Inhibition of erythrocyte superoxide dismutase by diethyldithiocarbamate also results in oxyhemoglobin-catalyzed glutathione depletion and methemoglobin production

N,N-Diethyldithiocarbamate (DDC), a copper-chelating agent, not only inhibits superoxide dismutase activity in the red cell, but also depletes glutathione and promotes the production of methemoglobin, sulfhemoglobin, and small amounts of lipid peroxidation products. DDC reacts with oxyhemoglobin to yield disulfiram, hydrogen peroxide, and methemoglobin. Disulfiram and hydrogen peroxide both convert GSH to GSSG, while DDC reduces methemoglobin to oxyhemoglobin. Although disulfiram also reacts with the hemoglobin sulfhydryl groups, this reaction does not play a role in the conversion of GSH to GSSG. Other hemoglobin derivatives, ferrous, and ferric ions do not catalyze the oxidation of GSH by DDC. These results support the conclusion that DDC reacts with the super-oxo-ferriheme complex of oxyhemoglobin to generate hydrogen peroxide and disulfiram and that the cyclic conversion of oxyhemoglobin to methemoglobin and DDC and disulfiram results in the net oxidation of GSH. Thus, damage to DDC-treated erythrocytes exposed to a putative superoxide-generating toxin, such as 1,4-naphthoquinone-2-sulfonate, may actually be due to diminished GSH concentration and hemoglobin oxidation rather than to superoxide radicals. Glucose added to the incubation medium of DDC-treated erythrocytes fully prevented glutathione depletion but not the oxidation of oxyhemoglobin to methemoglobin. Several other copper-chelating agents either failed to inhibit the activity of purified superoxide dismutase or when incubated with erythrocytes produced more extensive GSH depletion and hemoglobin oxidation than DDC. It is concluded that the interpretation of results with erythrocytes exposed to copper-chelating agents must consider their effects on GSH and hemoglobin as well as on superoxide dismutase inhibition. Moreover, one must be mindful of the interference by DDC in the analysis of GSH with 5,5'-dithiobis-(2-nitrobenzoic acid) in the absence of sufficient quantities of metaphosphoric acid to destroy DDC and that contamination of DDC with trace quantities of disulfiram may be a significant problem.     

Calorimetric studies of oxyhemoglobin ligand dissociation. III. Evidence for a hemoglobin-catalase-superoxide dismutase integrated system in the oxygen depletion by dithionite

Calorimetric studies of the effect of superoxide dismutase and/or catalase on the reduction of dioxygen into water by dithionite in oxyhemoglobin have been carried out and the results compared with those in red cell hemolysates. In the absence of the enzymes the stoichiometry (moles dithionite/mole dioxygen) is less than the value of 2:1 which was found previously in red cell hemolysates [Forlani et al., J. Inorg. Biochem. 20, 147-155 (1984)]. In the presence of either superoxide dismutase or catalase alone the stoichiometry increases but is still less than 2:1. In the presence of both enzymes the stoichiometry and the shape of the thermogram is that previously observed for hemolysates, suggesting the presence of a hemoglobin-catalase-superoxide dismutase integrated system. The absence of a calorimetric signal for hydrogen peroxide in the reduction of oxyhemoglobin in the presence of superoxide dismutase suggests a wider biological role of superoxide dismutase than previously thought.     

Inactivation of intracellular copper-zinc superoxide dismutase by copper chelating agents without glutathione depletion and methemoglobin formation

The copper chelator N,N'-diethyldithiocarbamate (DDC), is often used to inactivate intracellular copper-zinc superoxide dismutase in erythrocytes. However, in studies with red cells we found that the compound also reacted with oxyhemoglobin to produce oxygen radicals in addition to generating lipid peroxidation products, oxidized N,N'-diethyldithiocarbamate, methemoglobin, and sulfhemoglobin. Moreover, intracellular glutathione was depleted and vital cellular enzymes were susceptible to inactivation. We, and others, have confirmed these findings in nonerythrocytic cell lines. Thus, cells exposed to DDC are severely damaged before studies on the effects of added putative superoxide producing compounds can be performed with them. In this report, we have systematically investigated other copper chelators for their ability to inactivate intracellular copper-zinc superoxide dismutase without producing the deleterious effects mentioned above. Catechol, triethylenetetramine, and tetraethylenepentamine were found to be such agents when erythrocytes were dialyzed in the cold against dilute solutions of these chelators. In addition, with a myeloid leukemic cell line (HL-60), triethylenetetramine inhibited SOD without causing significant GSH oxidation. Examination of the affinity constants of chelators active against purified copper-zinc superoxide dismutase indicated that an affinity binding constant (log K1) between 12.6 and 13.8 was required for the chelator to successfully remove copper from the enzyme.     

Enhancement of heat sensitivity and modification of repair of potentially lethal heat damage in plateau-phase cultures of mammalian cells by diethyldithiocarbamate

Diethyldithiocarbamate (DDC) is active both in vivo and in vitro in reducing the levels of enzymes such as superoxide dismutase (SOD) and glutathione peroxidase whose role in respiring cells is to remove toxic superoxide radicals and organic hydroperoxides. Although DDC, a copper-chelating agent, has been used to treat benign diseases, its potential as a heat sensitizer has not been fully explored. We have recently shown that the presence of 10(-3) M DDC for 2 hr causes a threefold reduction in the level of SOD in plateau-phase cultures of mammalian cells. At this concentration, the drug causes minimal toxicity but markedly affects both the shoulder and the slope of the heat survival curves. To explore another pathway of DDC sensitization, other than through reduced levels of SOD, we examined the repair of potentially lethal damage with and without DDC following exposure for 1 hr and 40 min at 43 degrees C. The repair, which progressed with a T 1/2 of about 10 hr, in either full medium or Hank's balanced salt solution (HBSS), in the absence of DDC, was completely blocked when DDC was added to the monolayers on completion of the heat exposure. DDC, in view of its ability to potentiate the effects of heat, is a potentially useful drug that could be used in an adjunctive setting with clinical hyperthermia.     

Diffusion of peroxynitrite in the presence of carbon dioxide.

Peroxynitrite, the reactive species formed in vivo by the reaction of nitric oxide with superoxide anion, is capable of diffusing across erythrocyte membranes via anion channels and passive diffusion (A. Denicola, J. M. Souza, and R. Radi, Proc. Natl. Acad. Sci. USA 95, 3566-3571, 1998). However, peroxynitrite diffusion could be limited by extracellular targets, with the reaction with CO(2) (k(2) = 4.6 x 10(4) at 37 degrees C and pH 7.4) the most relevant. Herein, we studied the influence of physiological concentrations of CO(2) on peroxynitrite diffusion across intact red blood cells. The presence of CO(2) inhibited the oxidation of intracellular oxyhemoglobin by externally added peroxynitrite. However, the inhibition by CO(2) decreased at increasing red blood cell densities. At 45% hematocrit, 1.3 mM CO(2) (in equilibrium with 24 mM bicarbonate, at pH 7.4 and 25 degrees C) only inhibited 30% of intracellular oxyhemoglobin oxidation. This partial inhibition was also observed in red blood cells pretreated with the anion exchanger inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, ruling out a competition between peroxynitrite and bicarbonate for the transport through the anion channel. A theoretical model was developed to estimate the diffusion distance and half-life of extracellular peroxynitrite before reacting with intracellular oxyhemoglobin, at different red blood cell densities, and in the presence or absence of CO(2). The theoretical model correlated well with the experimental data. Our results indicate that, even in the presence of CO(2), peroxynitrite is able to diffuse and reach the inside of the erythrocyte.     

Superoxide levels and function of cerebral blood vessels after inhibition of CuZn-SOD

The goal of this study was to examine the role of endogenous copper/zinc (CuZn)-superoxide dismutase (SOD) on superoxide levels and on responses of cerebral blood vessels to stimuli that are mediated by nitric oxide (acetylcholine) and cyclooxygenase-dependent mechanisms (bradykinin and arachidonic acid). Levels of superoxide in the rabbit basilar artery were measured using lucigenin-enhanced chemiluminescence (5 microM lucigenin). Diethyldithiocarbamate (DDC; 10 mM), an inhibitor of CuZn-SOD, increased superoxide levels by approximately 2.4-fold (P < 0.05) from a baseline value of 1.0 +/- 0.2 relative light units x min(-1) x mm(-2) (means +/- SE). The diameter of cerebral arterioles (baseline diameter, 99 +/- 3 microm) was also measured using a closed cranial window in anesthetized rabbits. Topical application of DDC attenuated responses to acetylcholine, bradykinin, and arachidonate, but not nitroprusside. For example, 10 microM arachidonic acid dilated cerebral arterioles by 40 +/- 5 and 2 +/- 2 microm under control conditions and after DDC, respectively (P < 0.05). These inhibitory effects of DDC were reversed by the superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid (10 mM). Arachidonate increased superoxide levels in the basilar artery moderately under normal conditions and this increase was greatly augmented in the presence of DDC. These findings suggest that endogenous CuZn-SOD limits superoxide levels under basal conditions and has a marked influence on increases in superoxide in vessels exposed to arachidonic acid. The results also suggest that nitric oxide- and cyclooxygenase-mediated responses in the cerebral microcirculation are dependent on normal activity of CuZn-SOD.     

The interrelationship of superoxide dismutase and peroxidatic enzymes in the red cell.

Activities of superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) and catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) were determined during the course of incubation of red cell suspensions with 1,4-naphthoquinone-2-sulfonic acid. In the absence of glucose, incubation with napthoquinone sulfonate resulted in an inhibition of catalase and superoxide dismutase. The catalase inhibitor, 3-amino-1,2,4-triazole enhanced inactivation of catalase in the presence of naphthoquinone sulfonate and this in turn led to augmented inhibition of superoxide dismutase. The presence of glucose in the incubation medium prevented napthoquinone sulfonate-induced enzyme inhibition in the absence of aminotriazole, but had little effect in the presence of aminotriazole. The relevance of these findings to the cellular interrelationship of peroxidatic enzymes and superoxide dismutase is discussed.     

Generation of superoxide ion in human red blood cell lysates

The generation of superoxide ion in human red blood cell lysates was investigated by an experimental method employing Cu,Zn superoxide dismutase as O2- scavenger and EPR to probe the oxidation state of the enzyme. The average value of the O2- flux in the erythrocytes of 8 normal individuals was (2.02 +/- 0.97) X 10(-8) M S-1. A progressive saturation of the rate of O2- production was found increasing PO2, KM = 1.04 X 10(-4) M, while the autoxidation of oxyhemoglobin did not contribute significantly to the measured O2- production.     

Detection of hydrogen peroxide with Amplex Red: interference by NADH and reduced glutathione auto-oxidation

We report here that reduced pyridine nucleotides and reduced glutathione result in an oxidation of Amplex Red by dioxygen that is dependent on the presence of horseradish peroxidase (HRP). Concentrations of NADH and glutathione typically found in biological systems result in the oxidation of Amplex Red at a rate comparable to that produced, for example, by respiring mitochondria. The effects of NADH and glutathione in this assay system are likely to be the result of H(2)O(2) generation via a superoxide intermediate because both catalase and superoxide dismutase prevent the oxidation of Amplex Red. These results suggest caution in the assay of H(2)O(2) production in biological systems using the Amplex Red/HRP because the assay will also report the mobilization of NADH or glutathione. However, the interruption of this process by the addition of superoxide dismutase offers a simple and reliable method for establishing the source of the oxidant signal.     

Production of superoxide and activity of superoxide dismutase in rabbit epididymal spermatozoa

Mature rabbit spermatozoa from the cauda epididymidis suspended in potassium Tris phosphate buffer at 24 degrees C produced O2.-, as measured by reduction of acetylated ferricytochrome c, with an intrinsic rate of 0.20 nmol/min per 10(8) cells. This rate increased to 1.80 nmol/min per 10(8) cells in the presence of 10 mM cyanide. These spermatozoa contain 2.8 units per 10(8) cells of superoxide dismutase activity, 95% of which is sensitive, and 5% of which is insensitive, to cyanide inhibition. These activities correspond to the cytosolic Cu-Zn form and the mitochondrial Mn form of the dismutase, respectively. Only the cyanide-sensitive form is released from the sperm on hypo-osmotic treatment or sonication. Hypo-osmotically treated rabbit epididymal spermatozoa produced O2.- with an intrinsic rate of 0.24 nmol/min per 10(8) cells, which increased to 0.58 nmol/min per 10(8) cells in the presence of 10 mM cyanide. Both intact and hypo-osmotically treated cells react with O2.- in a second order reaction as inferred from the hyperbolic dependence on cell concentration of O2.- production rate in both the absence and presence of cyanide. The second order rate constant for this reaction with intact cells, kS, was calculated to be 22.9 X 10(-8) (cells/ml)-1 min-1 in its absence. For hypo-osmotically treated cells, the values of kS were 10.8 X 10(-8) (cells/ml)-1 min-1 and 8.2 X 10(-8) (cells/ml) -1 min-1, respectively. Since hypo-osmotically treated cells have lost much of their plasma membrane, the lower value of kS for the treated cells implies that this membrane is one site of reaction of O2.- with the cells. The increase in kS in the presence of cyanide, which inhibits superoxide dismutase and so increases O2.- production, suggests that the cells become more reactive with O2.- as its production rate increase, as would be expected for the occurrence of radical chain oxidation. This in turn suggests that superoxide dismutase plays a major role in protecting rabbit sperm against damage from lipid peroxidation.     

Superoxide anion and drug-induced hemolysis.

Superoxide anion, either generated during the autooxidation of dihydroxyfumaria acid or by the interaction of 1,4-naphthoquinone-2-sulfonate and intracellular hemoglobin in red cells pretreated with the intracellular superoxide dismutase inhibitor, diethyldithiocarbamate, produces structural changes in red cells hemoglobin and hypotonic lysis. No evidence for lipid peroxidation was found in red cells exposed to either 1,4 naphthoquinone-2-sulfonate in the presence of diethyldithiocarbamate or to dihydroxyfumaric acid, although the membranes of these cells exposed to either 1,4 naphthoquinone-2-sulfonate in the presence of diethyldithiocarbamate or to dihydroxyfumaric acid, although the membranes of these cells retained a green pigment. These results suggest that superoxide anion reacts with cellular hemoglobin to form hemoglobin breakdown products which bind to the red cell membrane and thereby increase the osmotic fragility of the cell.     

Photooxidation of Amplex red to resorufin: Implications of exposing the Amplex red assay to light

The Amplex Red assay, a fluorescent assay for the detection of H(2)O(2), relies on the reaction of H(2)O(2) and colorless, nonfluorescent Amplex Red with a 1:1 stoichiometry to form colored, fluorescent resorufin, catalyzed by horseradish peroxidase (HRP). We have found that resorufin is artifactually formed when Amplex Red is exposed to light. In the absence of H(2)O(2) and HRP, the absorption and fluorescence spectra of Amplex Red changed during exposure to ambient room light or instrumental excitation light, clearly indicating that the fluorescent product resorufin had formed. This photochemistry was initiated by trace amounts of resorufin that are present in Amplex Red stock solutions. ESR spin-trapping studies demonstrated that superoxide radical was an intermediate in this process. Oxygen consumption measurements further confirmed that superoxide and H(2)O(2) were artifactually produced by the photooxidation of Amplex Red. The artifactual formation of resorufin was also significantly increased by the presence of superoxide dismutase or HRP. This photooxidation process will result in a less sensitive assay for H(2)O(2) under ambient light exposure and potentially invalid measurements under high energy exposure such as UVA irradiation. In general, precautions should be taken to minimize exposure to light during measurement of oxidative stress with Amplex Red.     

Detection of oxygen radicals during reperfusion of intestinal cells in vitro

The nitroxide OXANO. (2-Ethyl-2,5,5-trimethyl-3-oxazolidinoxyl) which in its reduced form, OXANOH (2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine), is capable of reacting with short-lived radicals, forming a secondary stable radical, was used for ESR-detection of radical production in isolated cells. The properties of OXANO. and OXANOH in terms of stability in cellular and subcellular systems, membrane permeability and effects on cellular viability were evaluated. Ischemia and reperfusion was simulated in vitro in a preparation of cells from rat intestinal mucosa by incubation at high density (4 X 10(8) cells/ml) under an atmosphere of nitrogen for 25 min and resuspended with fresh oxygenated buffer containing 5 mM OXANOH. A significant increase in radical formation during the 15 min reperfusion period studied was obtained in cells exposed to ischemia compared to control cells incubated at normal density under an atmosphere of oxygen. The addition of 5 microM of the scavenging enzyme superoxide dismutase reduced the radical formation by 50%. The time sequence of the superoxide formation was calculated as the difference in radical production in the presence and absence of superoxide dismutase.     

特丁基氢过氧化物对乏硒大鼠红细胞的氧化损害

 本实验用特丁基氢过氧化物(t-BuOOH)分别与缺硒和不缺硒大鼠红细胞在体外作用后,观察了血红蛋白,脂质和膜蛋白的改变情况,并测定了抗氧化酶系的活力,以研究硒对t-BuOOH毒性的保护作用,结果指出:硒缺乏可使细胞抗氧化能力下降,缺乏组所受t-BuOOH的氧化损害大于硒不缺乏组。     

The interaction between nitrogen oxides and hemoglobin and endothelium-derived relaxing factor

Among nitrogen oxides, NO and NO₂ are free radicals and show a variety of biological effects. NO₂ is a strongly oxidizing toxicant, although NO, not oxidizing as NO₂, is toxic in that it interacts with hemoglobin to form nitrosyl-and methemoglobin. Nitrosylhemoglobin shows a characteristic electron spin resonance (ESR) signal due to an odd electron localized on the nitrogen atom of NO and reacts with oxygen to yield nitrate and methemoglobin, which is rapidly reduced by methemoglobin reductase in red cells. NO was found to inhibit the reductase activity. Part of NO inhaled in the body is oxidized by oxygen to NO₂, which easily dissolves in water and converts to nitrite. The nitrite oxyhemoglobin autocatalytically after a lag. The mechanism of the oxidation, particularly the involvement of superoxide, was controversial. The stoichiometry of the reaction has now been established using nitrate ion electrode and a methemoglobin free radical was detected by ESR during the oxidation. Complete inhibition of the autocatalysis by aniline or aminopyrine suggests that the radical catalyzes conversion of nitrite to NO₂, which oxidizes oxyhemoglobin. Recently NO was shown to be one of endothelium- derived relaxing factors and the relaxation induced by the factor was inhibited by hemoglobin and potentiated by superoxide dismutase.     

Apparent superoxide dismutase-like activity of immunoglobulin

Summary

Using various superoxide generating systems and nitroblue tetrazolium or cytochrome c as superoxide detector molecules it is possible to assess the superoxide dismutase activity of proteins. Intact antibodies raised to different antigens, the Fab’ fragment of anti-TNF [M632] and well-characterized recombinant Fv fragments of the murine antibody NQ11.7.22 appear to possess superoxide dismutase (SOD)-like activity.

Kinetic characteristics of the SOD-like activity of NQ11.7.22-Fv fragments suggest an enzymatic property and these fragments behave in an analogous manner to human erythrocyte Cu-Zn SOD. Furthermore, the SOD-like activity of the NQ11.7.22-Fv fragment is affected by certain single-point mutations in the amino acid composition and has a pH optimum of 6.2–6.6 which is unlike Cu-Zn SOD (pH 7.8–8.2). A change in tyrosine at the 32 position in the heavy chain and histidine at position 27 of the light chain of the NQ11.7.22-Fv fragment results in a profound reduction in SOD-like activity. Tyrosine at the 32 position in the heavy chain is known to play a significant role in antigen binding suggesting that the SOD-like activity occurs at the antigen-binding site itself. Single-point mutations at the periphery of the antigen combining site on the NQ11.7.22-Fv fragment had little or no effect on SOD-like activity.

Further studies show that immunoglobin (lgG-1), a commercially available murine monoclonal antibody, can also enhance the generation of hydrogen peroxide, the product of superoxide dismutation, when present in superoxide producing systems. The generation of hydrogen peroxide was increased by low pH (pH 6.25) with lgG-1 but reduced with Cu-Zn SOD.     

Biphasic radiosensitization of human lymphocytes by diethyldithiocarbamate: possible involvement of superoxide dismutase

A biphasic radiosensitization of human lymphocytes by diethyldithiocarbamate (DDC), a metal chelator, was observed. The first phase occurred at 10(-5) M and the second at 10(-3) MDDC. The biphasic radiosensitization coincided with the previously reported biphasic toxicity of DDC. Inhibition of superoxide dismutase (SOD) occurred only in the second phase, suggesting that it may be a contributing cause of this phase. The mechanism of the first radiosensitization phase is not known. The radiation survival curves indicated the presence of at least two lymphocyte populations differing in their radiosensitivity and representing 40 per cent and 60 per cent of the cells. Both cell populations were biphasically radiosensitized by DDC.     

An NMR study of the oxidation state of Cu,Zn superoxide dismutase in human red blood cells

The oxidation state of Cu,Zn superoxide dismutase was investigated by 19F-NMR spectroscopy in intact red blood cells and in their lysates. The superoxide dismutase concentration was determined in the red cells both by activity and by F- nuclear relaxation rate measurements and the results obtained showed that the high relaxation rate of F- in erythrocytes is mainly due to the presence of superoxide dismutase. The relaxation rate of F- was unaffected or slightly increased by the addition of a superoxide ion generating system to the cells or to their lysates so indicating that superoxide dismutase is fundamentally in steady state. The results are discussed in terms of the possible reactions of the enzyme in erythrocytes.     

Diethyldithiocarbamate inhibits in vivo Cu,Zn-superoxide dismutase and perturbs free radical processes in the yeast Saccharomyces cerevisiae cells

Copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in some model experiments in vitro demonstrated antioxidant as well as pro-oxidant properties. In the present study, yeast Saccharomyces cerevisiae lacking Mn-SOD were studied using Cu,Zn-SOD inhibitor N-N'-diethyldithiocarbamate (DDC) as a model system to study the physiological role of the yeast Cu,Zn-SOD. Yeast treatment by DDC caused dose-dependent inhibition of SOD in vivo, with 75% inhibition at 10mM DDC. The inhibition of SOD by DDC resulted in modification of carbonylprotein levels, indicated by a bell-shaped curve. The activity of glutathione reductase, isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase (enzymes associated with antioxidant) increased, demonstrating a compensatory effect in response to SOD inhibition by different concentrations of DDC. A strong positive correlation (R2=0.97) was found between SOD and catalase activities that may be explained by the protective role of SOD for catalase. All observed effects were absent in the isogenic SOD-deficient strain that excluded direct DDC influence. The results are discussed from the point of view that in vivo Cu,Zn-SOD of S. cerevisiae can demonstrate both anti- and pro-oxidant properties.     

Effects of diethyldithiocarbamate, an inhibitor of interferon antiviral activity, upon human natural killer cells

In support of a postulated role of the Cu++-dependent enzyme, superoxide dismutase (SOD), in antiviral effects of interferon (IFN), a close correspondence was previously shown to exist between inactivation of cellular SOD and concomitant blockade of IFN antiviral activity in fibroblasts by the Cu++-chelating agent, diethyldithiocarbamate (DDC). To further define the extent of "anti-IFN" activity, we initiated studies of DDC effects on IFN stimulation in the NK cell system. Unexpectedly, DDC directly inhibited cytotoxicity mediated by unstimulated NK cells. Pronounced inactivation occurred rapidly (less than 30 min), but was spontaneously reversible in the absence of DDC. Neither cell viability nor lymphocyte binding to target cells was detectably affected. Preincubation of DDC with Cu++ or Zn++ failed to neutralize its inhibitory effects nor could function be restored in DDC-pretreated NK cells by subsequent addition of Cu++, Zn++, Mg++, or Ca++. DDC treatment that inactivated NK cells did not detectably alter lymphocyte SOD activity. Thus, inhibition was probably not attributable to chelating properties of DDC. N-ethyl maleimide (NEM) and para-( hydroxymercuri ) benzoic acid ( PMBA ), enzyme inhibitors that preferentially react with sulfhydryl groups, both inactivated NK cells in a time- and dose-dependent manner similar to that of DDC. Preincubation with the sulfhydryl compound, cysteine, neutralized in parallel fashion the capacity of NEM, PMBA , and DDC to inhibit NK cell activity. Thus, a previously unreported reactivity of DDC with sulfhydryl groups appeared to be the basis of inhibition. NK cells incubated 1 hr with IFN and subsequently cultured 17 to 23 hr without IFN were activated to an extent comparable to cells continuously incubated 18 to 24 hr with IFN. Exposure to IFN for 1 hr was therefore sufficient to commit NK cells to acquisition of a fully activated state. Whether preactivated by a 1-hr or 18- to 24-hr IFN treatment, activated NK cells retained the DDC-sensitive phenotype characteristic of fresh unstimulated NK cells. Thus, prolonged IFN treatment did not render NK cells resistant to DDC or preferentially activate a DDC-sensitive NK cell subset. An 18- to 24-hr incubation of DDC-pretreated cells in the continual presence of IFN resulted in the boosting of NK cell activity. However, the 1-hr IFN pulse treatment protocol was consistently ineffective in boosting when IFN was added just after DDC-pretreatment. These results strongly suggested that DDC temporarily rendered NK cells unresponsive to what, under normal circumstances, approximated an optimally potentiating IFN stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)