Effect of normobaric hyperoxia on leukemic cell lines in vitro

Aims and Objectives: The objective of this study was to evaluate the molecular mechanisms following normobaric hyperoxia on acute lymphoblastic leukemic cell lines in vitro through assessment of oxidative stress, DNA-fragmentation, mitochondrial function and apoptosis induction. The study was aiming for a more detailed understanding of the basic molecular alterations in leukemic cells induced by reactive oxygen species. Methods: Acute lymphoblastic leukemic cell lines Jurkat and CCRF-SB were cultured according to standard operating procedures and exposed to a single bout of 65% oxygen for 2 or 18 hours respectively. The oxygen exposure protocol included a control sample before exposure, measurements immediately after exposure and after recovery intervals of 6 hours and 24 hours in normoxic conditions. DNA-fragmentation was assessed using single cell gel electrophoresis (comet assay), protein expression of oxidative stress markers, induction of nitric oxide Synthases and expression of pro- and anti-apoptotic proteins were assessed using western blot, electrophoretic mobility shift assay, or enzyme-linked immunosorbent assay. High-resolution respirometry was applied for measurement of mitochondrial respiratory capacity and complex activity within the electron transfer system. Results: The level of oxidative stress in both cell lines was increased after normobaric hyperoxia, the B-cell with a an immediate response after exposure but fast recovery and the T-cell with a delayed response up to 24 hours after the stimulus. Mitochondrial impairment of the intact cells could not be found, however, changes in the activities of complexes I and II were discovered after hyperoxia compared to normoxic conditions. Apoptosis induction and regulation of nitric oxide synthase expression was solely found in the T-cell. Discussion: Oxidative stress was induced with 65% oxygen in both cell lines and with both exposure schemes. However, molecular and metabolic reactions to reactive oxygen species in B- and T-cells varied greatly. While the B-cell was affected immediately but recovered within 24 hours and only showed a transient activation of apoptotic pathways, the T-cell responded later with DNA-fragmentation, apoptosis induction after long-term exposure, and regulation of nitric oxide synthase expression. Conclusion: Looking at the very different effects of normobaric oxygen on molecular and metabolic alterations in the two cell lines examined, it does seem inadequate to propose a general oxygen exposure scheme for acute lymphocytic leukemia. Sensitisation of each cell line will have to be considered individually. Additional research is needed to separately identify suitable intervals for each cell line before ex vivo or animal experiments can be initiated

Enriched-air/oxygen for pre-flight surface intervals

Introduction: Current guidelines for pre-flight surface intervals (PFSI) of 12-18 hours represent a delay if urgent medevac is required. This study seeks to investigate the potential use of enriched air mixtures and oxygen to decrease PFSI. Methods: Simulated dive profiles assumed a descent rate of 30m/min and an ascent rate of 10m/min. Dive times included descent plus time at maximum depth. Six single dive profiles were selected, at three popular recreational diver certification depths; three profiles at or near the no-decompression time limits for those depths, and three with shorter times. Inert gas pressures at the end of the dives and at 6, 12, 18 and 24 hrs breathing air post-dive were estimated for ZH-L16B compartments using the R package ‘scuba’. Then, still assuming the dives were made on air, surface interval times were estimated to reach equivalent (or less) pressures while breathing EANx32, EANx36, or 100% oxygen. Results: Oxygen-rich gases accelerated removal of inert gas, at least halving the 6h times with EANx32 (mean 2:59 vs 6:00 hours), and reducing the 24 hours PFSI across all tested dives to a mean of 1:07 hours with 100% oxygen (Table 1). Table 1. Pre-flight surface interval (hr:mins) required while breathing either EANx32, EANx36 or O2 before reaching equivalent tissue pressures as either 6, 12, 18 or 24 hours breathing normobaric air Dive Profile D’(~m)/ mins EANx32 EANx36 100% Oxygen 6 hr 12 hr 18 hr 24 hr 6 hr 12 hr 18 hr 24 hr 6 hr 12 hr 18 hr 24 hr 40(12) / 60 2:54 4:15 4:51 5:21 2:28 3:20 3:54 4:15 0:45 0:55 0:59 1:02 40(12) / 120 3:41 5:47 7:12 8:15 3:15 4:55 6:00 6:45 1:11 1:31 1:42 1:48 60(18) / 20 2:05 2:42 3:01 3:14 1:43 2:09 2:22 2:30 0:28 0:31 0:33 0:33 60(18) / 40 3:07 4:11 4:55 5:25 2:31 3:29 2:58 4:19 0:48 0:56 1:00 1:03 60(18) / 55 3:21 4:57 5:57 6:40 2:55 4:09 4:53 5:23 1:00 1:12 1:19 1:22 100(30) / 20 2:46 3:49 4:24 4:48 2:11 3:06 3:31 3:47 0:43 0.50 0:53 0:54 D’=depth in feet of seawater, ~m=approximate depth in metres of seawater, mins=total dive time in minutes from descent to start of ascent, EANx32=Enriched air nitrox containing 32% oxygen, EANx36=Enriched air nitrox containing 36% oxygen, hr=hours of surface interval breathing normobaric air Conclusions: The estimated PFSI from breathing EANx36 decreased to a mean of 42% the equivalent of 6 hours breathing air, and to a mean of 19% the equivalent time for 24 hours of post-dive breathing air. There was some variance with respect to the dive profile, with longer profiles showing less improvement due to greater saturation in slower tissues. This study presents required PFSI time estimates for breathing various gases to reach equivalent estimated compartment pressures after 6-24 hours of breathing normobaric air. These estimated times do not take into account physiological factors such oxygen-induced vasoconstriction, exercise, hydration, etc, and the results will need confirmation by actual human trial(s) before practical adoption

Health and wellbeing of recently active United States scuba divers

Introduction: This study aimed to describe recently active adult scuba divers in the United States (US) and compare their characteristics with other active adults. The research question was: do active scuba divers have different health and wellbeing characteristics, compared with adults active in other pursuits? Methods: The Behavioural Risk Factor Surveillance System (BRFSS) is a proportionally representative annual survey of adults in the US. It is the largest continuous population health survey in the world. Since 2011, data on scuba diving is collected biannually. A comparison group were matched on age, sex, being physically active and state of residence. Results: The dataset comprised 103,686,087 person-years of monthly behavioural data, including 14,360 person years of monthly scuba data. The median weekly frequency of recent scuba diving was 1.0 times per week and the median weekly duration was equivalent to two dives each of one hour. Compared with the comparison group, divers more often earned > USD$50,000 per year, were less frequently married, with fewer children in the house, which they more often owned. They reported being able to afford a doctor if needed within the previous year, but more often reported excellent/good health and excellent/good mental health, despite the divers being 16% more frequently overweight. Conclusions: The results demonstrate a relatively healthy cohort of active scuba divers, confirming previous survey results that active divers are commonly college-educated, unmarried, without children, home owning, often overweight, they often currently drink alcohol, and smoked tobacco in the past, but commonly gave up smoking ten years or more ago

Health and well-being of recently active U.S. scuba divers: potential implications for life expectancy

Introduction/Background Health care, income, education, mental health and housing have not been described for active scuba divers. This study characterizes the demography, health and well-being of active adult U.S. scuba divers, compared with matched U.S. adults active in other pursuits. Materials and Methods The Behavioral Risk Factor Surveillance System (BRFSS) is a proportionally representative annual survey of adults in the U.S. Scuba diving was included in 2011-2019 surveys. A comparison group of three per diver was physically active (in activities other than diving) and matched on survey year, age, sex, and state of residence. Results reported in this study are national estimates. Results The dataset comprised 2,307,980 telephone survey responses, yielding an estimated 103,686,087 person­years of data, including 14,360 person-years of scuba diving data. Diving in Florida and California was 4,626 person-years (32%), weekly frequency one occasion/week, and median minutes/week were 120. In diving there were 137,266 males (80%) and 35,058 (20%) females. Adults aged 2': 65 years com­prised 10% of the active divers. The table presents de­mography, personal circumstances and health status. Summary /Conclusion The picture these results paint is one in which it appears possible that active recreational scuba divers may enjoy additional anticipated life expectancy, though our results do not confirm (or even imply) this. The dive community awaits prospective birth cohort studies that include diving status, (or perhaps twin studies,in which one twin is a recreational scuba diver and the other is not), before we may eve

Rebreather divers’ responses to hyperalkaline hydroxide ingestion and aspiration (‘caustic cocktail’) events

Introduction/Background: Rebreathers recycle exhaled air using soda-lime scrubber material to remove carbon-dioxide and injecting oxygen into the normally closed breathing loop. When water enters the system, it generates a hyperalkaline hydroxide solution, commonly known as a “caustic cocktail”. Unexpectedly ingesting this can potentially result in serious harm. The gold standard first-aid treatment for hyperalkaline ingestion is to flush the mouth with water. This study surveyed caustic cocktail experiences of rebreather divers. Materials and Methods: Rebreather divers were recruited through DAN’s social media, website, or by research team members attending dive-related events. Participants were directed to an online survey that collected information on the participants’ training, equipment, diving history, caustic cocktail incidents, and consequential actions. Results: Of 413 respondents, 394 (95%) identified as male. Mean age was 46 years (SD 10) and median length of CCR certification six years (IQR 3-12). Reported rebreather diving experience ranged from 20 years (median=6 yrs, IQR 3-12). Median self-reported dives experience was 200 (IQR 100-500) and median reported hours of rebreather diving was 300 (IQR 120-750). Forty-four participants (11%) reported 50 hours experience or less. The three most common sources of information for treating caustic cocktail ingestion came from rebreather instructors (322), manufacturers (117), and other divers (112). Of the 249 first-aid treatments applied, 191 involved flushing with salt/fresh water. Other treatments include flushing with mildly acidic solutions such as soda (24), juice (7), or other mild acids (5). Pain scores of divers who sought medical attention (n=34, mean=4.9, SD=2.6,) was almost twice that of divers who did not (n=203, mean=2.6, SD=2.3). Summary/Conclusion: Although most divers treated caustic cocktail ingestion by flushing with water, many did not. Additionally, very few divers reported seeking professional medical advice, although those experiencing greater pain were more likely to seek medical assistance

Incidence of cardiac arrhythmias and left ventricular hypertrophy in recreational scuba divers

Introduction: The aims of this study were to investigate the potential impact of age, sex and body mass index (BMI) upon the incidence of arrhythmias pre- and post- diving, and to identify the prevalence of left ventricular hypertrophy (LVH) in older recreational divers. Methods: Divers aged ≥ 40 years participating in group dive trips had ECG rhythm and echocardiograph recordings before and after diving. Arrhythmias were confirmed by an experienced human reader. LVH was identified by two-dimensional echocardiography. Weighted (0.5 fractional) values were used to account for participation by seven divers in 14 trips. Results: Seventy-seven divers undertook 84 dive trips and recorded 677 dives. Among divers with no pre-trip arrhythmias (n = 55), we observed that 6.5 (12%) recorded post-trip arrhythmias and the median increase was 1.0 arrhythmia. In divers with pre-trip arrhythmias, 14.5 had a median of 1.0 fewer post-trip arrhythmias, 2.0 had no change and 5.5 had a median of 16.0 greater. Age, but neither sex nor BMI, was associated with change in the number of arrhythmias before and after dive trips (P = 0.02). The relative risk for experiencing a change in the frequency of arrhythmias after a diver trip, was 2.1 for each additional 10 years of age (95% CI 1.1, 4.0). Of the 60 divers with imaging of their heart, five had left ventricular hypertrophy. Conclusions: We observed a higher than expected prevalence of arrhythmias. Divers with pre-trip arrhythmias tended to be older than divers without pre-trip arrhythmias (P = 0.02). The prevalence of LVH in our cohort was one quarter of that found post-mortem in scuba fatalities

DecoBubbles.com: The first crowdsourcing initiative to expedite VGE counting in 2D-ultrasound recordings and provide labels for machine learning

Introduction/Background Venous gas emboli (VGE) detected with echocardi­ography post-dive are often used as a marker of decompression stress. The very time-consuming analysis of the acquired videos can help elucidate inter- and intrasubject variability in VGE, and DCS susceptibility. This collaborative project aims to reduce the time needed to count and analyze cardiac ultrasound videos and help train an Al to complete the task. To accomplish this, an interactive web platform 'www.decobubbles.com' was developed that allows crowdsourcing the counting process to lay persons without previous expertise in ultrasound analysis. Materials and Methods Volunteers from around the world are encouraged to register on the website to help with this initiative. After registration the volunteer is prompted to watch an eight-minute training tutorial teaching them how to choose suitable frames in the videos, count VGE and submit their assessment. A volunteer is allowed to participate in the project once they have succeeded in completing a number of training videos in which they need to agree with the counts of professional graders. A database of 204 de-identified post-dive ultrasound videos of variable quality have been uploaded to the application. Results The application on www.decobubbles.com is active since February 2021 and at the beginning of March 2021 had registered 153 unique raters, 587 submitted ratings, and 34% of the 204 videos in the database were fully rated, which means that three independent raters agreed in their counting. Summary/Conclusion This collaborative platform will allow refinement of an Al to count VGE in post-dive echocardiography recordings and advance the knowledge about inter­and intrasubjectVGE variability in divers. Future implications include the possibility for researchers to use the platform to directly analyze their own datasets - with no obligation to add these to our database, although we welcome more videos from other sources and collaborators. This research is funded through the Divers Alert Network (#DAN-UNC-1) and the Office of Naval Research (ONR #N00014-20-1-2590)

Analysis of Single- and Double-Stranded DNA Damage in Osteoblastic Cells after Hyperbaric Oxygen Exposure

(1) Background: Hyperbaric oxygen (HBO) exposure induces oxidative stress that may lead to DNA damage, which has been observed in human peripheral blood lymphocytes or non-human cells. Here, we investigated the impact of hyperbaric conditions on two human osteoblastic cell lines: primary human osteoblasts, HOBs, and the osteogenic tumor cell line SAOS-2. (2) Methods: Cells were exposed to HBO in an experimental hyperbaric chamber (4 ATA, 100% oxygen, 37 °C, and 4 h) or sham-exposed (1 ATA, air, 37 °C, and 4 h). DNA damage was examined before, directly after, and 24 h after exposure with an alkaline comet assay and detection of γH2AX+53BP1 colocalizing double-strand break (DSB) foci and apoptosis. The gene expression of TGFß-1, HO-1, and NQO1, involved in antioxidative functions, was measured with qRT-PCR. (3) Results: The alkaline comet assay showed significantly elevated levels of DNA damage in both cell lines after 4 h of HBO, while the DSB foci were similar to sham. γH2AX analysis indicated a slight increase in apoptosis in both cell lines. The increased expression of HO-1 in HOB and SAOS-2 directly after exposure suggested the induction of an antioxidative response in these cells. Additionally, the expression of TGF-ß1 was negatively affected in HOB cells 4 h after exposure. (4) Conclusions: in summary, this study indicates that osteoblastic cells are sensitive to the DNA-damaging effects of hyperbaric hyperoxia, with the HBO-induced DNA damage consisting largely of single-strand DNA breaks that are rapidly repaired

Pulsed high oxygen induces a hypoxic-like response in Human Umbilical Endothelial Cells (HUVECs) and in humans.

It has been proposed that relative changes of oxygen availability rather than steady state hypoxic or hyperoxic conditions, play an important role in HIF transcriptional effects. According to this hypothesis describing the "normobaric oxygen paradox", normoxia following a hyperoxic event is sensed by tissues as an oxygen shortage, upregulating HIF-1 activity. With the aim of confirming at cellular and at functional level that normoxia following an hyperoxic event is "interpreted" as a hypoxic event, we report a combination of experiments addressing the effects of an intermittent increase of oxygen concentration on HIF-1 levels and the activity level of specific oxygen-modulated proteins in cultured human umbilical vein endothelial cells (HUVECs), and the effects hemoglobin (Hb) levels after intermittent breathing normobaric high (100%) and low (15%) oxygen in vivo in humans. Our experiments confirm that during recovery after hyperoxia, an increase of HIF expression occurs in HUVECs, associated to an increase of matrix metalloproteinases activity. These data suggest that endothelial cells "interpret" the return to normoxia after hyperoxia as a hypoxic stimulus. At functional level, our data show that both breathing 15% and 100% oxygen 30 minutes every other day for a period of 10 days, induces an increase of Hb levels in humans. This effect was enhanced after the cessation of the oxygen breathing. These results indicate that a sudden decrease in tissue oxygen tension after hyperoxia, may act as a trigger for EPO synthesis so corroborating the hypothesis that "relative" hypoxia is a potent stimulator of HIF mediated gene expressions.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe