Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling

Mitochondria are proposed to play an important role in hypoxic cell signaling. One currently accepted signaling paradigm is that the mitochondrial generation of reactive oxygen species (ROS) increases in hypoxia. This is paradoxical, because oxygen is a substrate for ROS generation. Although the response of isolated mitochondrial ROS generation to [O(2)] has been examined previously, such investigations did not apply rigorous control over [O(2)] within the hypoxic signaling range. With the use of open-flow respirometry and fluorimetry, the current study determined the response of isolated rat liver mitochondrial ROS generation to defined steady-state [O(2)] as low as 0.1 microM. In mitochondria respiring under state 4 (quiescent) or state 3 (ATP turnover) conditions, decreased ROS generation was always observed at low [O(2)]. It is concluded that the biochemical mechanism to facilitate increased ROS generation in response to hypoxia in cells is not intrinsic to the mitochondrial respiratory chain alone but may involve other factors. The implications for hypoxic cell signaling are discussed.     

Nitroxides as antioxidants: Tempol protects against EO9 cytotoxicity

Nitroxide free radicals have been shown to be potent antioxidants in a variety of experimental models using diverse means of insults. Among other insults, nitroxides have been shown effective in inhibiting cytotoxicity of quinone-based drugs such as streptonigrin and mitomycin C. These drugs and other chemotherapeutic agents have the potential to undergo bioreductive activation by the normal reducing enzymes within a cell. In the present work we studied the effect of the nitroxide Tempol on the cytotoxicity induced by EO9, a mitomycin C analogue, in HT29 cells under aerobic and hypoxic conditions. The study was aimed to better understand the mechanism of EO9 cytotoxicity and the molecular level of the nitroxide's mode of protection. The reactions of Tempol with activated EO9, and the reactive species formed during EO9 activation were studied in a cell-free solution, using spin-trapping, and electron paramagnetic resonance (EPR) spectrometry. Our results indicate that EO9 induced similar cytotoxicity in HT29 cells under aerobic and hypoxic conditions while Tempol provided similar and almost complete protection to both aerobic and hypoxic cells. The results indicate that EO9 cytotoxicity is due to both 1- and 2-electron reductive activation processes, with aerobic toxicity caused by back-oxidation of the hydroquinone to the semiquinone, EO9.-. Tempol serves both as a useful tool in the study of the mechanisms of quinone-mediated cytotoxicity and as a potent antioxidant against the damaging effects of redox cycling quinones and semiquinones by scavenging of EO9.- or detoxification of O2.- and H2O2.     

Suppressive effects of flavonoids on dioxin toxicity

Dioxin type chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause a variety of toxicity. Most of the toxicity of TCDD has been attributed to a mechanism by which TCDD is bound to aryl hydrocarbon receptor (AhR) and transforms the receptor. Thus, suppression of the AhR transformation by food factors can suppress the dioxin toxicity. In this study, flavonoids at various concentrations were treated to a rat cytosolic fraction containing AhR before adding 1 nM TCDD. The transformed AhR was detected by an electrophoretic mobility shift assay with a DNA oligonucleotide consensus to dioxin response element. As the results, flavones and flavonols at dietary levels act as the antagonists for AhR and suppress the transformation. The antagonistic IC50 values were in a range between 0.14 and 10 microM, which are close to the physiological levels in human. These results suggest that a plant-based diet can prevent the dioxin toxicity.     

Reactive oxygen species attenuate nitric-oxide-mediated hypoxia-inducible factor-1alpha stabilization

Tissue hypoxia/ischemia are major pathophysiological determinants. Conditions of decreased oxygen availability provoke accumulation and activation of hypoxia-inducible factor-1 (HIF-1). Recent reports demonstrate a crucial role of HIF-1 for inflammatory events. Regulation of hypoxic responses by the inflammatory mediators nitric oxide (NO) and reactive oxygen species (ROS) is believed to be of pathophysiolgical relevance. It is reported that hypoxic stabilization of HIF-1alpha can be antagonized by NO due to its ability to attenuate mitochondrial electron transport. Likely, the formation of ROS could contribute to this effect. As conflicting results emerged from several studies showing either decreased or increased ROS production during hypoxia, we used experiments mimicking hypoxic intracellular ROS changes by using the redox cycling agent 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which generates superoxide inside cells. Treatment of A549, HEK293, HepG2, and COS cells with DMNQ resulted in a concentration-dependent raise in ROS which correlated with HIF-1alpha accumulation. By using a HIF-1alpha-von Hippel-Lindau tumor suppressor protein binding assay, we show that ROS produced by DMNQ impaired prolyl hydroxylase activity. When HIF-1alpha is stabilized by NO, low concentrations of DMNQ (<1 microM) revealed no effect, intermediate concentrations of 1 to 40 microM DMNQ attenuated HIF-1alpha accumulation and higher concentrations of DMNQ promoted HIF-1alpha stability. Attenuation of NO-induced HIF-1alpha stability regulation by ROS was mediated by an active proteasomal degradation pathway. In conclusion, we propose that scavenging of NO by ROS and vice versa attenuate HIF-1alpha accumulation in a concentration-dependent manner. This is important to fully elucidate HIF-1alpha regulation under inflammatory conditions.     

Induction of hypoxia in glass versus Permanox petri dishes

The survival of Chinese hamster ovary cells in culture following graded doses of X rays delivered under aerobic and hypoxic conditions, or treatment with the bioreductive drug SR 4233 under hypoxic conditions, was evaluated as a function of whether cells were plated onto glass or Permanox plastic petri dishes. In the case of treatment with SR 4233, the influence of varying the total volume of medium in the dishes was also studied. While the Permanox petri dishes were sufficient to yield "radiobiological" hypoxia, that is, oxygen enhancement ratios of approximately 3.0 were obtained for X irradiation, they were inferior to glass petri dishes with respect to the hypoxia-selective cytotoxicity of SR 4233. For a 90-min hypoxic exposure to 40 microM SR 4233, the surviving fraction of cells plated on plastic dishes averaged about 50-fold higher than that of cells plated on glass dishes. Although varying the total medium volume did affect the extent of SR 4233-induced cytotoxicity for glass dishes--drug toxicity decreased slightly with increasing medium volume--this was not the case for the plastic dishes, in which the cell survival following a fixed SR 4233 exposure was essentially constant as a function of medium volume. These results suggest, at least for SR 4233, and under these experimental conditions, that Permanox petri dishes are not satisfactory for such studies.     

Functional role of AhR in the expression of toxic effects by TCDD

Cytochrome P450 1A1 (CYP1A1) is one of the xenobiotic metabolizing enzymes (XMEs), which is induced by polycyclic aromatic hydrocarbons (PAHs). The most potent inducer of CYP1A1 is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, TCDD induces a broad spectrum of biochemical and toxic effects, such as teratogenesis, immunosuppression and tumor promotion. Most, if not all, of the effects caused by TCDD and other PAHs are known to be mediated by AhR (aryl hydrocarbon receptor or dioxin receptor) which has a high binding affinity to TCDD. The liganded AhR translocates from cytoplasm to nuclei where it switches its partner molecule from Hsp90 to Arnt. Thus formed AhR/Arnt heterodimer binds a specific DNA sequence designated XRE in the promoter region of the target genes including CYP1A1, UDP-glucuronosyl transferase and others to enhance their expression. Although it remains to be studied how AhR is involved in the other TCDD-induced biological effects such as teratogenesis and immunosuppression than induction of XMEs, it is believed that these adverse TCDD effects are caused by untimely activation of gene expression by ligand-activated AhR in the biological process. We summarize the present knowledge about functional role of AhR in TCDD-induced biological effects.     

Production of reactive oxygen by mitochondria from normoxic and hypoxic rat heart tissue.

Reactive oxygen species (ROS), which may be involved in ischemic or reperfusion heart injury, can be produced by mitochondria. Previous work indicated that coupled mitochondria from ischemic heart tissue incubated in calcium-free medium produced less ROS than normal. The effects of calcium, which may be elevated in hypoxic or ischemic tissue, were not examined. The relative production of ROS by mitochondria from normoxic or hypoxic rat heart tissue was estimated by measuring the oxidation of dichlorofluorescin to the fluorescent compound, dichlorofluorescein. ROS were detectable during succinate-stimulated State 4 respiration. In the absence of calcium, mitochondria from hypoxic (60 min) heart tissue produced less ROS than mitochondria from normoxic heart tissue. In the presence of 0.1, 1 or 10 microM calcium, ROS produced by hypoxic mitochondria were increased to normoxic levels. While function was depressed in mitochondria from hypoxic tissue, the presence of 0.1 and 1 microM calcium had no further effect. Respiration was uncoupled in the presence of 10 microM calcium in mitochondria from both normoxic and hypoxic heart tissue. ROS production was increased in mitochondria from hypoxic tissue with both increasing concentrations of calcium and increasing duration of exposure. ROS production in mitochondria from normoxic heart tissue was only stimulated after 200 or more seconds of exposure to 1 or 10 microM calcium. Production of ROS in mitochondria from hypoxic tissue in the presence of 1 microM calcium was inhibited by rotenone (80%), ruthenium red (69%), and a combination of these agents (96%). In contrast, ruthenium red had no effect on ROS production by mitochondria from normoxic heart tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Omeprazole attenuates hyperoxic injury in H441 cells via the aryl hydrocarbon receptor

Hyperoxia contributes to the development of bronchopulmonary dysplasia in premature infants. Earlier we observed that aryl hydrocarbon receptor (AhR)-deficient mice are more susceptible to hyperoxic lung injury than AhR-sufficient mice, and this phenomenon was associated with a lack of expression of cytochrome P450 1A enzymes. Omeprazole, a proton pump inhibitor used in humans with gastric acid-related disorders, activates AhR in hepatocytes in vitro. However, the effects of omeprazole on AhR activation in the lungs and its impact on hyperoxia-induced reactive oxygen species (ROS) generation and inflammation are unknown. In this study, we tested the hypothesis that omeprazole attenuates hyperoxia-induced cytotoxicity, ROS generation, and expression of monocyte chemoattractant protein-1 (MCP-1) in human lung-derived H441 cells via AhR activation. Experimental groups included cells transfected with AhR small interfering RNA (siRNA). Hyperoxia resulted in significant increases in cytotoxicity, ROS generation, and MCP-1 production, which were significantly attenuated with the functional activation of AhR by omeprazole. The protective effects of omeprazole on cytotoxicity, ROS production, and MCP-1 production were lost in H441 cells whose AhR gene was silenced by AhR siRNA. These findings support the hypothesis that omeprazole protects against hyperoxic injury in vitro via AhR activation that is associated with decreased ROS generation and expression of MCP-1.     

Beryllium-stimulated reactive oxygen species and macrophage apoptosis

Beryllium (Be), the etiologic agent of chronic beryllium disease, is a toxic metal that induces apoptosis in human alveolar macrophages. We tested the hypothesis that Be stimulates the formation of reactive oxygen species (ROS) which plays a role in Be-induced macrophage apoptosis. Mouse macrophages were exposed to 100 microM BeSO4 in the absence and presence of the catalytic antioxidant MnTBAP (100 microM). Apoptosis was measured as the percentage of TUNEL+ and caspase-8+ cells. ROS production was measured by flow cytometry using the fluorescence probes, dihydroethidine (DHE) and dichlorofluorescein diacetate (DCFH-DA). Be-exposed macrophages had increased TUNEL+ cells (15+/-1% versus controls 1+/-0.2%, P<0.05) and increased caspase-8+ cells (18.7+/-2% versus controls 1.8+/-0.4%, P<0.05). Be-induced caspase-8 activation, and a 4-fold increase in ROS formation, was ameliorated by exposure to MnTBAP. Hydrogen peroxide (30 microM) exposure potentiated Be-induced caspase-8 activation, and was also attenuated by MnTBAP. Our data are the first to demonstrate that Be stimulates macrophage ROS formation which plays an important role in Be-induced macrophage apoptosis.     

Selenite or selenomethionine interaction with methylmercury on uptake and toxicity showing a weak selenite protection: studies on cultured K-562 cells

Selenium and methylmercuric chloride (MMC) interactions regarding cellular uptake and selenium protection on MMC toxicity have been studied. Human K-562 cells were pretreated or simultaneously treated with either selenite (5 or 50 μM) or selenomethionine (10 or 50 μM) together with (3.5 or 5 μM) MMC. Cells simultaneously treated with selenite or selenomethionine and 3.5 μM MMC showed a decreased mercury concentration with increased selenium dose especially seen in the selenite combinations. The simultaneous selenite and MMC 3.5 μM combinations showed growth curves with an increasing number of viable cells with increased selenite dose. All combinations with 5 μM MMC were toxic to the cells. Interactions between selenite or selenomethionine and MMC regarding cellular uptake of mercury and selenium were observed and indications of selenite protection against MMC toxicity in human K-562 cells were noticed.     

Preclinical studies of porfiromycin as an adjunct to radiotherapy

The bioreductive alkylating agent porfiromycin (POR) is more toxic to EMT6 cells that are hypoxic at the time of treatment than to aerobic cells. The toxicity of POR to hypoxic EMT6 cells in vitro was similar to that of mitomycin C (MC): the aerobic toxicity of POR was considerably less than that of MC. Treatment of cells in vitro with POR before and during irradiation did not sensitize either hypoxic or aerobic cells to X rays; instead, only additive cytotoxicity was produced. In contrast, treatment of solid EMT6 tumors in vivo with POR plus radiation produced supra-additive cytotoxicity, as assessed by analyses of the complete dose-response curves for the killing of tumor cells by radiation alone or by POR alone. The supra-additivity of the combination regimens appeared to reflect the preferential killing by each agent of those tumor cells which were in an environment conferring resistance to the other agent. In contrast, combinations of POR and X rays produced only additive cytotoxicities to marrow CFU-GM. Supra-additive antineoplastic effects were obtained at doses of POR which produced little hematologic or other host toxicity. The complementary cytotoxicities of radiation and POR to cells in different microenvironments in solid tumors and the absence of a similar effect in normal tissue make optimized regimens combining radiotherapy and POR unusually promising for the treatment of solid tumors.     

Synthesis and cytotoxicity of pyranonaphthoquinone natural product analogues under bioreductive conditions

We have synthesised a focused library of derivatives of natural products containing the pyranonaphthoquinone moiety including the first report of such a scaffold with an appended tetrazole functionality. Examples include kalafungin derivatives as well as analogues of nanaomycin and eleutherin. These compounds were assessed for cytotoxic activation by breast cancer cell lines engineered to express the prototypic human one- and two-electron quinone bioreductive enzymes, NADPH: cytochrome P450 oxidoreductase (POR) and NAD(P)H: quinoneoxidoreductase 1 (NQO1; DT-diaphorase), respectively. Several compounds were observed to be cytotoxic at sub-micromolar level and a pattern of increased aerobic potency was observed in cells over expressing POR. A subset of analogues was assessed under anoxic conditions, where cytotoxicity was reduced, implicating redox cycling as a major mechanism of toxicity. The substrate specificity for reductive enzymes is relevant to the future design of bioreductive prodrugs to treat cancer.     

Preliminary Studies with a New Hypoxia-Selective Cytotoxin, KS119W, In Vitro and In Vivo

Agents with selective toxicity to hypoxic cells have shown promise as adjuncts to radiotherapy. Our previous studies showed that the bioreductive alkylating agent KS119 had an extremely large differential toxicity to severely hypoxic and aerobic cells in cell culture, and was effective in killing the hypoxic cells of EMT6 mouse mammary tumors in vivo. However, the limited solubility of that compound precluded its development as an anticancer drug. Here we report our initial studies with KS119W, a water-soluble analog of KS119. The cytotoxicity of KS119W to EMT6 cells in vitro was similar to that of KS119, with both agents producing only minimal cytotoxicity to aerobic cells even after intensive treatments, while producing pronounced cytotoxicity to oxygen-deficient cells. This resulted in large differentials in the toxicities to hypoxic and aerobic cells (>1,000-fold at 10 μM). Low pH had only minimal effects on the cytotoxicity of KS119W. Under hypoxic conditions, EMT6 cells transfected to express high levels of either human or mouse versions of the repair protein O(6)-alkylguanine-DNA alkyltransferase, which is also known as O(6)-methylguanine DNA-methyltransferase, were much more resistant to KS119W than parental EMT6 cells lacking O(6)-alkylguanine-DNA alkyltransferase, confirming the importance of DNA O-6-alkylation to the cytotoxicity of this agent. Studies with EMT6 tumors in BALB/c Rw mice using both tumor cell survival and tumor growth delay assays showed that KS119W was effective as an adjunct to irradiation for the treatment of solid tumors in vivo, producing additive or supra-additive effects in most combination regimens for which the interactions could be evaluated. Our findings encourage additional preclinical studies to examine further the antineoplastic effects of KS119W alone and in combination with radiation, and to examine the pharmacology and toxicology of this new bioreductive alkylating agent so that its potential for clinical use as an adjuvant to radiotherapy can be evaluated.     

A quinoxaline 1,4-di-N-oxide derivative induces DNA oxidative damage not attenuated by vitamin C and E treatment

Some anticancer compounds are pro-drugs which give rise to toxic species through enzymatic reduction. The quinoxaline-di-N-oxide derivative Q-85 HCl (7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride) is a bioreductive compound selectively toxic in hypoxia. Due to the possibility of secondary tumors the study of the genotoxic capability of antitumoral drugs is very important. The aim of this study was to assess the ability of Q-85 HCl to produce reactive oxygen species (ROS) and oxidative DNA damage in Caco-2 cells, both in hypoxia and in well-oxygenated conditions. Secondly, we attempted to evaluate the effect of vitamins C and E under hypoxic and normoxic conditions, in order to determine if these antioxidant substances modify Q-85 HCl effect in hypoxic cells or possibly exert a protective action in normal cells. Caco-2 cells were treated with Q-85 HCl for 2h, at high concentrations in normoxia (0.1-5 microM) and at low concentrations in hypoxia (0.002-0.1 microM). In normoxia, a dose-related significant increase in intracellular ROS level was evident; in hypoxia all the concentrations produced very high level of ROS. Just after the treatment and 24h later, oxidative DNA damage was evaluated by the modified comet assay after post-digestion of the cells with formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (Endo III). Q-85 HCl treatment evoked a significant dose-dependent increase in the total comet score of the cells both in hypoxia and normoxia, indicating that this compound or some metabolite is able to oxidize purine and pyrimidine bases. After 24h DNA damage caused by the compound was completely repaired with only one exception: cells treated with the highest concentration of Q-85 HCl in hypoxia and post-digested with FPG. Vitamin C (5-100 microM) and vitamin E (500-400 microM) did not have a pro-oxidant effect in Caco-2 cells. Treatment of cells with vitamin C (10 microM) or vitamin E (100 microM) did not significantly reduce oxidative DNA damage in hypoxia and normoxia. In conclusion, the use of these vitamins would not hinder toxicity against hypoxic cells, but a protective effect in normoxic cells was not evident.     

Characterization of the effects of oxygen on xanthine oxidase-mediated nitric oxide formation

Under anaerobic conditions, xanthine oxidase (XO)-catalyzed nitrite reduction can be an important source of nitric oxide (NO). However, questions remain regarding whether significant XO-mediated NO generation also occurs under aerobic conditions. Therefore, electron paramagnetic resonance, chemiluminescence NO-analyzer, and NO-electrode studies were performed to characterize the kinetics and magnitude of XO-mediated nitrite reduction as a function of oxygen tension. With substrates xanthine or 2,3-dihydroxybenz-aldehyde that provide electrons to XO at the molybdenum site, the rate of NO production followed Michaelis-Menten kinetics, and oxygen functioned as a competitive inhibitor of nitrite reduction. However, with flavin-adenine dinucleotide site-binding substrate NADH as electron donor, aerobic NO production was maintained at more than 70% of anaerobic levels, and binding of NADH to the flavin-adenine dinucleotide site seemed to prevent oxygen binding. Therefore, under aerobic conditions, NADH would be the main electron donor for XO-catalyzed NO production in tissues. Studies of the pH dependence of NO formation indicated that lower pH values decrease oxygen reduction but greatly increase nitrite reduction, facilitating NO generation. Isotope tracer studies demonstrated that XO-mediated NO formation occurs in normoxic and hypoxic heart tissue. Thus, XO-mediated NO generation occurs under aerobic conditions and is regulated by oxygen tension, pH, nitrite, and reducing substrate concentrations.     

Nitroxides as antioxidants: Tempol protects against EO9 cytotoxicity

Nitroxide free radicals have been shown to be potent antioxidants in a variety of experimental models using diverse means of insults. Among other insults, nitroxides have been shown effective in inhibiting cytotoxicity of quinone-based drugs such as streptonigrin and mitomycin C. These drugs and other chemotherapeutic agents have the potential to undergo bioreductive activation by the normal reducing enzymes within a cell. In the present work we studied the effect of the nitroxide Tempol on the cytotoxicity induced by EO9, a mitomycin C analogue, in HT29 cells under aerobic and hypoxic conditions. The study was aimed to better understand the mechanism of EO9 cytotoxicity and the molecular level of the nitroxide's mode of protection. The reactions of Tempol with activated EO9, and the reactive species formed during EO9 activation were studied in a cell-free solution, using spin-trapping, and electron paramagnetic resonance (EPR) spectrometry. Our results indicate that EO9 induced similar cytotoxicity in HT29 cells under aerobic and hypoxic conditions while Tempol provided similar and almost complete protection to both aerobic and hypoxic cells. The results indicate that EO9 cytotoxicity is due to both 1- and 2-electron reductive activation processes, with aerobic toxicity caused by back-oxidation of the hydroquinone to the semiquinone, EO9^–. Tempol serves both as a useful tool in the study of the mechanisms of quinone-mediated cytotoxicity and as a potent antioxidant against the damaging effects of redox cycling quinones and semiquinones by scavenging of EO9^– or detoxification of O₂ ^– and H₂O₂.