Spindle disturbances in mammalian cells. IV. The action of some glutathione-specific agents in V79 Chinese hamster cells, changes in levels of free sulfhydryls and ATP, c-mitosis and effects on DNA metabolism

The glutathione-specific agents diamide, diethyl maleate and 1-chloro-2,4-dinitrobenzene were found to induce a low frequency of c-mitosis (15%) at non-toxic concentrations concomitant with a 30-40% decrease of non-protein sulfhydryls. The frequency of c-mitosis did not increase further with increased concentrations until non-protein sulfhydryl levels were obtained suggesting depletion of reduced glutathione. The observed shape of the concentration-response curve for c-mitosis is particular to these 3 agents and caffeine among 22 different compounds being tested under comparable conditions. This suggests a similar mechanism of action and from what is known about caffeine this mechanism probably involves an impaired control of cytoplasmic free Ca2+. It is speculated that this impairment with the glutathione-specific agents is primarily due to depletion of a particular pool of reduced glutathione. Tertiary butylhydroperoxide which is a substrate for glutathione peroxidase(s) also causes c-mitosis when there is no significant decrease of non-protein sulfhydryls. The c-mitotic response was found to be biphasic with maintained control levels at an intermediate concentration. The humps in the concentration-response curve for c-mitosis appeared coincident with a mitogenic response (G1----S). Since the latter type of effect most probably is Ca2+ dependent and since the spindle is sensitive to Ca2+ it is tentatively suggested that the c-mitotic effect of tertiary butylhydroperoxide is due to an increase of cytoplasmic Ca2+. Measurements performed imply that an increase of glutathione disulfide (diamide) is more inhibitory to uptake and incorporation of thymidine than a decrease of reduced glutathione per se (diethyl maleate). This difference is probably due to secondary effects on pertinent protein sulfhydryls with diamide, one possible target being the ribonucleotide reductase. All compounds were found to cause an increase of ATP with some of the applied concentrations. The results with diethyl maleate suggest that an increase of ATP is favored by an attack on mitochondrial reduced glutathione. The possible analogy between this effect and an increase of ATP and Ap4A in bacteria during oxidative stress is considered.     

Thiol-dependent K∶Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide

Summary The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO₃ or methane sulfonate (CH₃SO₃). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the KCl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983.J. Membrane Biol..73:237–246). Like NEM, diamide activated KCl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L₁, the allo-antibody known to inhibit OR K fluxes.     

Sulfhydryls and the in vitro polymerization of tubulin

The free sulfhydryls of brain tubulin prepared by cyclic polymerization procedures both with and without glycerol have been examined. The average free sulfhydryl titer of tubulin prepared with glycerol (7.0 sulfhydryls/55,000 mol wt) is greater than that of tubulin prepared without glycerol (4.0 sulfhydryls/55,000 mol wt). Diamide, a sulfhydryl- oxidizing agent, inhibits the polymerization of tubulin. Diamide also disperses the 20S and 30S oligomers of tubulin seen in analytical ultracentrifuge patterns of tubulin solutions and, depending on the temperature at which diamide is added, converts all or part of the oligomeric material to 6S dimers. Electron microscopy demonstrates that diamide also destroys the 450-A ring structures characteristic of tubulin solutions. All diamide effects are reversible by the addition of 10 mM dithioerythreitol, a sulfhydryl-reducing agent. That diamide interacts with sulfhydryls on tubulin is directly demonstrated by a 50% decrease in the free sulfhydryl titer of tubulin measured after diamide treatment. Concentrations of CaCl2 which inhibit polymerization also decrease the free sulfhydryl titer of tubulin.     

Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide

The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO3 or methane sulfonate (CH3SO3). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the K:Cl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983. J. Membrane Biol. 73:237-246). Like NEM, diamide activated K:Cl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L1, the allo-antibody known to inhibit OR K fluxes.     

The action of carbaryl and its metabolite alpha-naphthol on mitosis in V79 Chinese hamster fibroblasts. Indications of the involvement of some cholinester in cell division

Carbaryl (1-naphthyl-N-methylcarbamate) is a spindle-disturbing agent. A number of effects probably contribute to this activity. Carbaryl efficiently lowers the intracellular level of glutathione, thus endangering the integrity of the spindle. Carbaryl also causes lipid peroxidation. With anti-oxidant pretreatment, the frequency of c-mitotic cells is lowered which indicates that lipid peroxidation is a partial cause of the spindle-disturbing activity. At high concentrations of carbaryl, monopolar configurations in combination with cleavage are frequent and when tested, alpha-naphthol, which is thought to be formed from carbaryl in significant amounts at these concentrations, also gave monopolar configurations but with significantly lower frequencies of concomitant cleavage. Carbaryl inhibits cholinesterases and when tested, another cholinesterase inhibitor, diisopropylfluorophosphate, in combination with alpha-naphthol also increased the frequency of monopolar configuration in combination with cleavage. We therefore propose the involvement of some cholinester in the process of cell division.     

The effect of diamide on amino acid transport by rat renal cortex slices

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of α-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4°C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.     

Spindle disturbances in mammalian cells. III. Toxicity, c-mitosis and aneuploidy with 22 different compounds. Specific and unspecific mechanisms

Early investigations have shown that many chemically different compounds can cause disturbances of the spindle function (c-mitosis) in eukaryotic cells and that there is an unspecific (physical) mechanism based on the partitioning of the compound into cellular hydrophobic compartments. This suggests that the approach should be quantitative when testing compounds for this type of activity in vitro; effect/no effect is not the most pertinent question. The present study demonstrates how a set of reference compounds can be used in attempts to identify compounds that act by a more specific (chemical) mechanism to disturb the spindle function. All experiments were performed with an established cell line (V79 Chinese hamster). The results suggest that there is a good qualitative coupling in these cells between c-mitosis and aneuploidy with chemical treatment. Among compounds that are particularly active in relation to their lipophilic character are some chlorophenols, caffeine, diamide, diethyl maleate, 1-chloro-2,4-dinitrobenzene and tertiary butylhydroperoxide. This points to Ca2+-sequestering by mitochondria and/or cellular pH regulation (chlorophenols), Ca2+ release and sequestering by the endoplasmic reticulum (caffeine), enzymatic conjugation to glutathione (diethyl maleate, chlorodinitrobenzene) and hydroperoxide metabolism (t-butylhydroperoxide) as important target functions for specific activity.     

巯基参与胃粘膜防御机制

本工作研究了胃粘膜非蛋白质巯基物质(NPSH)在粘膜防御功能中的作用。结果表明,酸性乙醇灌胃或冷冻加束缚应激引起大鼠胃粘膜 NPSH 含量显著下降;补充含-SH 的化合物半胱胺或还原型谷胱甘肽可防止酸性乙醇引起的胃粘膜损伤;在酸性乙醇灌胃或应激后,胃粘膜谷胱甘肽还原酶活性明显降低,并与 NPSH 含量的下降在时间上一致;丙二醛含量在酸性乙醇灌胃后显著升高,自由基清除剂二甲亚砜可减轻胃粘膜损伤。上述结果提示,胃粘膜NPSH 可能通过对自由基的清除作用参与粘膜的局部防御机制;谷胱甘肽还原酶活性下降和自由基生成增加所导致的粘膜 NPSH 含量下降可能是损伤发生过程的重要环节。     

Changes in organ nonprotein sulfhydryl and glutathione concentrations during acute and chronic administration of inorganic lead to chicks

The effects of dietary and injected lead (Pb) on organ nonprotein sulfhydryl (NPSH) and glutathione (GSH) concentrations in the chick were studied. Lead acetate·3H₂O was administered either in the diet for 3 wk at 2000 ppm Pb or by intraperitoneal (ip) injection of 3-wkold chicks with 52 mg Pb/100 g body wt. In Exp. 1, NPSH concentrations in liver and kidney were increased by both dietary and injected Pb in comparison to chicks not receiving Pb. Thigh muscle NPSH was decreased by injected Pb, whereas dietary Pb had no effect. In Expt. 2, whole blood and plasma NPSH were measured at 0, 0.5, 1.0, 2.0, and 4.0 h following ip Pb injection. Both whole blood and plasma NPSH were increased by 30 min. Whole blood NPSH concentrations plateaued at 30 min, and plasma NPSH continued to rise for 2 h. In Expt. 3, injected Pb increased hepatic NPSH, but not GSH concentrations. The ratio of GSH/NPSH was therefore lowered. The incorporation of [1-¹⁴C]glycine into hepatic GSH was stimulated by injected Pb. Buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, reduced hepatic NPSH and [¹⁴C]glycine incorporation in Pb-treated chicks to below control (non-Pb injected) values. In Expt. 4, dietary Pb fed for 3 wk increased the hepatic concentrations of both NPSH and GSH such that the ratio of GSH/NPSH was unchanged in comparison to chicks not fed Pb. The data suggest that the initial response to acute Pb intoxication may involve a mobilization of nonprotein thiols via the interorgan translocation system for GSH. Such a response would help to maintain adequate levels of GSH in organs crucial to detoxification.     

Thiol modification in H2O2- and thromboxane-induced vaso- and bronchoconstriction in rat perfused lung.

Hydrogen peroxide (H2O2), arachidonic acid (AA), and U-44069, a thromboxane analogue, all induced vaso- and bronchoconstriction in the isolated perfused rat lung. The role of protein sulfhydryl modifications in these processes was investigated. The thiol oxidizing agent diamide inhibited both vaso- and bronchoconstriction induced by H2O2, AA, or U-44069. Diamide had only a marginal effect on glutathione and protein thiol levels and no effect on lung mechanics. The diamide inhibition was reversible, and H2O2-induced vaso- and bronchoconstriction was almost maximal after 10 min of perfusion with buffer. The recovery was more rapid if dithiothreitol, a thiol reducing agent, was used in the buffer. H2O2- and AA-induced vaso- and bronchoconstriction is caused by thromboxane release. Diamide did not influence H2O2- or AA-dependent thromboxane formation, indicating that neither AA release nor AA metabolism to thromboxane is sensitive to thiol oxidation. Thus our results indicate that the site of diamide-induced thiol oxidation is the thromboxane receptor or its signal transduction.     

Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs. Involvement of NADP and glutathione

Methylxanthines (MX) inhibit cell division in sea urchin and clam eggs. This inhibitory effect is not mediated via cAMP. MX also inhibit respiration in marine eggs, at concentrations which inhibit cleavage. Studies showed that no changes occurred in ATP and ADP levels in the presence of inhibitory concentrations of MX, indicating an extra-mitochondrial site of action for the drug. Subsequent studies revealed decreased levels of NADP+ and NADPH, when eggs were incubated with inhibitory concentrations of MX, but no change in levels of NAD+ and NADH. MX did not affect the pentose phosphate shunt pathway and did not have any effect on the enzyme NAD+ -kinase. Further studies showed a marked inhibitory effect on the glutathione reductase activity of MX-treated eggs. Reduced glutathione (GSH) could reverse the cleavage inhibitory effect of MX. Moreover, diamide, a thiol-oxidizing agent specific for GSH in living cells, caused inhibition of cell division in sea urchin eggs. Diamide added to eggs containing mitotic apparatus (MA) could prevent cleavage by causing a dissolution of the formed MA. Both MX and diamide inhibit a Ca2+-activated ATPase in whole eggs. The enzyme can be reactivated by sulfhydryl reducing agents added in the assay mixture. In addition, diamide causes an inhibition of microtubule polymerization, reversible with dithioerythritol. All experimental evidence so far suggests that inhibition of mitosis in sea urchin eggs by MX is mediated by perturbations of the in vivo thiol-disulfide status of target systems, with a primary effect on glutathione levels.     

The effect of diamide on amino acid transport by rat renal cortex slices.

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of alpha-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4 degrees C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.     

Stimulation of the active transport of serotonin into mouse platelets by the sulfhydryl oxidizing agent diamide

The purpose of this study was to determine the effects of diamide, a reversible sulfhydryl oxidizing agent, on the transport of serotonin (5-HT) by mouse platelets. Diamide produced a concentration-dependent (10–200 μM) stimulation of 5-HT transport that was rapid and sustained over 0–10 minutes of incubation. When platelets were incubated with diamide (10–200 μM) in the presence of glucose, the content of reduced glutathione was significantly decreased only at a final concentration of 200 μM, while washed platelets incubated with diamide (10–200 μM), in the absence of glucose, had a significant concentration-dependent decrease in their content of reduced glutathione. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked diamide-induced stimulation of 5-HT transport. The kinetics of 5-HT transport showed that diamide caused a marked increase in the maximal rate of transport (V_max control = 28.4 ± 1.4 vs. V_max diamide = 60.9 ± 4.1 pM/10⁸ platelets/4 min) but did not significantly alter the K_m values. Ouabain, an inhibitor of platelet Na⁺-K⁺ ATPase, blocked the stimulation by diamide in a concentration-dependent manner. Dithiothreitol, a disulfide reducing agent, was able to partially reverse the stimulation of platelet 5-HT transport caused by diamide. This study has shown that diamide can stimulate the active transport of 5-HT by mouse platelets and suggests a possible role for free sulfhydryl groups in the regulation of this process.     

Diamide reversibly induces a stress response in the sea urchin, Arbacia punctulata

Sea urchin blastulae were treated with two concentrations (0.54 and 0.72 mM) of diamide, a sulfhydryl oxidant, after hatching. These treatments increased the relative synthesis of one set of embryonic proteins while decreasing that of another. This was demonstrated by quantitating the incorporation of [35S]methionine into polypeptides separated by 2-dimensional polyacrylamide gel electrophoresis (2D PAGE). These shifts were dose dependent and apparently reversible after the embryos had regenerated reduced sulfhydryls. Those proteins showing increased incorporation migrated at the same position by 2D PAGE as heat shock proteins, suggesting that diamide was inducing a stress response. Diamide also caused some developmental aberrations at low frequency, and reversibly inhibited ciliary beating.     

Diamide inhibits pulmonary vasoconstriction induced by hypoxia or prostaglandin F2 alpha

Diamide oxidizes glutathione and other cellular sulfhydryl groups. It decreases calcium ATPase activity and alters mitochondrial calcium flux, probably as a result of the sulfhydryl oxidation. We examined the effect of diamide (5 mg/kg, iv) on pulmonary vascular reactivity in 12 anesthetized dogs. Diamide reversed the pulmonary vasoconstriction caused by hypoxia in seven dogs (control delta PVR + 2.5 +/- 0.6 mm Hg/liter/min; postdiamide delta PVR - 0.1 +/- 0.4 mm Hg/liter/min; P less than 0.01). The pulmonary pressor response to prostaglandin F2 alpha (5 micrograms/kg/min, iv) was also reduced (control delta PVR + 3.8 +/- 0.5 mm Hg/liter/min; postdiamide delta PVR + 1.1 +/- 0.7 mm Hg/liter/min; P less than 0.01). However, in a further five dogs, diamide had only a small effect on the pulmonary vasoconstriction caused by angiotensin II, while the pressor response to hypoxia was again inhibited. The mechanism by which diamide reverses pulmonary vasoconstriction is not certain but the effect is rapid, consistent, and reversible. Because the intravenous infusion of diamide does not produce systemic hypotension, during its period of action on the pulmonary vasculature, unlike the drugs currently available for the clinical treatment of pulmonary hypertension, further studies of its mechanism of action are indicated.     

Differential sensitivity of metaphase to diamide and ouabain in HeLa cells

We have examined the effects of diamide, an oxidizer of glutathione, on the progress of HeLa cells through the cell cycle. At concentrations which do not significantly alter generation time, anaphase or cytokinesis, diamide causes a two-fold increase in the duration of metaphase. At 3 X 10(-8) M, ouabain also prolongs metaphase without effect on anaphase or cytokinesis, though with a different time course. The data suggest that the metaphase stage of mitosis is particularly sensitive to alterations in both sulfhydryl groups and Na+ levels but that the effects of diamide are probably not primarily due to the oxidation of sulfhydryl groups of the Na+/K+-ATPase.     

Reversible activation of soluble guanylate cyclase by oxidizing agents.

Soluble guanylate cyclase of human platelets was stimulated by thiol oxidizing compounds like diamide and the reactive disulfide 4, 4'-dithiodipyridine. Activation followed a bell-shaped curve, revealing somewhat different optimum concentrations for each compound, although in both cases, higher concentrations were inhibitory. Diamide at a concentration of 100 microM transiently activated the enzyme. In the presence of moderate concentrations of diamide and 4,4'-dithiodipyridine, causing a two- to fourfold activation by themselves, the stimulatory activity of NO-releasing compounds like sodium nitroprusside was potentiated. In contrast, higher concentrations of thiol oxidizing compounds inhibited the NO-stimulated activation of soluble guanylate cyclase. Activation of guanylate cyclase was accompanied by a reduction in reduced glutathione and a concomitant formation of protein-bound glutathione (protein-SSG). Both compounds showed an activating potency as long as reduced glutathione remained, leading to inhibition of the enzyme just when all reduced glutathione was oxidized. Activation was reversible while reduced glutathione recovered and protein-SSG disappeared. We propose that diamide or reactive disulfides and other thiol oxidizing compounds inducing thiol-disulfide exchange activate soluble guanylate cyclase. In this respect partial oxidation is associated with enzyme activation, whereas massive oxidation results in loss of enzymatic activity. Physiologically, partial disulfide formation may amplify the signal toward NO as the endogenous activator of soluble guanylate cyclase.     

Alterations of the glutathione-redox balance induced by metals in CHO-K1 cells

The effects of cadmium (Cd(2+)), mercury (Hg(2+)), lead (Pb(2+)), copper (Cu(2+)) and nickel (Ni(2+)) on the glutathione (GSH)-redox cycle were assessed in CHO-K1 by the neutral red uptake inhibition (NR) assay (NR(6.25), NR(12.5) and NR(25)). Mercury proved to be the most and lead the least toxic of the metals tested. The effects on GSH content and intracellular specific activities of enzymes involved in the GSH-redox balance were measured after a 24-h exposure. Total GSH content increased significantly in cultures exposed to the lowest metal concentration assayed (NR(6.25)), but fell to below control values when exposed to concentrations equivalent to NR(25). Oxidised glutathione content dropped significantly at NR(6.25), while somewhat higher values were obtained for cultures exposed to higher doses. Glutathione peroxidase (Gpx) activities were 1.2-, 1.5-, 1.6-, 2.0- and 2.5-fold higher than untreated controls for cadmium, copper, mercury, nickel and lead, respectively, at concentrations equivalent to NR(6.25). Gpx activity declined at metal concentrations equivalent to NR(12.5) and NR(25). Glutathione reductase activity remained almost unchanged except at low doses of mercury, nickel and lead. Glutathione-S-transferase activity decreased at rising metal concentrations. The results suggest that a homeostatic defence mechanism was activated when cells were exposed to doses equivalent to NR(6.25) while the ability of the cells to respond weakened as the dose increased. A close relationship was also observed between metal cytotoxicity, total GSH content and the dissociation energy of the sulphur-metal bonds. These facts confirm the involvement of antioxidant defence mechanisms in the toxic action of these ions.     

比较不同类型白内障形成过程中非蛋白质巯基与蛋白质巯基的动态变化及关系

本文报道了在亚硒酸钠、平阳霉素及半乳糖诱发大鼠产生白内障过程中晶状体中非蛋白质巯基及蛋白质巯基的动态变化,并探讨了其变化机理及相互关系。在亚硒酸钠诱发白内障过程中,给药24h后晶状体中非蛋白质巯基减少到正常的二分之一,以后又逐渐回升,但始终未达到正常水平,至第7天,非蛋白质巯基又再度减少。在平阳霉素及半乳糖诱发白内障过程中,晶状体中非蛋白质巯基分别在给药后的第7天及第3天开始大量减少,以后继续减少,至第15天时,其含量分别为正常的十分之一及五分之一。在体外,亚硒酸钠有促进还原型谷胱甘肽自氧化的作用,半乳糖对此作用无影响,而平阳霉素可阻止其进行,但能加强亚硒酸钠的促进作用。在三种白内障晶状体中,蛋白质巯基开始减少的时间均较非蛋白质巯基为晚,这表明只有非蛋白质巯基减少到一定程度后蛋白质巯基才会被大量氧化,同时也说明非蛋白质巯基具有保护蛋白质巯基免受氧化的作用。只有这种保护作用减弱后,才会使蛋白质巯基遭受氧化而导致白内障。     

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.