Influence of oxygen inhibition on the surface free-energy and dentin bond strength of self-etch adhesives.

We compared the surface free-energies and dentin bond strengths of single-step self-etch adhesives with and without an oxygen-inhibited layer. The labial dentin surfaces of bovine mandibular incisors were wet ground with #600-grit silicon carbide paper. The adhesives were applied to the ground dentin, light-irradiated, and the oxygen-inhibited layer was either retained or removed with ethanol. The surface free-energies were determined by measuring the contact angles of three test liquids placed on the cured adhesives. The dentin bond strengths of specimens with and without the oxygen-inhibited layer were measured. For all surfaces, the value of the estimated surface tension component was relatively constant at 35.5-39.8 mJ m(-2) . The value of the , Lewis acid component increased slightly when the oxygen-inhibited layer was removed, whereas that of the , Lewis base component decreased significantly. The bond strengths of the self-etch adhesives were significantly lower in specimens without an oxygen-inhibited layer (13.2-13.6 MPa) than in those with an oxygen-inhibited layer (17.5-18.4 MPa). These results indicate that the presence of an oxygen-inhibited layer in single-step self-etch adhesives with advanced photoinitiators promotes higher dentin bond strength.

Effect of VEGF trap on normal retinal vascular development and oxygen-induced retinopathy in the dog.

Purpose. To evaluate the effects of a vascular endothelial growth factor trap (VEGF Trap) on retinal vascular development and pathologic neovascularization (NV) in the canine model of oxygen-induced retinopathy (OIR). Methods. Newborn dogs (postnatal day [P]1) were exposed to 100% O(2) and then returned to room air on P5. VEGF Trap (5, 25, or 250 µg) was injected intravitreally in one eye and human FC (hFc) injected in the fellow eye of air control and oxygen-treated dogs on P8. The retinal vasculature and NV were evaluated on P21. Other oxygen-exposed animals received 5 µg of VEGF Trap or hFc on P22 after confirmation of retinopathy of prematurity (ROP)-like pathology and were evaluated at P45. Results. In air controls, both the vascularized area of the retina and the density of superficial capillaries were reduced in 250 or 25 µg VEGF Trap-injected eyes, and deep capillaries were absent. Eyes that received the 5 µg dose were indistinguishable from controls. In oxygen-treated animals, all eyes injected with VEGF Trap exhibited markedly less intravitreal NV than that of hFc-injected fellow eyes, irrespective of dose. Retinal vascular area in OIR animals was significantly reduced in eyes injected with 250 or 25 µg of VEGF Trap, but the 5 µg dose did not inhibit retinal revascularization. Eyes with existing NV that received 5 µg VEGF Trap at P22 exhibited substantial resolution of OIR pathology at P45. Conclusions. The VEGF Trap inhibited the formation of NV, but higher doses also inhibited revascularization of retina when injected at P8. In contrast, the lowest dose tested effectively blocked NV and caused regression of existing NV, without appreciably affecting vasculogenesis or retinal revascularization. These findings suggest that dose selection is an important variable when considering the use of VEGF-targeting agents for the treatment of ROP.

Early suppressive mechanisms and the negative blood oxygenation level-dependent response in human visual cortex.

Functional magnetic resonance imaging (fMRI) studies of early sensory cortex often measure stimulus-driven increases in the blood oxygenation level-dependent (BOLD) signal. However, these positive responses are frequently accompanied by reductions in the BOLD signal in adjacent regions of cortex. Although this negative BOLD response (NBR) is thought to result from neuronal suppression, the precise relationship between local activity, suppression, and perception remains unknown. By measuring BOLD signals in human primary visual cortex while varying the baseline contrast levels in the region affected by the NBR, we tested three physiologically plausible computational models of neuronal modulation that could explain this phenomenon: a subtractive model, a response gain model, and a contrast gain model. We also measured the ability of isoluminant contrast to generate an NBR. We show that the NBR can be modeled as a pathway-specific contrast gain modulation that is strongest outside the fovea. We found a similar spatial bias in a psychophysical study using identical stimuli, although these data indicated a response gain rather than a contrast gain mechanism. We reconcile these findings by proposing (1) that the NBR is associated with a long-range suppressive mechanism that hyperpolarizes a subset of magnocellularly driven neurons at the input to V1, (2) that this suppression is broadly tuned to match the spatial features of the mask region, and (3) that increasing the baseline contrast in the suppressed region drives all neurons in the input layer, reducing the relative contribution of the suppressing subpopulation in the fMRI signal.

Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria.

The assembly of vital reactive iron-sulfur (Fe-S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial-type Fe-S cluster assembly proteins and possess instead an analogous bacterial-type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial-type Fe-S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10-fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe-S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe-S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe-S protein-mediated oxygen detoxification.

Reactions of the flavin mononucleotide in complex I: a combined mechanism describes NADH oxidation coupled to the reduction of APAD+, ferricyanide, or molecular oxygen.

NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory chain enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. Alternatively, NADH oxidation, by the flavin mononucleotide in complex I, can be coupled to the reduction of hydrophilic electron acceptors, in non-energy-transducing reactions. The reduction of molecular oxygen and hydrophilic quinones leads to the production of reactive oxygen species, the reduction of nicotinamide nucleotides leads to transhydrogenation, and "artificial" electron acceptors are widely used to study the mechanism of NADH oxidation. Here, we use a combined modeling strategy to accurately describe data from three flavin-linked electron acceptors (molecular oxygen, APAD(+), and ferricyanide), in the presence and absence of a competitive inhibitor, ADP-ribose. Our combined ping-pong (or ping-pong-pong) mechanism comprises the Michaelis-Menten equation for the reactions of NADH and APAD(+), simple dissociation constants for nonproductive nucleotide-enzyme complexes (defined for specific flavin oxidation states), and second-order rate constants for the reactions of ferricyanide and oxygen. The NADH-dependent parameters are independent of the identity of the electron acceptor. In contrast, a further flavin-linked acceptor, hexaammineruthenium(III), does not obey ping-pong-pong kinetics, and alternative sites for its reaction are discussed. Our analysis provides kinetic and thermodynamic information about the reactions of the flavin active site in complex I that is relevant to understanding the physiologically relevant mechanisms of NADH oxidation and superoxide formation.

Differential sensitivity and mechanism of inhibition of COX-2 oxygenation of arachidonic acid and 2-arachidonoylglycerol by ibuprofen and mefenamic acid.

Ibuprofen and mefenamic acid are weak, competitive inhibitors of cyclooxygenase-2 (COX-2) oxygenation of arachidonic acid (AA) but potent, noncompetitive inhibitors of 2-arachidonoylglycerol (2-AG) oxygenation. The slow, tight-binding inhibitor, indomethacin, is a potent inhibitor of 2-AG and AA oxygenation whereas the rapidly reversible inhibitor, 2'-des-methylindomethacin, is a potent inhibitor of 2-AG oxygenation but a poor inhibitor of AA oxygenation. These observations are consistent with a model in which inhibitors bind in one subunit of COX-2 and inhibit 2-AG binding in the other subunit of the homodimeric protein. In contrast, ibuprofen and mefenamate must bind in both subunits to inhibit AA binding.

Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions.

A new technique for immobilizing H2-photoproducing green algae within a thin (<400 microm) alginate film has been developed. Alginate films with entrapped sulfur/phosphorus-deprived Chlamydomonas reinhardtii, strain cc124, cells demonstrate (a) higher cell density (up to 2,000 microg Chl mL(-1) of matrix), (b) kinetics of H2 photoproduction similar to sulfur-deprived suspension cultures, (c) higher specific rates (up to 12.5 micromol mg(-1) Chl h(-1)) of H2 evolution, (d) light conversion efficiencies to H2 of over 1% and (e) unexpectedly high resistance of the H2-photoproducing system to inactivation by atmospheric O2. The algal cells, entrapped in alginate and then placed in vials containing 21% O2 in the headspace, evolved up to 67% of the H2 gas produced under anaerobic conditions. The results indicate that the lower susceptibility of the immobilized algal H2-producing system to inactivation by O2 depends on two factors: (a) the presence of acetate in the medium, which supports higher rates of respiration and (b) the capability of the alginate polymer itself to effectively separate the entrapped cells from O2 in the liquid and headspace and restrict O2 diffusion into the matrix. The strategy presented for immobilizing algal cells within thin polymeric matrices shows the potential for scale-up and possible future applications.

Quinoid radio-toxin (QRT) induced metabolic changes in mice: an ex vivo and in vivo EPR investigation.

Radio-toxins are toxic metabolites produced by ionizing irradiation and have toxic effects similar to those caused by direct irradiation. We have investigated the effect of a quinoid radio-toxin (QRT) obtained from gamma-irradiated potato tuber on various organs in mice using ex vivo and in vivo EPR spectroscopy. Results indicate a decrease in the activity of ribonucleotide reductase enzyme in spleen of mice treated with 0.2mg QRT. A dose of 2mg QRT was fatal to mice within 45-60 min of treatment. Nitrosyl hemoglobin complexes alpha-(Fe(2+)-NO)alpha-(Fe(2+))beta-(Fe(2+))(2) were detected from spleen, blood, liver, kidney, heart, and lung tissue samples of mice treated with lethal doses of QRT. A significant decrease of pO(2) in liver and brain was observed after administration of QRT at the lethal dose. The time of the appearance of the nitrosyl hemoglobin complex and its intensity varied with the dose of QRT and the type of tissue. These results indicate that the effect of the QRT is more prominent in spleen and to a lesser extent in liver and blood. The QRT action at the lethal doses resulted in an increased hypoxia over time with disruption of compensatory adaptive response. The results indicate similar outcome of QRT as observed with gamma-irradiation.

Mitochondrial toxicity and antioxidant activity of a prenylated flavonoid isolated from Dalea elegans.

The prenylated flavanone 2'-4'-dihidroxy-5'-(1" '-dimethylallyl)-6-prenylpinocembrin) (6PP), isolated from the roots of Dalea elegans, shows antimicrobial activity. The aim of this study was to evaluate mitochondrial toxicity and antioxidant properties of 6PP. Addition of micromolar concentrations of 6PP to rat liver mitochondria, stimulated O2 uptake in state 4 and inhibited it in state 3 when malate-glutamate was the respiratory substrate, and inhibited O2 uptake in state 3 when succinate was the substrate. Highest concentration of 6PP also inhibited O2 uptake in state 4 in the latter case; in both conditions, respiratory control index values were decreased. This flavanone collapsed the mitochondrial membrane potential in a concentration-dependent manner. 6PP also inhibited F0F1-ATPase activity in coupled mitochondria and in submitochondrial particles. In the latter, this compound also inhibited NADH oxidase and succinate dehydrogenase activities. HEp-2 cells were incubated for 24 h with 6PP in presence or absence of 0.5% albumin. As measured by reduction of the mitochondrial-related probe MTT, in the albumin-free condition, 6PP was cytotoxic in a concentration-dependent manner; on the other hand, albumin decreased 6PP effect. In addition, in rat liver microsomes 6PP: (1) inhibited the enzymatic lipid peroxidation, (2) exhibited significant scavenging activity, measured by DPPH reduction assay and (3) demonstrated significant antioxidant activity by decreasing the reduction of Mo(VI) to Mo(V). We suggest that 6PP impairs the hepatic energy metabolism by acting as mitochondrial uncoupler and by inhibiting enzymatic activities linked to the respiratory chain. 6PP also exerts both antioxidant and antiradical activities. Due to its cytotoxicity, this molecule, and its future structure developments, can be considered as a potentially promising therapeutic agent, for instance in cancer chemotherapy.

Antioxidant properties and free radical-scavenging reactivity of a family of hydroxynaphthalenones and dihydroxyanthracenones.

This study was undertaken to investigate the free radical-scavenging and antioxidant activities of various structurally related hydroquinones including hydroxynaphthalenones and dihydroxyanthracenones. Electron spin resonance spectroscopy and spin trapping techniques were used to evaluate the ability of hydroquinones to scavenge hydroxyl, diphenylpicrylhydrazyl, and galvinoxyl radicals. In addition, the oxygen radical absorbing capacity assay using fluorescein (ORAC-FL) was used to obtain the relative antioxidant capacity of these radicals. The rate constants of the first H atom abstraction by 2,2-diphenyl-2-picrylhydrazyl (k(2)), were obtained under pseudo-first-order conditions. The free radical-scavenging activities and k(2) values discriminate well between hydroxynaphthalenones and dihydroxyanthracenones, showing that the latter have better antioxidant properties. The aforementioned experimental data agree with quantum-chemical results demonstrating the relevance of intramolecular H bonding to radical-scavenging activities.

Effect of oxygen inhibition on composite repair strength over time.

The study was aimed at examining whether an oxygen inhibition layer is required for bonding a repairing to a pre-existing composite, and to determine the time required for free radicals within a composite substrate to decay to the extent that the composite repair strength drops significantly. Ten slabs of Gradia Direct Anterior (GC Corp.) were divided into (1) control group: an interfacial oxygen inhibition layer was created by applying and light-curing two layers of bonding resin (D/E Resin, Bisco) to the slabs surface in atmospheric air; (2) experimental group: the absence of an interfacial oxygen inhibition layer was obtained by light-curing the second bonding resin layer in a nitrogen atmosphere. After 1 and 2 h, 1, 14, and 30 days of air storage, a composite repair was layered over the bonding resin. Microtensile bond strengths were measured and statistically analyzed. The curing atmosphere was not a significant factor for bond strength (p = 0.82), and time and curing atmosphere-time interaction were significant (p < 0.001). The 30 day-strengths were the lowest (p < 0.05). An oxygen-inhibited layer is not initially required for bonding to resin composite, and it takes more than 14 days before the bond strength between a pre-existing and a fresh composite drops.

Increased prolyl 4-hydroxylase domain proteins compensate for decreased oxygen levels. Evidence for an autoregulatory oxygen-sensing system.

Prolyl 4-hydroxylase domain (PHD) proteins are oxygen-dependent enzymes that hydroxylate hypoxia-inducible transcription factor (HIF) alpha-subunits, leading to their subsequent ubiquitination and degradation. Paradoxically, the expression of two family members (PHD2 and PHD3) is induced in hypoxic cell culture despite the reduced availability of the oxygen co-substrate, and it has been suggested that they become functionally relevant following re-oxygenation to rapidly terminate the HIF response. Here we show that PHDs are also induced in hypoxic mice in vivo, albeit in a tissue-specific manner. As demonstrated under chronically hypoxic conditions in vitro, PHD2 and PHD3 show a transient maximum but remain up-regulated over more than 10 days, suggesting a feedback down-regulation of HIF-1alpha which then levels off at a novel set point. Indeed, hypoxic induction of PHD2 and PHD3 is paralleled by the attenuation of endogenous HIF-1alpha. Using an engineered oxygen-sensitive reporter gene in a cellular background lacking endogenous HIF-1alpha and hence inducible PHD expression, we could show that increased exogenous PHD levels can compensate for a wide range of hypoxic conditions. Similar data were obtained in a reconstituted cell-free system in vitro. In summary, these results suggest that due to their high O2 Km values, PHDs have optimal oxygen-sensing properties under all physiologically relevant oxygen concentrations; increased PHDs play a functional role even under oxygen-deprived conditions, allowing the HIF system to adapt to a novel oxygen threshold and to respond to another hypoxic insult. Furthermore, such an autoregulatory oxygen-sensing system would explain how a single mechanism works in a wide variety of differently oxygenated tissues.

R-state haemoglobin with low oxygen affinity: crystal structures of deoxy human and carbonmonoxy horse haemoglobin bound to the effector molecule L35.

Although detailed crystal structures of haemoglobin (Hb) provide a clear understanding of the basic allosteric mechanism of the protein, and how this in turn controls oxygen affinity, recent experiments with artificial effector molecules have shown a far greater control of oxygen binding than with natural heterotropic effectors. Contrary to the established text-book view, these non-physiological compounds are able to reduce oxygen affinity very strongly without switching the protein to the T (tense) state. In an earlier paper we showed that bezafibrate (BZF) binds to a surface pocket on the alpha subunits of R state Hb, strongly reducing the oxygen affinity of this protein conformation. Here we report the crystallisation of Hb with L35, a related compound, and show that this binds to the central cavity of both R and T state Hb. The mechanism by which L35 reduces oxygen affinity is discussed, in relation to spectroscopic studies of effector binding.

Effect of light-cure time of adhesive resin on the thickness of the oxygen-inhibited layer and the microtensile bond strength to dentin.

A thick oxygen-inhibited layer (OIL) on a cured adhesive layer (AL) is believed to result in both good adaptation of composite resin (CR) and high bond strength. A high degree of conversion (DC) of the AL is also needed for durable bonding. This study evaluated the hypothesis that increasing the DC by prolonging the light-curing time of adhesive bonding resin might decrease the bond strength of the adhesive to dentin because of the subsequent thinning of the OIL thickness. The OIL thickness and the DC of solvent-removed One Step and D/E bonding resin of All Bond 2 (Bisco, USA) were measured simultaneously with FT-NIR spectroscopy according to increasing light-cure times (10, 20, 30, and 60 s) so as to evaluate their effect on the microtensile bond strength. The bonded interfaces were evaluated using scanning electron microscopy. Excessive irradiation of light-curing adhesives increased the DC, but decreased the OIL thickness. When the OIL was significantly thin by curing the adhesives for 30 or 60 s, defects were observed at the interface between the AL and the CR, as well as at the interface between the AL and the hybrid layer. When the OIL was thick, free radicals from the overlying CR may have diffused into the unreacted monomer mixtures of the OIL, chemically connecting the cured AL and the newly curing composite. It was found that to obtain maximum dentin bond strength, light-curing adhesives should be cured for the irradiation time recommended by the manufacturer.

Exogenous manganous ion at millimolar levels rescues all known dioxygen-sensitive phenotypes of yeast lacking CuZnSOD.

Yeasts lacking copper-zinc superoxide dismutase (sod1Delta) exhibit a broad range of phenotypes, many of which can be rescued by growth in the presence of high levels of ionic manganese. We undertook a comprehensive survey of the effects of manganese on wild-type and sod1Delta yeasts and found that 5 mM Mn2+ rescued all known growth-related phenotypes, such as slow growth in air, temperature sensitivity, specific amino acid auxotrophies, no growth in high oxygen, poor growth in nonfermentable carbon sources, and decreased stationary-phase survival. Iron-related phenotypes-elevated electron paramagnetic resonance detectable ("free") iron, decreased aconitase activity, and fragmenting vacuoles-as well as zinc sensitivity were also rescued. The activity of manganese superoxide dismutase remained constant or was reduced when the yeasts were grown in the presence of MnCl2, indicating that induction of this alternative superoxide dismutase is not the explanation. In contrast to MnCl2 treatment, addition of two manganese-containing superoxide dismutase mimetic compounds to the growth medium did not provide any rescue of sod1Delta yeast growth but rather had an sod1Delta-selective inhibitory effect at micromolar concentrations. Mechanisms by which ionic manganese can effect this rescue, while the mimetic compounds do not, are discussed.

Characteristics of iodide activation and inhibition of oxygen evolution by photosystem II.

Oxygen evolution by photosystem II (PSII) is activated by chloride and other monovalent anions. In this study, the effects of iodide on oxygen evolution activity were investigated using PSII-enriched membrane fragments from spinach. In the absence of Cl(-), the dependence of oxygen evolution activity on I(-) concentration showed activation followed by inhibition in both intact PSII and NaCl-washed PSII, which lacked the PsbP and PsbQ subunits. Using a substrate inhibition model, the range of values of the Michaelis constant K(M) in intact PSII (0.5-1.5 mM) was smaller than that in NaCl-washed PSII (1.5-5 mM), whereas values of the inhibition constant K(I) in intact PSII (9-17 mM) were larger than those in NaCl-washed PSII (1-4 mM). Studies of I(-) inhibition of Cl(-)-activated oxygen evolution in intact PSII revealed that I(-) was primarily an uncompetitive inhibitor, with uncompetitive constant K(i)' = 37 mM and Cl(-)-competitive constant K(i) > 200 mM. This result indicated that the activating Cl(-) must be bound for inhibition to take place, which is consistent with the substrate inhibition model for I(-) activation. The S(2) state multiline and g = 4.1 EPR signals in NaCl-washed PSII were examined in the presence of 3 and 25 mM NaI, corresponding to I(-)-activated and I(-)-inhibited conditions, respectively. The two S(2) state signals were observed at both I(-) concentrations, indicating that I(-) substitutes for Cl(-) in formation of the signals and that advancement to the S(2) state was not prevented by high I(-) concentrations. A model is presented that incorporates the results of this study, including the action of both chloride and iodide.

Reduction-induced inhibition and Mn(II) release from the photosystem II oxygen-evolving complex by hydroquinone or NH2OH are consistent with a Mn(III)/Mn(III)/Mn(IV)/Mn(IV) oxidation state for the dark-adapted enzyme.

Hydroxylamine and hydroquinone were used to probe the oxidation states of Mn in the oxygen-evolving complex of dark-adapted intact (hydroxylamine) and salt-washed (hydroquinone) photosystem II. These preparations were incubated in the dark for 24 h in the presence of increasing reductant/photosystem II ratios, and the loss of oxygen evolution activity and of Mn(II) was determined for each incubation mixture. Monte Carlo simulations of these data yielded models that provide insight into the structure, reactivity, and oxidation states of the manganese in the oxygen-evolving complex. Specifically, the data support oxidation states of Mn(III)(2)/Mn(IV)(2) for the dark stable S(1) state of the O(2)-evolving complex. Activity and Mn(II) loss data were best modeled by assuming an S(1) --> S(-)(1) conversion of intermediate probability, a S(-)(1) --> S(-)(3) reaction of high probability, and subsequent step(s) of low probability. This model predicts that photosystem II Mn clusters that have undergone an initial reduction step become more reactive toward a second reduction, followed by a slower third reduction step. Analysis of the Mn(II) release parameters used to model the data suggests that the photosystem II manganese cluster consists of three Mn atoms that exhibit a facile reactivity with both reductants, and a single Mn that is reducible but sterically trapped at or near its binding site. Activity assays indicate that intact photosystem II centers reduced to S(-)(1) can evolve oxygen upon illumination, but that these centers are inactive in preparations depleted of the extrinsic 23 and 17 kDa polypeptides. Finally, it was found that a substantial population of the tyrosine D radical is reduced by hydroxylamine, but a smaller population reacts with hydroquinone over the course of a 24 h exposure to the reductant.

Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related protein exhibit selective target specificities in response to different forms of DNA damage.

The ataxia telangiectasia mutated (ATM) and ATR (ATM and Rad3-related) protein kinases exert cell cycle delay, in part, by phosphorylating Checkpoint kinase (Chk) 1, Chk2, and p53. It is well established that ATR is activated following UV light-induced DNA damage such as pyrimidine dimers and the 6-(1,2)-dihydro-2-oxo-4-pyrimidinyl-5-methyl-2,4-(1H,3H)-pyrimidinediones, whereas ATM is activated in response to double strand DNA breaks. Here we clarify the activation of these kinases in cells exposed to IR, UV, and hyperoxia, a condition of chronic oxidative stress resulting in clastogenic DNA damage. Phosphorylation on Chk1(Ser-345), Chk2(Thr-68), and p53(Ser-15) following oxidative damage by IR involved both ATM and ATR. In response to ultraviolet radiation-induced stalled replication forks, phosphorylation on Chk1 and p53 required ATR, whereas Chk2 required ATM. Cells exposed to hyperoxia exhibited growth delay in G1, S, and G2 that was disrupted by wortmannin. Consistent with ATM or ATR activation, hyperoxia induced wortmannin-sensitive phosphorylation of Chk1, Chk2, and p53. By using ATM- and ATR-defective cells, phosphorylation on Chk1, Chk2, and p53 was found to be ATM-dependent, whereas ATR also contributed to Chk1 phosphorylation. These data reveal activated ATM and ATR exhibit selective substrate specificity in response to different genotoxic agents.