diethyl-maleate and Hypoxia

The effects of hydrogen sulfide (H(2)S) and acute hypoxia are similar in isolated pulmonary arteries from various species. However, the involvement of H(2)S in hypoxic pulmonary vasoconstriction (HPV) has not been studied in the intact lung. The present study used an intact, isolated, perfused rat lung preparation to examine whether adding compounds essential to H(2)S synthesis or to its inhibition would result in a corresponding increase or decrease in the magnitude of HPV. Western blots performed in lung tissue identified the presence of the H(2)S-synthesizing enzymes, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), but not cystathionine β-synthase (CBS). Adding three H(2)S synthesis precursors, cysteine and oxidized or reduced glutathione, to the perfusate significantly increased peak arterial pressure during hypoxia compared with control (P < 0.05). Adding α-ketoglutarate to enhance the 3-MST enzyme pathway also resulted in an increase (P < 0.05). Both aspartate, which inhibits the 3-MST synthesis pathway, and propargylglycine (PPG), which inhibits the CSE pathway, significantly reduced the increases in arterial pressure during hypoxia. Diethylmaleate (DEM), which conjugates sulfhydryls, also reduced the peak hypoxic arterial pressure at concentrations >2 mM. Finally, H(2)S concentrations as measured with a specially designed polarographic electrode decreased markedly in lung tissue homogenate and in small pulmonary arteries when air was added to the hypoxic environment of the measurement chamber. The results of this study provide evidence that the rate of H(2)S synthesis plays a role in the magnitude of acute HPV in the isolated perfused rat lung.

Topics: Alkynes; Animals; Aspartic Acid; Blood Pressure; Cystathionine beta-Synthase; Cystathionine gamma-Lyase; Cysteine; Glutathione; Glutathione Disulfide; Glycine; Hydrogen Sulfide; Hypoxia; Ketoglutaric Acids; Lung; Male; Maleates; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Sulfurtransferases; Vasoconstriction

Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers with different substrate specificity and sensitivity to environmental stimuli. We reported previously that transport activities of cystine and arginine in the macrophages were induced markedly by low concentrations of bacterial lipopolysaccharide (LPS). It is known that a variety of macrophage functions are affected by ambient oxygen tension. In this study, we have investigated the effects of oxygen on the induction of amino acid transport activity by LPS and found that the induction of cystine, but not arginine, transport activity was dependent on the ambient oxygen tension. When the macrophages were cultured with 2% O(2) in the presence of 1 ng/ml LPS, induction of cystine transport activity was reduced by approximately 70% compared with cells cultured under normoxic conditions. In macrophages, transport of cystine is mediated by a Na(+)-independent anionic amino acid transporter named system x(c)(-). System x(c)(-) is composed of two protein components, xCT and 4F2hc, and the expression of xCT was closely correlated with system x(c)(-) activity. A putative NF-kappaB binding site was found in the 5'-flanking region of the xCT gene, but the enhanced expression of xCT by LPS and oxygen was not mediated by NF-kappaB binding. An increase in intracellular GSH in macrophages paralleled induction of xCT, but not gamma-glutamylcysteine synthetase. These results suggest the importance of system x(c)(-) in antioxidant defense in macrophages exposed to LPS and oxidative stress.

Topics: Amino Acids; Animals; Antioxidants; Base Sequence; Binding Sites; Biological Transport; Blotting, Northern; Cell Nucleus; Cystine; Dipeptides; Dose-Response Relationship, Drug; Female; Glutamate-Cysteine Ligase; Glutathione; Hypoxia; Lipopolysaccharides; Macrophages; Macrophages, Peritoneal; Maleates; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; NF-kappa B; Oxidative Stress; Oxygen; RNA, Messenger; Signal Transduction; Time Factors

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-hr exposure to 0.5 mM DEM or a 4- or 24-hr exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under air or hypoxic conditions. When GSH levels are lowered to less than 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater X-ray sensitization than hypoxic BSO-treated cells. The D0's for hypoxic survival curves were as follows: control, 4.87 Gy; DEM, 3.22 Gy; and BSO, 4.30 Gy for the V79 cells and 5.00 Gy versus 4.02 Gy for BSO-treated A549 cells. The D0's for aerobic V79 cells were 1.70 Gy versus 1.13 Gy, DEM, and 1.43 Gy for BSO-treated cells. The D0's for the aerobic A549 were 1.70 and 1.20 for BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.

Topics: Animals; Buthionine Sulfoximine; Carcinoma; Cell Line; Cells, Cultured; Cricetinae; Dose-Response Relationship, Radiation; Glutathione; Humans; Hypoxia; Lung; Lung Neoplasms; Maleates; Methionine Sulfoximine; Oxygen Consumption; Radiation-Sensitizing Agents; Time Factors

Misonidazole (MIS) is a hypoxic cell radiosensitizer currently undergoing Phase III clinical trials in the treatment of cancer by radiation. It is also a cytotoxic agent with specificity toward hypoxic cells, and consequently has a tumoricidal effect in laboratory animals. This tumoricidal effect has not been clinically applicable, in part because the initial resistance to the cytotoxic action of MIS (the nonexponential portion, or shoulder, of the semilogarithmic plot of the surviving fraction of the cell population versus the time of exposure to MIS, referred to hereafter as the shoulder of the survival curve) for cells treated with MIS under hypoxic conditions is too large to be overcome at clinically tolerable doses of MIS. We report here that pretreatment of Chinese hamster ovary cells in vitro with diethylmaleate to deplete intracellular glutathione results in a substantial decrease in the shoulder of the survival curve for MIS-treated hypoxic cells. Restoration of glutathione results in restoration of the shoulder of the survival curve and a slight extension beyond that seen with control cells. These results demonstrate that glutathione protects against the cytotoxic effect of MIS. However, glutathione depletion does not significantly affect the rate of binding of MIS metabolites to cellular macromolecules, indicating that the cytotoxicity of MIS is not simply a reflection of massive binding of MIS metabolites to cellular constituents. We propose that the cytotoxicity of MIS toward hypoxic cells is a result of hydrogen abstraction from target molecules by free radicals formed in the reduction of the nitro group.

Topics: Animals; Antineoplastic Agents; Cell Line; Cell Survival; Cricetinae; Cricetulus; Female; Glutathione; Glutathione Transferase; Hypoxia; Maleates; Misonidazole; Nitroimidazoles; Ovary

Depletion of glutathione in Chinese hamster ovary cells in vitro by diethyl maleate resulted in enhancement of the effect of x-rays on cell survival under hypoxic conditions but not under oxygenated conditions. Hypoxic EMT6 tumor cells were similarly sensitized in vivo. The action of diethyl maleate is synergistic with the effect of the electron-affinic radiosensitizer misonidazole, suggesting that the effectiveness of misonidazole in cancer radiotherapy may be improved by combining it with drugs that deplete intracellular glutathione.

Topics: Animals; Cell Survival; Cells, Cultured; Cricetinae; Cricetulus; Drug Synergism; Glutathione; Hypoxia; Maleates; Mice; Mice, Inbred BALB C; Misonidazole; Neoplasms, Experimental; Oxygen Consumption