Regions Of Low Oxygen Tension Research Articles

An Ethacrynic Acid‐Brominated BODIPY Photosensitizer (EA‐BPS) Construct Enhances the Lethality of Reactive Oxygen Species in Hypoxic Tumor‐Targeted Photodynamic Therapy

AbstractDespite being a clinically approved intervention for cancer, photodynamic therapy (PDT) still suffers from limitations. Prime among these is a therapeutic response that is mostly oxygen dependent. This limits the utility of PDT in treating hypoxic tumors since lower levels of cytotoxic reactive oxygen species (ROS) are generated in regions of low oxygen tension. Glutathione‐pi (GST‐pi) is a key enzyme that militates against ROS‐mediated apoptosis. We report herein a new construct, EA‐BPS, that contains both a brominated BODIPY photosensitizer (BPS) and an ethacrynic acid (EA) GST‐pi inhibitor. Photoirradiation of EA‐BPS induces a synergistic antitumor effect that results from the combination of ROS production and GST‐pi inhibition. Relative to BPS alone, an enhanced cell‐killing effect is seen under hypoxic conditions both in vitro and in vivo. We conclude that by making better use of the available oxygen in tumor environments, improved therapeutic PDT outcomes should be achievable even under hypoxic conditions.

An Ethacrynic Acid-Brominated BODIPY Photosensitizer (EA-BPS) Construct Enhances the Lethality of Reactive Oxygen Species in Hypoxic Tumor-Targeted Photodynamic Therapy.

Despite being a clinically approved intervention for cancer, photodynamic therapy (PDT) still suffers from limitations. Prime among these is a therapeutic response that is mostly oxygen dependent. This limits the utility of PDT in treating hypoxic tumors since lower levels of cytotoxic reactive oxygen species (ROS) are generated in regions of low oxygen tension. Glutathione-pi (GST-pi) is a key enzyme that militates against ROS-mediated apoptosis. We report herein a new construct, EA-BPS, that contains both a brominated BODIPY photosensitizer (BPS) and an ethacrynic acid (EA) GST-pi inhibitor. Photoirradiation of EA-BPS induces a synergistic antitumor effect that results from the combination of ROS production and GST-pi inhibition. Relative to BPS alone, an enhanced cell-killing effect is seen under hypoxic conditions both in vitro and in vivo. We conclude that by making better use of the available oxygen in tumor environments, improved therapeutic PDT outcomes should be achievable even under hypoxic conditions.

5-(Hydroxymethyl)furfural restores low-oxygen rheology of sickle trait blood in vitro.

Sickle cell trait (SCT) is the benign heterozygous carrier state for the sickle variant of the HBB gene. Most of the ~300million people with SCT worldwide will not experience any significant complications. However, accumulating evidence finds SCT associated with increased risk for the common conditions of chronic kidney disease and venous thromboembolism, and severe but rare renal medullary carcinoma and exercise-induced rhabdomyolysis. The mechanism is uncertain, but probably involves pathological rheology of SCT blood in regions of low oxygen tension, resulting from sickle haemoglobin polymerization in SCT red cells and leading to reduced blood flow and further tissue hypoxia and damage. Here, we used an in vitro microfluidic flow system to study the oxygen-dependent rheology of SCT blood and show that 5-(hydroxymethyl)furfural, a natural breakdown product of glucose and fructose-containing foods, such as fruit juices, can reduce the effects of hypoxia on SCT blood rheology in vitro, restoring near-normal flow velocities at very low oxygen. While opinions regarding the clinical significance of the risks associated with SCT are still evolving, these results suggest that a compound present in some food may provide a potential approach for managing risks that may be associated with SCT.

Hypoxia Preconditioning of Bone Marrow Mesenchymal Stem Cells Before Implantation in Orthopaedics.

Human bone marrow mesenchymal stem cells (hBM-MSCs) have been widely used for tissue engineering applications because of their abundance in adults, relatively easy isolation, ability to differentiate into multiple mesodermal lineages, and capacity to secrete a spectrum of bioactive/therapeutic/trophic factors with chemoattractive, immunomodulatory, angiogenic, antiscarring, and antiapoptotic functions.1–6 Despite the notable advances in the field, their ex vivo expansion remains challenging, often associated with phenotypic drift and senescence, imposing the need for engineering more appropriate/bioinspired in vitro microenvironments to maintain their function and therapeutic potential.7,8 Among the various in vitro microenvironment modulators, physiologically relevant (to the cells) low oxygen tension (frequently termed hypoxia) has been characterized as a critical component of the stem cell niche.9–11 It has even been suggested that hypoxia preconditioning of BM-MSCs should become a clinical standard before their implantation.12 This can be rationalized considering that in vivo tissues and organs undergo a broad range of oxygen tensions, depending on their distance from the capillaries, which is markedly lower than the inhalation oxygen tension (∼20%). After entering the lungs, oxygen travels through the bloodstream, and when it reaches the different organs, oxygen tension ranges from 2% to 9%.13 In the bone marrow, for example, BM-MSCs reside in regions of low oxygen tension, ranging from 1% to 6%.10,11 Herein, the influence of low oxygen tension in hBM-MSC in vitro and in vivo fate, with emphasis to orthopaedic clinical indications, is briefly discussed. Several studies have advocated the beneficial effects of low oxygen tension in hBM-MSCs fate. Hypoxia has been shown to maintain hBM-MSCs undifferentiated state and multipotency,14 and to suppress senescence, typically observed at 20% oxygen tension, via the downregulation of the expression of p16 and extracellular signal regulated kinase.15 In addition, 2% oxygen tension in combination with macromolecular crowding, a biophysical phenomenon known to enhance and accelerate (over 80-fold) extracellular matrix deposition,16–18 resulted in a microenvironment capable of maintaining the phenotype of hBM-MSCs and their multilineage potential.19 It has been reported that the hypoxia-inducible factor-1α is activated under low oxygen conditions, which in turn activates the Twist-related protein and downregulates the cyclin-dependent kinase inhibitor 1 (p21), which stimulates cell proliferation.20 Moreover, hypoxia-activated hypoxia-inducible factor-1α significantly increased hBM-MSCs migration through downregulation of integrin α4 and upregulation of Rho-associated kinase ROCK1 and serine/threonine kinase (Rac1/2/3) pathways.21 Furthermore, hBM-MSCs cultured at 1% oxygen and supplemented with fibroblast growth factor-2 exhibited enhanced cell proliferation, chondrogenesis, osteogenesis, migration, collagen formation and regenerative potential.22 Regarding chondrogenic induction, a wealth of studies has demonstrated the beneficial effect of low oxygen tension. For example, isolation and expansion of hBM-MSCs at 2%23 and 3%24 oxygen tension have been shown to promote chondrogenesis, compared with cells cultured at 20% to 21% oxygen tension, as revealed by the increased expression of chondrogenic-related genes and higher glycosaminoglycan content. Notably, in vivo implantation of hypoxia preconditioning hBM-MSCs embedded in alginate beads enhanced the chondrocyte phenotype of cells, producing a cartilage-like tissue in athymic mice.25 Hypoxia preconditioning of BM-MSCs has also been shown to promote osteogenesis in both in vitro and in vivo settings. Cells cultured at 1% oxygen tension in Arg-Gly-Asp-alginate hydrogels were shown to promote osteogenic and angiogenic responses.26 Two percent oxygen tension has been shown to increase the proliferation and the osteogenic differentiation of stem cells from donors with osteonecrosis of the femoral head, suggesting hypoxia preconditioning as an effective treatment to stimulate bone healing.27 In immunocompromised mice, preconditioned at 1% oxygen tension, as opposed to 20 % oxygen tension, hBM-MSCs that were seeded on hydroxyapatite/tricalcium phosphate-based scaffolds produced soluble and insoluble collagen that brought about a stable extracellular matrix and resulted in improved regenerative potential of the bone.28 Physiologically, for the hBM-MSCs, low oxygen tension has been shown to be beneficial to their proliferation, migration, plasticity, and differentiation in vitro and to augment their therapeutic potential in vivo. Yet again, only a handful of studies have assessed the therapeutic potential of hypoxia preconditioned stem cells in clinic. For example, intracoronary administration of hypoxia preconditioned of hBM-MSCs after acute myocardial infarction has been shown to be safe and feasible.29 It is imperative to conduct more clinical trials to assess the efficacy and efficiency of low oxygen tension-treated hBM-MSCs in orthopaedic clinical indications.

Hypoxia imaging and radiotherapy: bridging the resolution gap.

Oxygen distribution is a major determinant of treatment success in radiotherapy, with well-oxygenated tumour regions responding by up to a factor of three relative to anoxic volumes. Conversely, tumour hypoxia is associated with treatment resistance and negative prognosis. Tumour oxygenation is highly heterogeneous and difficult to measure directly. The recent advent of functional hypoxia imaging modalities such as fluorine-18 fluoromisonidazole positron emission tomography have shown promise in non-invasively determining regions of low oxygen tension. This raises the prospect of selectively increasing dose to hypoxic subvolumes, a concept known as dose painting. Yet while this is a promising approach, oxygen-mediated radioresistance is inherently a multiscale problem, and there are still a number of substantial challenges that must be overcome if hypoxia dose painting is to be successfully implemented. Current imaging modalities are limited by the physics of such systems to have resolutions in the millimetre regime, whereas oxygen distribution varies over a micron scale, and treatment delivery is typically modulated on a centimetre scale. In this review, we examine the mechanistic basis and implications of the radiobiological oxygen effect, the factors influencing microscopic heterogeneity in tumour oxygenation and the consequent challenges in the interpretation of clinical hypoxia imaging (in particular fluorine-18 fluoromisonidazole positron emission tomography). We also discuss dose-painting approaches and outline challenges that must be addressed to improve this treatment paradigm.

Targeting the hypoxic fraction of tumours using hypoxia-activated prodrugs

The presence of a microenvironment within most tumours containing regions of low oxygen tension or hypoxia has profound biological and therapeutic implications. Tumour hypoxia is known to promote the development of an aggressive phenotype, resistance to both chemotherapy and radiotherapy and is strongly associated with poor clinical outcome. Paradoxically, it is recognised as a high-priority target and one of the therapeutic strategies designed to eradicate hypoxic cells in tumours is a group of compounds known collectively as hypoxia-activated prodrugs (HAPs) or bioreductive drugs. These drugs are inactive prodrugs that require enzymatic activation (typically by 1 or 2 electron oxidoreductases) to generate cytotoxic species with selectivity for hypoxic cells being determined by (1) the ability of oxygen to either reverse or inhibit the activation process and (2) the presence of elevated expression of oxidoreductases in tumours. The concepts underpinning HAP development were established over 40 years ago and have been refined over the years to produce a new generation of HAPs that are under preclinical and clinical development. The purpose of this article is to describe current progress in the development of HAPs focusing on the mechanisms of action, preclinical properties and clinical progress of leading examples.

Pronounced hypoxia in the subventricular zone following traumatic brain injury and the neural stem/progenitor cell response.

Traumatic brain injury (TBI) elicits identifiable changes within the adult subventricular zone (SVZ). Previously, we demonstrated that EphB3/ephrinB3 interaction inhibits neural stem/progenitor cell (NSPC) proliferation and downregulating this pathway following TBI plays a pivotal role in the expansion of the SVZ neurogenic compartment. It remains unclear, however, what early initiating factors may precede these changes. Using hypoxyprobe-1 (HPb) to identify regions of low oxygen tension or hypoxia (<1%), we found HPb uptake throughout the cortex (CTX), corpus callosum (CC) and SVZ within the first 24 h following controlled cortical impact (CCI) injury. At this early time point, HPb co-localized with EphB3 in the SVZ. NSPC specific markers also co-localized with HPb staining throughout the lateral wall of the ventricle. To determine the cell autonomous effects of hypoxia on EphB3/ephrinB3 signaling in NSPCs, we used an in vitro model of hypoxia to mimic 1% oxygen in the presence and absence of soluble aggregated ephrinB3 (eB3). As expected, hypoxia stimulated the uptake of 5-bromo-2'-deoxyuridine (BrdU) and reduced cell death. Coincident with these proliferative changes, both Hif1-α and phospho (p)-AKT were increased while EphB3 expression was decreased. Stimulation of EphB3 attenuated hypoxia-induced proliferation and prevented phosphorylation of AKT. Hif1-α accumulation, on the other hand, was not affected by EphB3/ephrinB3 signaling. These findings indicate that this pathway limits the NSPC response to hypoxic stimuli. These studies also suggest that early transient changes in oxygen tension following localized cortical injury may initiate a growth-promoting response in the SVZ.

Mitochondrial Bioenergetics of Metastatic Breast Cancer Cells in Response to Dynamic Changes in Oxygen Tension: Effects of HIF-1α

Solid tumors are characterized by regions of low oxygen tension (OT), which play a central role in tumor progression and resistance to therapy. Low OT affects mitochondrial function and for the cells to survive, mitochondria must functionally adapt to low OT to maintain the cellular bioenergetics. In this study, a novel experimental approach was developed to examine the real-time bioenergetic changes in breast cancer cells (BCCs) during adaptation to OT (from 20% to <1% oxygen) using sensitive extracellular flux technology. Oxygen was gradually removed from the medium, and the bioenergetics of metastatic BCCs (MDA-MB-231 and MCF10CA clones) was compared with non-tumorigenic (MCF10A) cells. BCCs, but not MCF10A, rapidly responded to low OT by stabilizing HIF-1α and increasing HIF-1α responsive gene expression and glucose uptake. BCCs also increased extracellular acidification rate (ECAR), which was markedly lower in MCF10A. Interestingly, BCCs exhibited a biphasic response in basal respiration as the OT was reduced from 20% to <1%. The initial stimulation of oxygen consumption is found to be due to increased mitochondrial respiration. This effect was HIF-1α-dependent, as silencing HIF-1α abolished the biphasic response. During hypoxia and reoxygenation, BCCs also maintained oxygen consumption rates at specific OT; however, HIF-1α silenced BCC were less responsive to changes in OT. Our results suggest that HIF-1α provides a high degree of bioenergetic flexibility under different OT which may confer an adaptive advantage for BCC survival in the tumor microenvironment and during invasion and metastasis. This study thus provides direct evidence for the cross-talk between HIF-1α and mitochondria during adaptation to low OT by BCCs and may be useful in identifying novel therapeutic agents that target the bioenergetics of BCCs in response to low OT.

Abstract 1864: Mitochondrial bioenergetics of metastatic breast cancer cells in response to decreasing oxygen tension.

Abstract Solid tumors are characterized by regions of low oxygen tension, which play a central role in tumor progression and resistance to therapy. The major organelle affected by reduced oxygen tension is the mitochondrion, which has to functionally adapt to maintain cellular bioenergetics for the cell survival. In the present study, a novel experimental approach was developed to examine the real-time bioenergetic changes during adaptation to various oxygen tensions ranging from 20% to &amp;lt; 1% oxygen using highly sensitive extracellular flux technology. Oxygen was gradually removed from the culture medium, and bioenergetic changes were measured in normal breast epithelial (MCF10A) and metastatic cancer cell lines (MDA-MB-231 and MCF10CA clones). We found that breast cancer cells, but not MCF10A cells, rapidly responded to low oxygen tension by stabilizing HIF-1α, increasing hypoxia responsive gene expression, and stimulating cellular uptake of glucose, confirming previous observations. In addition, breast cancer cells increased extracellular acidification rate (ECAR) or glycolysis during adaptation to low oxygen tension, and this effect was markedly lower in MCF10A cells. Interestingly, breast cancer cells exhibited a biphasic response in oxygen consumption rate (OCR) as the oxygen tension was reduced gradually from 20% to &amp;lt;1.0%, a response not previously described. This effect is HIF-1α-dependent, as silencing HIF-1α function in cancer cells completely abolished the biphasic response in OCR and increase in ECAR. Moreover, cancer cells demonstrated the ability to restore and maintain its OCR steadily at specific oxygen tension either during deoxygenation or reoxygenation, an effect not observed in HIF-1α silenced cells. Additional studies confirmed that the initial stimulation of OCR is due to increased mitochondrial respiration. In conclusion, our results suggest that HIF-1α provides high degree of bioenergetic flexibility under different oxygen tensions which may confer an adaptive advantage in the ever-changing tumor microenvironment as well as during invasion and metastasis. Moreover, these differences may be useful in screening novel therapeutic agents that target the bioenergetics of cancer cells in response to low oxygen tension. Citation Format: Praveen K. Vayalil, Anne R. Diers, Claudia R. Olivia, Corinne E. Griguer, Victor Darley-Usmar, Douglas R. Hurst, Danny R. Welch, Aimee Landar. Mitochondrial bioenergetics of metastatic breast cancer cells in response to decreasing oxygen tension. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1864. doi:10.1158/1538-7445.AM2013-1864

Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro

In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34(+) cells and mesenchymal stromal cells with regard to different spatial localizations. Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay. In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner. We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.

Oxygen and cytokine-dependent changes in choline phospholipid saturation in hematopoietic progenitor cells detected by MALDI-TOF mass spectrometry

The adaptation of cells to a changing environment is normally accompanied by rapid and/or chronic remodeling of membrane lipids. In order to understand the role played by membrane lipid metabolism in such responses, it is necessary to characterize in more detail the changes in membrane composition occurring in response to defined stimuli. There has been intense interest in characterizing the “stem cell niche” in recent years and an emerging consensus that stem cells are located in regions of low oxygen tension and probably well-isolated from the blood supply. We report here the use of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry to monitor changes in the composition and saturation degree of choline phospholipids of hematopoietic progenitor (FDCPmix) cells under standard nutrient-rich culture conditions and at low oxygen and low glucose concentrations. We found that the increase in proliferation rate driven by high concentrations of interleukin-3 (IL-3) is associated with a decrease in membrane phosphatidylcholine (PC) 18:0/20:4 and sphingomyelin (SM) together with an increase in PC 18:0/18:2 and dihydro SM. Furthermore, this effect is most pronounced under low oxygen and low glucose conditions, independent of cell proliferation rates.

Mycobacterium tuberculosis infection induces hypoxic lung lesions in the rat

Hypoxia is believed to influence the metabolic state of Mycobacterium tuberculosis and cause phenotypic drug resistance. Using pimonidazole adduct staining, we show that lung lesions of infected rats contain regions of low oxygen tension. Our results support the use of the rat model for evaluating anaerobic drug activity invivo.

Oxygen transport to muscle tissue where regions of low oxygen tension exist

Most studies devoted to mathematical modelling of oxygen transport to tissue have assumed a constant oxygen consumption, independent of oxygen tension, whilst oxygen concentration remains positive. However, it is more physiologically realistic to allow the oxygen consumption to fall continuously to zero as the oxygen tension falls towards zero for low oxygen tensions. We show that using this more physiologically realistic oxygen consumption in a mathematical model of oxygen transport to tissue gives a significantly different solution to the governing equations when areas of low oxygen tension exist. We then use this method of modelling oxygen consumption to compare the location of regions of low tissue oxygen tension predicted by two previously published mathematical models of oxygen transport. The two models are a partial differential equation (PDE) model, and an ordinary differential equation (ODE) model that is a simplification of the PDE model. We show that most of the predictions of the ODE model are almost identical to those of the PDE model, but there are some significant differences.

Hypoxia and hypoxia inducible factors (HIF) as important regulators of tumor physiology.

Regions of low oxygen tension are common findings in malignant tumors and are associated with increased frequency of tumor invasion and metastasis. Indeed, the ability to initiate homeostatic responses and adapt to hypoxia, e.g. by induction of angiogenesis, represents an important and crucial aspect in solid tumor growth. A significant advance in our understanding of the hypoxia response stems from the discovery of the hypoxia inducible factors (HIF) which act as key regulators of hypoxia-induced gene expression. Both, low levels of oxygen, apparently via reduced activity of a recently identified class of 2-oxoglutarate dependent oxygenases, and various tumor specific genetic alterations synergistically act to induce the HIF system. A widespread HIF activation can be observed in a variety of malignant tumors including brain tumors. The HIF system induces adaptive responses including angiogenesis, glycolysis, and pH regulation which confer increased resistance towards the hostile tumor microenvironment. Apart from protumorigenic the wide-ranging HIF pathway is known to harbor antitumorigenic components, which may, however, be disabled by tumor specific genetic alterations. Thus, mounting evidence has identified HIF as a crucial regulator of tumor growth and progression constituting an intriguing and novel target for therapeutic intervention.

NADPH:cytochrome c (P450) reductase activates tirapazamine (SR4233) to restore hypoxic and oxic cytotoxicity in an aerobic resistant derivative of the A549 lung cancer cell line.

Tirapazamine (TPZ, SR4233, WIN 59075) is a bioreductive drug that is activated in regions of low oxygen tension to a cytotoxic radical intermediate. This labile metabolite shows high selective toxicity towards hypoxic cells, such as those found in solid tumours. Under aerobic conditions, redox cycling occurs with subsequent generation of superoxide radicals, which are also cytotoxic. NADPH:cytochrome c (P450) reductase (P450R) is a one-electron reducing enzyme that efficiently activates TPZ. Recently a derivative of the A549 non-small cell lung cancer cell line (A549c50) was generated that showed substantially reduced P450R activity compared to its parental line (Elwell et al (1997) Biochem Pharmacol54: 249–257). Here, it is demonstrated that the A549c50 cells are markedly more resistant to TPZ under both aerobic and hypoxic conditions. In addition, these cells have a dramatically impaired ability to metabolize TPZ to its two-electron reduction product, SR4317, under hypoxic conditions when compared to wild-type cells. P450R activity in the A549c50 cells was reintroduced to similar levels as that seen in the parental A549 cells by transfection of the full-length cDNA for human P450R. These P450R over-expressing cells exhibit restored sensitivity to TPZ under both aerobic and hypoxic conditions, comparable to that found in the original parental A549 cells. Further, the ability of the transfected cells to metabolize TPZ to SR4317 under hypoxic conditions is also shown to be restored. This provides further evidence that P450R can play an important role in the activation, metabolism and toxicity of this lead bioreductive drug. © 2000 Cancer Research Campaign

EVALUATION OF THE CYTOTOXICITY OF THE INDOLOQUINONE EO9 IN A HUMAN COLON ADENOCARCINOMA MODEL

The development of anti-cancer drugs that are active in regions of low oxygen tension within solid tumours is an important goal for the chemotherapist. Equally as important is the utilization of appropriate model systems for their selection. This study describes morphological characteristics of multicellular spheroids derived from the human carcinoma cell line, DLD-1 and the evaluation of an investigational bioreductive alkylating agent (EO9) in monolayers, spheroids and xenografts. Histological examination of the cell line in vitro revealed typical features of glandular epithelium with microvilli on free surfaces and cell junction formation. Spheroids had acina formation, extensive necrosis and hypoxia at the time of treatment suggesting the spheroid model to be more representative of solid tumour geometry than more conventional in vitro test systems. EO9 is active against this cell line grown as a monolayer (IC50=0.76 mug ml-1) but is inactive against spheroids or established solid tumours in vivo. The suitability of this system for evaluating bioreductive drugs is discussed.

Studies on Plastron Respiration

ABSTRACT Aphelocheirus lives in swiftly flowing streams, an environment in which, chiefly because of its method of plastron respiration, it must be able to maintain itself in a particular stretch of river and must especially have some safeguard against sudden and uncontrolled descent into regions of low oxygen tension. These necessary powers of orientation are provided : (a) by the ordinary type of tactile trichoid sensillum, (b) by light-sensitive organs, and (c) by specialized pressure receptors. When stationary or when climbing on weeds and stones the insect should have no difficulty in appreciating direction and rate of flow of the water by means of its antennae and trichoid sensilla. When swimming the dorsal light response, light compass orientation and probably also rudimentary form sight enable the animal to maintain position except when in deep smoothly flowing water under conditions of very low illumination. A certain gross sensitivity to pressure changes probably resides in the proprioceptors of the cuticle, but, in addition, there are specialized pressure receptors which consist of a pair of oval depressions on the sterna of the second abdominal segment closely associated with the spiracles of that segment and bearing a pile of long recumbent hydrofuge hairs, approximately 60,000 per sq.mm., holding a film of gas. Special tactile sensilla are interspersed among the hydrofuge hairs. The hairs move with the movements of the air-water interface which result from changes in pressure. Experiments have shown that these two organs are exceedingly sensitive both to uniform and differential pressure changes, and that by their means the animal is enabled to direct its swimming upward or downward and also to maintain an even keel irrespective of directional light stimulation. The adult bug responds immediately by jerky upward swims when exposed to sudden increase of pressure, and the same response is also elicited independently by oxygen lack. But the insect cannot fully distinguish between the two stimuli and is unable to distinguish loss of gas by the plastron due to unsaturation of the medium from an actual pressure increase. Thus bugs placed in water deficient in nitrogen but containing 20% saturation in oxygen give persistent pressure-increment responses. The possibility is considered that stimulation of the pressure receptors caused by changes in the gas pressure in the tracheal system, due to muscle or body movements or to changes in the gas tension of the medium, might interfere with their efficiency as organs of balance. It is suggested that the small, very compressible, concertina-like air sacs associated with each sense organ serve to damp out such fluctuations and so avoid this difficultv. Aphelocheirus nymphs lack the specific pressure receptors. They are accordingly more dependent on directional light for orientation, and in the dark behave as do adults with the pressure receptors extirpated. Aquatic insects which carry substantial air bubbles with them and so are lighter than water have a ready means of perceiving pressure changes by volume changes of the bubble. Aphelocheirus being heavier than water and carrying no bubble therefore needs the special pressure receptors. Similarly, Nepa, which is also heavier than water, has specialized pressure receptors. The organs of the latter are redescribed and their mode of action compared with those of Aphelocheirus. The action of the Nepa organs is not finely graduated as in Aphelocheirus but is of the ‘all-or-none’ type. The three pairs of organs in Nepa are co-ordinated into a single differential manometer system giving no response to changes in absolute pressure but conferring high sensitivity to relative changes as between different parts of the body and correspondingly accurate orientation in the vertical axis. It is shown how the two types of sense organ are exactly suited to the peculiar contingencies to which the insects are exposed by their particular environments. The experiments of Baunacke on Nepa are repeated and extended and his conclusions confirmed.