Transmembrane protein oxygen content and compartmentalization of cells

Recently, there was a report that explored the oxygen content of transmembrane proteins over macroevolutionary time scales where the authors observed a correlation between the geological time of appearance of compartmentalized cells with atmospheric oxygen concentration. The authors predicted, characterized and correlated the differences in the structure and composition of transmembrane proteins from the three kingdoms of life with atmospheric oxygen concentrations in geological timescale. They hypothesized that transmembrane proteins in ancient taxa were selectively excluding oxygen and as this constraint relaxed over time with increase in the levels of atmospheric oxygen the size and number of communication-related transmembrane proteins increased. In summary, they concluded that compartmentalized and non-compartmentalized cells can be distinguished by how oxygen is partitioned at the proteome level. They derived this conclusion from an analysis of 19 taxa. We extended their analysis on a larger sample of taxa comprising 309 eubacterial, 34 archaeal, and 30 eukaryotic complete proteomes and observed that one can not absolutely separate the two groups of cells based on partition of oxygen in their membrane proteins. In addition, the origin of compartmentalized cells is likely to have been driven by an innovation than happened 2700 million years ago in the membrane composition of cells that led to the evolution of endocytosis and exocytosis rather than due to the rise in concentration of atmospheric oxygen.     

LATE PALEOZOIC FUSULINOIDEAN GIGANTISM DRIVEN BY ATMOSPHERIC HYPEROXIA

Atmospheric hyperoxia, with pO(2) in excess of 30%, has long been hypothesized to account for late Paleozoic (360-250 million years ago) gigantism in numerous higher taxa. However, this hypothesis has not been evaluated statistically because comprehensive size data have not been compiled previously at sufficient temporal resolution to permit quantitative analysis. In this study, we test the hyperoxia-gigantism hypothesis by examining the fossil record of fusulinoidean foraminifers, a dramatic example of protistan gigantism with some individuals exceeding 10 cm in length and exceeding their relatives by six orders of magnitude in biovolume. We assembled and examined comprehensive regional and global, species-level datasets containing 270 and 1823 species, respectively. A statistical model of size evolution forced by atmospheric pO(2) is conclusively favored over alternative models based on random walks or a constant tendency toward size increase. Moreover, the ratios of volume to surface area in the largest fusulinoideans are consistent in magnitude and trend with a mathematical model based on oxygen transport limitation. We further validate the hyperoxia-gigantism model through an examination of modern foraminiferal species living along a measured gradient in oxygen concentration. These findings provide the first quantitative confirmation of a direct connection between Paleozoic gigantism and atmospheric hyperoxia.     

The terrestrial biota prior to the origin of land plants (embryophytes): a review of the evidence

It is often assumed that life originated and diversified in the oceans prior to colonizing the land. However, environmental constraints in chemical evolution models point towards critical steps leading to the origin of life as having occurred in subaerial settings. The earliest fossil record does not include finds from terrestrial deposits, so much of our understanding about the presence of a terrestrial microbial cover prior to the Proterozoic is based on inference and geochemical proxies that indicate biospheric carbon cycling during the Archaean. Our assessment is that by 2.7 Ga, microbial ecosystems in terrestrial settings were driven by oxygen‐generating, photosynthetic cyanobacteria. Studies of modern organisms indicate that both the origin and primary diversification of the eukaryotes could have occurred in terrestrial settings, shortly after 2.0 Ga, but there is no direct fossil evidence of terrestrial eukaryotes until about 1.1 Ga. At this time, it appears that the diversity of life in non‐marine habitats exceeded that found in marine settings where sulphidic seas may have impaired eukaryotic physiology and retarded evolution. Geochemical proxies indicate the establishment of an extensive soil‐forming microbial cover by 850 Ma, and it is possible that a rise in atmospheric oxygen at this time was due to the evolutionary expansion of green algae into terrestrial habitats. Direct fossil evidence of the earliest terrestrial biotas in the Phanerozoic consists of problematical palynomorphs from the Cambro‐Ordovician of Laurentia. These indicate that the evolution of the first land plants (embryophytes) during the Middle Ordovician took place within a landscape that included aeroterrestrial algae which were actively adapting to selection in subaerial settings.     

Impacts of Paleo-Oxygen Levels on the Size, Development, Reproduction, and Tracheal Systems of Blatella germanica

Atmospheric oxygen has varied substantially over the Phanerozoic (the last 500 million years) with periods of both hyperoxia and hypoxia relative to today. Unlike some insect groups, cockroaches have not been reported to exhibit gigantism during the late Paleozoic period of hyperoxia. Studies with modern insects have shown a diversity of developmental responses to oxygen, suggesting that evaluation of historical hypotheses should focus on groups most closely related to those present in the Paleozoic. Here we investigated the impacts of Paleozoic oxygen levels (12–31%) on the development of Blatella germanica cockroaches. Body size decreased strongly in hypoxia, but was only mildly affected by hyperoxia. Development time, growth rate and fecundity were negatively impacted by both hypoxia and hyperoxia. Tracheal volumes were inversely proportional to rearing oxygen, suggesting developmental responses aimed at regulating internal oxygen level. The results of these experiments on a modern species are consistent with the fossil record and suggest that changes in atmospheric oxygen would be challenging for many insects, despite plastic compensatory responses in the tracheal system.     

Sterol biosynthesis and prokaryotes-to-eukaryotes evolution

It has been widely accepted that there exists a correlation between prokaryotes-to-eukaryotes evolution and atmospheric oxygen rise. However, it is a great challenge to elucidate the mechanisms underlying the correlation. Considering the facts that cellular communication of eukaryotes depends largely on membrane functions (e.g., endo- and exocytosis) and sterols play a key role in fulfilling these functions, we propose that the biosynthesis of sterols represents a critical step in the prokaryotes-to-eukaryotes evolution. Indeed, sterol biosynthesis is nearly ubiquitous among eukaryotes, but is generally excluded by prokaryotes. More importantly, during the biosynthesis of sterols, oxygen is absolutely required. Therefore, the missing link between prokaryotes-to-eukaryotes evolution and atmospheric oxygen rise is likely to reside in, at least in part, sterol biosynthesis, i.e., high atmospheric oxygen concentration facilitates the generation of sterols and thus benefits the birth of complex organisms.     

Geological constraints on the origin of oxygenic photosynthesis

This article examines the geological evidence for the rise of atmospheric oxygen and the origin of oxygenic photosynthesis. The evidence for the rise of atmospheric oxygen places a minimum time constraint before which oxygenic photosynthesis must have developed, and was subsequently established as the primary control on the atmospheric oxygen level. The geological evidence places the global rise of atmospheric oxygen, termed the Great Oxidation Event (GOE), between ~2.45 and ~2.32 Ga, and it is captured within the Duitschland Formation, which shows a transition from mass-independent to mass-dependent sulfur isotope fractionation. The rise of atmospheric oxygen during this interval is closely associated with a number of environmental changes, such as glaciations and intense continental weathering, and led to dramatic changes in the oxidation state of the ocean and the seawater inventory of transition elements. There are other features of the geologic record predating the GOE by as much as 200–300 million years, perhaps extending as far back as the Mesoarchean–Neoarchean boundary at 2.8 Ga, that suggest the presence of low level, transient or local, oxygenation. If verified, these features would not only imply an earlier origin for oxygenic photosynthesis, but also require a mechanism to decouple oxygen production from oxidation of Earth’s surface environments. Most hypotheses for the GOE suggest that oxygen production by oxygenic photosynthesis is a precondition for the rise of oxygen, but that a synchronous change in atmospheric oxygen level is not required by the onset of this oxygen source. The potential lag-time in the response of Earth surface environments is related to the way that oxygen sinks, such as reduced Fe and sulfur compounds, respond to oxygen production. Changes in oxygen level imply an imbalance in the sources and sinks for oxygen. Changes in the cycling of oxygen have occurred at various times before and after the GOE, and do not appear to require corresponding changes in the intensity of oxygenic photosynthesis. The available geological constraints for these changes do not, however, disallow a direct role for this metabolism. The geological evidence for early oxygen and hypotheses for the controls on oxygen level are the basis for the interpretation of photosynthetic oxygen production as examined in this review.     

Strukturiert erhaltene Fossilien aus dem Archaikum von Südafrika

All organic remains known from the Archean are in such a poor state of preservation, that their biogenicity has been repeatedly doubted. Structures of unquestionable organismic origin have been recently detected in cherts of the Onverwacht group in South Africa. The finds are preserved in a detailed three-dimensional condition. A radiometric age of more than 3 350 mio. y. for the stratum is indicated. With this date, the finds represent the oldest certain evidences of life on earth. Moreover the well-preserved details yield information on the principles of structure and growth of a primeval organism. The body is interpreted as a ramificational system of tiny droplike subunits and appears to be constructed according to the principle of consequent homonomy. It seems possible, that the finds represent an initial form of organismic evolution. With this statement and with the data presently known from the early tellural evolution, it seems possible and credible that life originated on Earth.     

A SHIFT IN THE LONG‐TERM MODE OF FORAMINIFERAN SIZE EVOLUTION CAUSED BY THE END‐PERMIAN MASS EXTINCTION

Size is among the most important traits of any organism, yet the factors that control its evolution remain poorly understood. In this study, we investigate controls on the evolution of organismal size using a newly compiled database of nearly 25,000 foraminiferan species and subspecies spanning the past 400 million years. We find a transition in the pattern of foraminiferan size evolution from correlation with atmospheric pO₂ during the Paleozoic (400–250 million years ago) to long‐term stasis during the post‐Paleozoic (250 million years ago to present). Thus, a dramatic shift in the evolutionary mode coincides with the most severe biotic catastrophe of the Phanerozoic (543 million years ago to present). Paleozoic tracking of pO₂ was confined to Order Fusulinida, whereas Paleozoic lagenides, miliolids, and textulariids were best described by the stasis model. Stasis continued to best describe miliolids and textulariids during post‐Paleozoic time, whereas random walk was the best supported mode for the other diverse orders. The shift in evolutionary dynamics thus appears to have resulted primarily from the selective elimination of fusulinids at the end of the Permian Period. These findings illustrate the potential for mass extinction to alter macroevolutionary dynamics for hundreds of millions of years.     

The early atmosphere: a new picture

Over the last several years, many of the fundamental ideas concerning the composition and chemical evolution of the Earth's early atmosphere have changed. While many aspects of this subject are clouded--either uncertain or unknown, a new picture is emerging. We are just beginning to understand how astronomical, geochemical, and atmospheric processes each contributed to the development of the gaseous envelope around the third planet from the sun some 4.6 billion years ago and how that envelope chemically evolved over the history of our planet. Simple compounds in that gaseous envelope, energized by atmospheric lightning and/or solar ultraviolet radiation, formed molecules of increasing complexity that eventually evolved into the first living systems on our planet. This process is called "chemical evolution" and immediately preceded biological evolution; once life developed and evolved, it began to alter the chemical composition of the atmosphere that provided the very essence of its creation. Photosynthetic organisms which have the ability to biochemically transform carbon dioxide and water to carbohydrates, which they use for food, produce large amounts of molecular oxygen (O2) as a by-product of the reaction. Atmospheric oxygen photochemically formed ozone, which absorbs ultraviolet radiation from the sun and shields the Earth's surface from this biologically lethal radiation. Once atmospheric ozone levels increased sufficiently, life could leave the safety of the oceans and go ashore for the first time. Throughout the history of our planet, there has been strong interaction between life and the atmosphere. Understanding our cosmic roots is particularly relevant as we embark on a search for life outside the Earth. At this very moment, several radio telescopes around the world are searching for extraterrestrial intelligence (SETI).     

Insect eggs exert rapid control over an oxygen-water tradeoff

In terrestrial environments, the exchange of respiratory gases exacts a water cost: obtaining oxygen or carbon dioxide requires losing water. Insect eggs should be especially sensitive to this tradeoff-because they are unable to forage for water, have high surface area-to-volume ratios, and experience large temperature-driven changes in oxygen demand. Previous work from our laboratory, on eggs of a common hawk-moth, Manduca sexta, has shown that, during development, metabolic rate and water loss rates rise in parallel. These correlative data suggest that eggshell conductance increases to accommodate increasing metabolic demand. Here, we test this idea experimentally by subjecting eggs of M. sexta to 15, 21 (normoxia) and 35% oxygen for 24h, while measuring rates of metabolism (as carbon dioxide emission) and water loss. Hypoxia depressed egg metabolic rates, but led to pronounced, rapid increases in water loss. By contrast, hyperoxia had no significant effect on metabolism or water loss. These data demonstrate that insect eggs actively participate in balancing oxygen gain and water loss, and that they use tissue oxygen status, or some correlate of it, as a cue for increasing eggshell conductance. Rapid control over conductance may allow eggs to conserve water during an initial period of low metabolic demand, thereby deferring water costs of respiratory gas exchange until late in development.     

Relationship between postillumination burst of CO2 and enhancement of respiration in tall fescue leaves

The postillumination burst (PIB) of CO₂ and light-enhanced dark respiration (LEDR) depending on oxygen concentration, temperature, respiratory substrates and photorespiratory inhibitor aminoacetonitrile (AAN) were investigated in detached leaves of tall fescue (Festuca arundinacea) using a closed circuit system with an infrared gas analyzer. No PIB was observed in 1 % O₂ under temperature over the range from 15 °C to 35 °C. The rate of LEDR was about twice as low in 1 % O₂ as that in 21 and 50 % O₂ under all temperatures applied. The PIB was absent and LEDR decreased at 21 % O₂ following illumination of leaves for 1 hour at 1 % O₂. When 200 mM glycine or malate solutions were introduced into the leaves of tall fescue, the magnitudes of PIB increased by about 60 and 40 % and rate of LEDR by about 70 % and 40 %, respectively. Pyruvate and succinate were less effective in promotion of PIB and LEDR. AAN had a small stimulatory effect on PIB and LEDR (about 20 % and 10 %, respectively). The dependences between magnitudes of PIB and rates of LEDR were highly correlated (r=0.94). The results presented indicate that atmospheric concentration of oxygen during the period of photosynthesis of tall fescue leaves was necessary not only for occurrence of PIB and LEDR but also for production of substrate(s) (glycine and/or malate) for these phenomena.     

The Metabolism of Oat Leaves during Senescence: I. Respiration, Carbohydrate Metabolism, and the Action of Cytokinins

When the detached first leaves of green or etiolated oat (Avena sativa cv. Victory) seedlings senesce in the dark, their oxygen consumption shows a large increase, beginning after 24 hours and reaching a peak of up to 2.5 times the initial rate by the 3rd day. This effect takes place while the chlorophyll of green leaves, or the carotenoid of etiolated leaves, is steadily decreasing. Kinetin, at a concentration which inhibits the decrease in pigment, completely prevents the respiratory rise; instead, the oxygen consumption drifts downwards. Lower kinetin concentrations have a proportional effect, 50% reduction of respiration being given by about 0.1 mg/l. About one-fifth of the respiratory rise may be attributed to the free amino acids which are liberated during senescence; several amino acids are shown to cause increases of almost 50% in the oxygen consumption when supplied at the concentrations of total amino acid present during senescence. A smaller part of the rise may also be due to soluble sugars liberated during senescence, largely coming from the hydrolysis of a presumptive fructosan. The remainder, and the largest part, of the increase is ascribed to a natural uncoupling of respiration from phosphorylation. This is deduced from the fact that dinitrophenol causes a similar large rise in the oxygen consumption of the fresh leaves or of leaf segments kept green with kinetin, but causes only a very small rise when the oxygen consumption is near its peak in senescent controls. The respiration of these leaves is resistant to cyanide, and 10 mm KCN even increases it by some 30%; in contrast, etiolated leaves of the same age, which undergo a similar rise in oxygen consumption over the same time period, show normal sensitivity to cyanide. The respiratory quotient during senescence goes down as low as 0.7, both with and without kinetin, though it is somewhat increased by supplying sugars or amino acids; glucose or alanine at 0.3 m bring it up to 1.0 and 0.87, respectively.     

The photochemistry of the paleoatmosphere

Summary The ideas of Harold Urey on the origin and evolution of the atmosphere have dominated thinking in this area for 3 decades. Recent progress in this area is reviewed, with particular emphasis on photochemical modeling studies of atmospheric evolution. Research into the paleoatmosphere can be divided into 3 distinct areas: (1) The photochemistry/chemistry of the prebiological paleoatmosphere, (2) the evolution of oxygen and the transition to an oxidizing atmosphere, and (3) the origin and evolution of ozone. Photochemical calculations indicate that the stability of a heavily reducing paleoatmosphere of CH₄—NH₃ was extremely shortlived, if such a prebiological atmosphere ever existed at all. A more mildly reducing early atmosphere of CO₂—N₂ is favored by photochemical considerations. Recent calculations of O₂ in the prebiological paleoatmosphere vary from less than 10^–14 of present atmospheric level (PAL) to 10^–1 PAL. Clearly, additional work is indicated. The evolution of O₃ as a function of O₂ level has been investigated with increasingly detailed photochemical models that have included the photochemistry/chemistry of the oxygen, hydrogen, nitrogen, carbon, and chlorine species, as well as the effects of eddy transport, the rainout of water-soluble species, dry deposition and lightning as a source of trace atmospheric gases.     

Methanogenesis, fires and the regulation of atmospheric oxygen

The Gaia hypothesis states that the composition, oxidation-reduction potential and the temperature of the Earth's lower atmosphere are modulated by and for the biota living on the surface (Lovelock, 1972; Margulis and Lovelock, 1974). A corollary is that atmospheric oxygen is presently regulated at about 21% for the dominant life forms today: vascular plants and metazoa. We suggest that the enormous annual production of methane (of the order of 10¹⁴ mol) is directly related to the short term modulation of oxygen concentration. Atmospheric oxygen results from the burial of reduced carbon; methanogenesis and subsequent atmospheric oxidation of methane prevents that burial. We also present experimental work on the probability of ignition of vegetation as a function of increasing oxygen concentration (Watson, 1978). Both the experiments and consideration of the fossil record lead us to conclude that oxygen has been regulated by methane (and perhaps by N₂O and others) at about 10–25% for very long periods relative to the atmospheric residence times of these reactive gases.     

A paleosol record of the evolution of Cr redox cycling and evidence for an increase in atmospheric oxygen during the Neoproterozoic

Atmospheric oxygen levels control the oxidative side of key biogeochemical cycles and place limits on the development of high‐energy metabolisms. Understanding Earth's oxygenation is thus critical to developing a clearer picture of Earth's long‐term evolution. However, there is currently vigorous debate about even basic aspects of the timing and pattern of the rise of oxygen. Chemical weathering in the terrestrial environment occurs in contact with the atmosphere, making paleosols potentially ideal archives to track the history of atmospheric O₂ levels. Here we present stable chromium isotope data from multiple paleosols that offer snapshots of Earth surface conditions over the last three billion years. The results indicate a secular shift in the oxidative capacity of Earth's surface in the Neoproterozoic and suggest low atmospheric oxygen levels (<1% PAL pO₂) through the majority of Earth's history. The paleosol record also shows that localized Cr oxidation may have begun as early as the Archean, but efficient, modern‐like transport of hexavalent Cr under an O₂‐rich atmosphere did not become common until the Neoproterozoic.     

Hypoxia or in situ normoxia: The stem cell paradigm

Although O₂ concentrations are considerably lowered in vivo, depending on the tissue and cell population in question (some cells need almost anoxic environment for their maintenance) the cell and tissue cultures are usually performed at atmospheric O₂ concentration (20–21%). As an instructive example, the relationship between stem cells and micro‐environmental/culture oxygenation has been recapitulated. The basic principle of stem cell biology, “the generation‐age hypothesis,” and hypoxic metabolic properties of stem cells are considered in the context of the oxygen‐dependent evolution of life and its transposition to ontogenesis and development. A hypothesis relating the self‐renewal with the anaerobic and hypoxic metabolic properties of stem cells and the actual O₂ availability is elaborated (“oxygen stem cell paradigm”). Many examples demonstrated that the cellular response is substantially different at atmospheric O₂ concentration when compared to lower O₂ concentrations which better approximate the physiologic situation. These lower O₂ concentrations, traditionally called “hypoxia” represent, in fact, an in situ normoxia, and should be used in experimentation to get an insight of the real cell/cytokine physiology. The revision of our knowledge on cell/cytokine physiology, which has been acquired ex vivo at non physiological atmospheric (20–21%) O₂ concentrations representing a hyperoxic state for most primate cells, has thus become imperious. J. Cell. Physiol. 219: 271–275, 2009. © 2009 Wiley‐Liss, Inc.     

Free oxygen and evolutionary progress.

A conception of the origin of multicellularity and respiration is presented. It is assumed that the evolution of aerobic life was a phased process. The most important milestones of the process were the appearance, at first, of aerotolerance factors (catalase, peroxidase, Superoxide dismutase), and later, of an aerobic-type energy system (respiratory chain). The consecutive steps of morphophysiological progress are associated with the origin of these enzymes and enzyme systems. The advent of catalase could have given rise to the phenomenon of multicellularity on the basis of complementation of the catalase containing (deprived of another, initial enzyme, essential for the survival of cells) and catalase non-containing (preserving this initial “wild” enzyme) cells. The presence of catalase in multicellular heterotrophic fermenters led to their acquisition of photosynthesizing symbionts—protoplastids (which did not have their own catalase). The occurrence of energetically or physiologically expedient reactions in aerotolerant organisms utilizing free oxygen, did not balance the global effects of photosynthesis (an excessive accumulation of organic matter and oxygen, and a shortage of carbon dioxide). An equilibrium between organic synthesis and destruction processes was restored in the biosphere after the emergence of phosphorylative respiration. The great resemblance of enzyme systems and phosphorylation mechanisms in photosynthesis and respiration suggests that the respiratory assemblies were not created by nature anew, but evolved by way of inversion of the photosynthesizing apparatus in a part of protoplastids which had lost their capacity for photosynthesis (because of the termination of the supply of the radiant energy). The complication of the symbiosis of heterotrophs and photosynthesizers by the rise of a “third element”—protomitochondria —opened up new opportunities, the use of which could have led to a great diversification of cellular forms and thus promoted evolutionary progress.     

Physiological intestinal oxygen modulates the Caco-2 cell model and increases sensitivity to the phytocannabinoid cannabidiol

The Caco-2 cell model is widely used as a model of colon cancer and small intestinal epithelium but, like most cell models, is cultured in atmospheric oxygen conditions (～21%). This does not reflect the physiological oxygen range found in the colon. In this study, we investigated the effect of adapting the Caco-2 cell line to routine culturing in a physiological oxygen (5%) environment. Under these conditions, cells maintain a number of key characteristics of the Caco-2 model, such as increased formation of tight junctions and alkaline phosphatase expression over the differentiation period and maintenance of barrier function. However, these cells exhibit differential oxidative metabolism, proliferate less and become larger during differentiation. In addition, these cells were more sensitive to cannabidiol-induced antiproliferative actions through changes in cellular energetics: from a drop of oxygen consumption rate and loss of mitochondrial membrane integrity in cells treated under atmospheric conditions to an increase in reactive oxygen species in intact mitochondria in cells treated under low-oxygen conditions. Inclusion of an additional physiological parameter, sodium butyrate, into the medium revealed a cannabidiol-induced proliferative response at low doses. These effects could impact on its development as an anticancer therapeutic, but overall, the data supports the principle that culturing cells in microenvironments that more closely mimic the in vivo conditions is important for drug screening and mechanism of action studies.     

Effect of prolonged running on physiological responses to subsequent exercise

The purpose of this study was to compare metabolic and cardiopulmonary responses for submaximal and maximal exercise performed several days preceding (pre-test) and 45 min after (post-test) 21 miles of high intensity (70% VO2 max) treadmill running. Seven aerobically trained subjects' oxygen uptake, oxygen pulse, respiratory exchange ratio, heart rate, pulmonary ventilation, ventilatory equivalent of oxygen, and blood lactate concentration were determined for exercise during the pre- and post-test sessions. No differences were found for submaximal oxygen uptake, oxygen pulse, pulmonary ventilation and ventilatory equivalent of oxygen between the pre- and post-test values. Generally, submaximal heart rate responses were higher, and respiratory exchange ratio values were lower during the post-test. Reductions of maximal work time (12%), maximal oxygen uptake (6%) and maximal blood lactate concentration (47%) were found during the post-test. Thermal stress and glycogen depletion are possible mechanisms which may be responsible for these observed differences.     

Teriflunomide – The common drug with underestimated oxygen - Dependent anticancer potential

Leflunomide (LFN) is a well-known immunomodulatory and anti-inflammatory prodrug of teriflunomide (TFN). Due to pyrimidine synthesis inhibition TFN also exhibits potent anticancer effect. Because, there is the strict coupling between the pyrimidine synthesis and the mitochondrial respiratory chain, the oxygen level could modify the cytostatic TNF effect.The aim of the study was to evaluate the cytostatic effect of pharmacologically achievable teriflunomide (TFN) concentrations at physiological oxygen levels, i.e. 1% hypoxia and 10% tissue normoxia compared to 21%oxygen level occurred in routine cell culture environment.The TFN effect was evaluated using TB, MTT and FITC Annexin tests for human primary (SW480) and metastatic (SW620) colon cancer cell lines at various oxygen levels.We demonstrated significant differences between proliferation, survival and apoptosis at 1, 10 and 21% oxygen in primary and metastatic colon cancer cell lines (SW480, SW620) under TFN treatment. The cytostatic TFN effect was more pronounced at hypoxia compared to tissue and atmospheric normoxia in both cancer cell lines, however metastatic cells were more resistant to antiproliferative and proapoptotic TFN action. The early apoptosis was predominant in physiological oxygen tension while in atmospheric normoxia the late apoptosis was induced.Our findings showed that anticancer TFN effect is more strong in physiological oxygen compared to atmospheric normoxia. It suggests that results obtained from in vitro studies could be underestimated. Thus, it gives assumption for future comprehensive studies at real oxygen environment involving TNF use in combination with other antitumor agents affecting oxygen-dependent pyrimidine synthesis.