each cell line. Interestingly, most of the top differentially expressed genes are involved in cancer biology, which highlights the importance of O2 levels in cancer cell research. Further, we observed several hypoxia-inducible factor (HIF) targets, HIF-2α targets particularly, upregulated at 5% O2, consistent with a role for HIFs in physioxia. O2 levels also differentially induced the transcription of mitochondria-encoded genes in most cell lines. Finally, by comparing our transcriptomic data from LNCaP and PC-3 with datasets from the Prostate Cancer Transcriptome Atlas, a correlation between genes upregulated at 5% O2 in LNCaP cells and the in vivo prostate cancer transcriptome was found. We conclude that the transcriptional response to O2 over the range from 5-18% is robust and highly cell-type specific. This latter finding indicates that the effects of O2 levels are difficult to predict and thus highlights the importance of regulating O2 in cell culture.

Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives.

Most conventional incubators used in cell culture do not regulate O2 levels, making the headspace O2 concentration ~18%. In contrast, most human tissues are exposed to 2-6% O2 (physioxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in standard cell culture practices affect a variety of biological processes. In this review, we discuss how supraphysiological O2 levels affect reactive oxygen species (ROS) metabolism and redox homeostasis, gene expression, replicative lifespan, cellular respiration, and mitochondrial dynamics. Furthermore, we present evidence demonstrating how hyperoxic cell culture conditions fail to recapitulate the physiological and pathological behavior of tissues in vivo, including cases of how O2 alters the cellular response to drugs, hormones, and toxicants. We conclude that maintaining physioxia in cell culture is imperative in order to better replicate in vivo-like tissue physiology and pathology, and to avoid artifacts in research involving cell culture.

Alteration in liver function tests among patients hospitalized for COVID-19: a multicentric study in Peru

Introduction: COVID-19 affects the liver, causing alteration in liver biochemistry tests such as aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), total bilirubin and albumin. Objective: To determine the prevalence of alteration in liver functions tests and associated factors for severity among Peruvian COVID-19 patients. Materials and methods: A descriptive, retrospective and cross-sectional study was performed in 4 public hospitals in Peru. Patients admitted to hospitalization wards and intensive care units with a diagnosis COVID-19 were enrolled. The evaluation of AST, ALT, ALP, total bilirubin and albumin was performed. Associations with demographic and medical data were assessed. Results: 1,100 patients were enrolled, of which 81.7% had altered liver function tests. Only 2.8% of the patients had cirrhosis and 2.1% hepatitis B/C virus. AST and ALT were altered at admission in 64.7% and 63.7%, of the patients respectively. Factors associated with liver injury were: being female OR=0.53 (95% CI: 0.39-0.73; p<0.01), dyslipidemia OR=1.72 (95% CI: 1.10-2.70; p=0.01), previous medication OR=1.56 (95% CI: 1.12 -2.16, p<0.01) and fever OR=1.43 (95% CI: 1.03-1.199, p=0.03). Disease severity was associated with levels of AST and ALT (p<0.01). Patients taking self-medication OR=1.56 (95% CI: 1.12-2.16; p<0.01) and paracetamol OR= 1.41 (95% CI:1.01-1.98; p=0.04) had higher risk of liver injury. Meanwhile, corticosteroids OR=0.55 (95% CI: 0.38-0.78; p<0.01) and enoxaparin OR=0.53 (95% CI: 0.35- 0.81; p<0.01) were protective factors. Conclusions: Peruvian patients with COVID-19 presented high prevalence of alteration in liver function tests, high levels of AST and ALT were related to disease severity.

Introducción: La COVID-19 afecta al hígado, provocando alteración en las pruebas de función hepática como aspartato aminotransferasa (AST), alanina aminotransferasa (ALT), fosfatasa alcalina (FA), bilirrubina total y albúmina. Objetivo: Determinar la prevalencia de alteración en las pruebas de función hepática y su asociación con la severidad en pacientes peruanos con COVID-19. Materiales y métodos: Se realizó un estudio descriptivo, retrospectivo y transversal en 4 hospitales públicos del Perú. Se incluyeron pacientes admitidos en hospitalización y unidades de cuidados intensivos con diagnóstico de COVID-19. Se realizó la evaluación de AST, ALT, FA, bilirrubina total y albúmina. Se evaluaron las asociaciones con datos demográficos y médicos. Resultados: Se incluyeron 1,100 pacientes, de los cuales el 81,7% presentaba alteraciones en las pruebas de función hepática. Solo el 2,8% de los pacientes tenía cirrosis y el 2,1% infección por virus de la hepatitis B / C. Se encontraron niveles alterados de AST y ALT al ingreso en el 64,7% y 63,7% de los pacientes, respectivamente. Los factores asociados con alteración en pruebas de función hepáticas fueron: ser mujer OR = 0,53 (IC 95%: 0,39-0,73; p <0,01), dislipidemia OR=1,72 (IC 95%: 1,10-2,70; p=0,01), uso de medicación previa OR = 1,56 (IC del 95%: 1,12 -2,16, p <0,01) y fiebre OR = 1,43 (IC del 95%: 1,03-1,199, p = 0,03). La gravedad de la enfermedad se asoció con los niveles de AST y ALT (p <0,01). Los pacientes que se automedicaban OR = 1,56 (IC 95%: 1,12-2,16; p <0,01) y tomaban paracetamol OR = 1,41 (IC 95%: 1,01-1,98; p =0,04) tenían mayor riesgo de injuria hepática. Mientras tanto, los corticosteroides OR=0,55 (IC del 95%: 0,38-0,78; p <0,01) y la enoxaparina OR=0,53 (IC del 95%: 0,35-0,81; p <0,01) fueron factores protectores. Conclusiones: los pacientes peruanos con COVID-19 presentaron alta prevalencia de alteración en las pruebas de función hepática, niveles elevados de AST y ALT se relacionaron con la gravedad de la enfermedad.