Interactions of the carcinogen 4-nitroquinoline 1-oxide with the non-protein thiols of mammalian cells.

The carcinogen 4-nitroquinoline 1-oxide (4-NQO) was found to rapidly deplete non-protein thiols (NPSH) from Ehrlich ascites tumor cells and V79 Chinese hamster fibroblasts. The effects of NPSH on 4-NQO metabolism were studied by measuring 4-hydroxyaminoquinoline 1-oxide formation, CN- -insensitive oxygen consumption, and reduction of ferricytochromes c + c1 in normal cells and in cells pretreated with the thiol reagent N-ethylmaleimide. Removal of thiols before treatment with 4-NQO resulted in increased production of 4-hydroxyaminoquinoline 1-oxide and increased production of nitro radicals. The NPSH thus appeared to play a significant role in 4-NQO detoxification. Glutathione, when present in culture medium during 4-NQO treatment, protected V79 cells from 4-NQO toxicity. Several mechanisms for reaction of 4-NQO with intracellular NPSH were indicated. Both V79 and Ehrlich cells contained appreciable amounts of glutathione S-transferase (EC 2.5.1.18), which catalyzes the nucleophilic substitution of the nitro group of 4-NQO with thiols. Greater thiol loss under oxic than under hypoxic conditions suggested oxidation by superoxide, peroxide, or hydroxyl radical formed in the course of 4-NQO reduction. In addition, reaction of thiols with nitro radicals or with nitrosoquinoline 1-oxide was indicated by the inhibitory effect of glutathione on oxygen consumption in solutions of 4-NQO and sodium ascorbate.

Metabolism of benzo(a)pyrene by guinea pig pancreatic microsomes.

The in vitro microsomal metabolism of the strain 13 guinea pig pancreas was investigated by determining the benzo(a)pyrene (BP) hydroxylase activity in the 9000 x g supernatant and microsomal pellet. BP hydroxylase activity in both 9000 x g supernatant and microsomal pellet of the pancreas was less than 1% of the activity in the respective liver fractions, However, pretreatment of animals with methylcholanthrene or BP at 20 mg/kg, for either 1 day or 3 consecutive days, markedly enhanced the BP hydroxylase activity of pancreatic microsomes over that of controls; the induction in the liver microsomes was less than 2-fold over that of controls. The hydroxylation of BP by pancreatic microsomes was linear with time over a 30-min period, with the rate of hydroxylation dependent on both the enzyme and substrate concentrations.

Enhancement of photodynamic cell killing (with chloroaluminum phthalocyanine) by treatment of V79 cells with the ionophore nigericin.

The K+/H+ ionophore nigericin dramatically increases killing of V79 cells by photodynamic therapy (PDT), when cells pretreated with 1 microM chloroaluminum phthalocyanine are incubated with nigericin before exposure to red light. Nigericin affects primarily the shoulder of the PDT dose-response curve, reducing the surviving fraction from 0.90 to 0.02 after a fluence of 7 kJ/m2 and from 0.80 to 0.0003 after a fluence of 12 kJ/m2. Optimal enhancement of PDT occurs when cells are incubated with 2 microM nigericin, at pHe 6.7, for 30 to 60 min before irradiation. However, significant enhancement of PDT also occurs when nigericin is added immediately before irradiation. Treatments with chloroaluminum phthalocyanine and nigericin, nigericin alone, or nigericin and red light are not toxic to cells. Cells treated with the combined agents display a rounded morphology 2 h after light exposure and lyse within 12 h. However, rounding of cells is not accompanied by severe depletion of ATP or by permeabilization of the plasma membrane to trypan blue. These results, together with known metabolic effects of nigericin, suggest that nigericin potentiates PDT by perturbing ion transport across either mitochondrial or plasma membranes.

Effect of the K+/H+ ionophore nigericin on response of A549 cells to photodynamic therapy and tert-butylhydroperoxide.

The K+/H+ ionophore nigericin dramatically increases killing of V79 cells and A549 cells by photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Previous studies suggested that the interaction between PDT and nigericin is related to the ability of this ionophore to reduce intracellular pH (pHi). The present study was undertaken to test the possibility that nigericin, by lowering pHi, inhibits reductive detoxification of PDT-produced peroxides by enzymes of the glutathione (GSH) redox cycle and the pentose cycle. To test this possibility we examined the effects of nigericin on the toxicity and metabolism of a model peroxide, tert-butylhydroperoxide (tert-BOOH), in A549 cells, a cell line in which the GSH redox cycle is known to be the principal pathway for reduction and detoxification of tert-BOOH. We found that nigericin equilibrates pHi of A549 cells with extracellular pH (pHe) in a time-dependent manner. It increases the toxicity of tert-BOOH toward A549 cells, inhibits loss of tert-BOOH from the buffer overlying the cells, and reduces the rate of 14CO2 release from radiolabelled glucose, which is a measure of pentose cycle activity. These effects are significantly greater at pHe 6.40 than at 7.40. Monensin, a Na+/H+ ionophore which does not reduce pHi, does not enhance the toxicity of tert-BOOH and has only a minimal effect on tert-BOOH reduction. These data suggest that nigericin-induced inhibition of peroxide detoxification is at least a plausible mechanism by which the ionophore might interact with PDT.

Misonidazole-induced biochemical alterations of mammalian cells: effects on glycolysis.

Incubation of Ehrlich ascites tumor cells with misonidazole under aerobic conditions causes a stimulation of glucose consumption which is probably related to stimulation of hexose monophosphate shunt activity. Incubation of Ehrlich cells with 5 mM misonidazole under hypoxic conditions results in a time-dependent inhibition of glycolysis, seen when treated cells are washed and resuspended in fresh buffered saline or media. This inhibition does not appear to be related to breakdown and loss of pyridine nucleotides from the cells during misonidazole treatment, nor is it a consequence of cell death. Post-incubation in buffer containing cysteine or cysteamine restores cellular glycolytic activity to near control levels. Partial inhibition of glucose consumption after incubation with misonidazole also occurs with V79-379A, V79-171B, EMT6 and A549 cell lines. The extent of inhibition varies among the lines, but is accompanied by approximately a 50% reduction in intracellular non-protein thiol levels in all cases. Ehrlich cells incubated anaerobically with misonidazole lose their response to the uncoupler 2,4-dinitrophenol, which normally increases glucose consumption, and exhibit less ability to metabolize pyruvate. Cells incubated under anaerobic conditions but in the absence of misonidazole do not show these effects.

Inhibition of recovery from potentially lethal radiation damage in A549 cells by the K+/H+ ionophore nigericin.

A549 cells held for 4 hr in Hank's balanced salt solution, after 10 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is dependent on extracellular pH (pHe). Recovery factors of 2.2 to 3.5 are observed when pHe is 6.40 to 7.30, but recovery factors of less than 1.0 are found when pHe is reduced to 6.20 or 6.00. The K+/H+ ionophore nigericin, when added to cells post-irradiation, inhibits PLDR in a pHe-dependent manner; it is increasingly more effective as pHe is reduced from 6.80 to 6.40. The presence of nigericin thus causes inhibition of PLDR at pHe's that normally promote recovery. The drug does not affect radiation response of A549 cells when present only during irradiation. Effects of low pHe buffer, with and without nigericin, on intracellular pH (pHi) and on ATP levels were examined in an effort to elucidate the mechanisms for inhibition of PLDR and enhancement of radiation response. Incubation of cells in pHe 6.00 buffer results in a slight decrease in pHi and does not induce a drop in ATP levels. In contrast, post-irradiation incubation of cells in pHe 6.40 buffer containing 2 microM nigericin causes an immediate and dramatic decrease in pHi, and a gradual loss of ATP to 30% of control levels by 4 hr. The data obtained so far suggest that a very slight lowering of pHi may influence post-irradiation holding recovery, and that the mechanisms by which pHe 6.00 buffer alone, or pHe 6.40 buffer containing nigericin, affect holding recovery are different.

Chemosensitization: do thiols matter?

It is well known that endogenous sulfhydryls are radioprotective in mammalian cells. Their comparable role in chemotherapeutic drug toxicity has been known for almost as long but less well defined. Thiol depletion as a mechanism responsible for enhanced cytotoxicity of melphalan was assayed by pretreatment of cells in vitro with misonidazole and buthionine sulfoximine (BSO). Hypoxic cell sensitizers, such as MISO, deplete endogenous thiols by metabolic activation under hypoxic conditions to thiol reactive intermediates, whereas BSO specifically inhibits a key enzyme in the synthesis of glutathione. For a given level of thiol reduction, sensitization to melphalan was far greater by preincubation with MISO than it was for BSO. This indicated that thiol reduction itself was not the sole factor involved in chemosensitization by MISO. As evidence that the method of thiol depletion predisposes to the expression of biological damage, it was shown that cells preincubated with MISO were appreciably more vulnerable to oxidative stress than those exposed to BSO. BSO was shown to totally inhibit the repair of damage from a preincubation treatment with MISO, demonstrating that recovery is dependent upon thiol regeneration. Thiol depletion "per se" is a good qualitative but not necessarily a quantitative indicator of chemosensitization--the biological and biochemical function of the thiol depleting agents used influences further drug interactions. The results of the study with these two agents suggest that thiols may play a potentially more critical role in the repair rather than the initiation of drug-induced damage.

Factors involved in depletion of glutathione from A549 human lung carcinoma cells: implications for radiotherapy.

We have measured the rate of GSH resynthesis in plateau phase cultures of A549 human lung carcinoma cells subjected to a fresh medium change. Buthionine sulfoximine (BSO) blocks this resynthesis. Diethyl maleate (DEM) causes a decrease in accumulation of GSH. If DEM is added concurrently with BSO there is a rapid decline in GSH that is maximal in the presence of 0.5 mM DEM. GSH depletion rapidly occurs when BSO is added to log phase cultures which initially are higher in GSH content. Twenty-four hr treatment of A549 cells with BSO results in cells that are more radiosensitive in air and show a slight hypoxic radiation response. A 2 hr treatment with either 0.25 mM or 0.5 mM DEM results in some hypoxic sensitization and little increase in the aerobic radiation response. The 24 hr BSO + 2 hr DEM treatment sensitizes hypoxic cells to a greater degree than either agent alone but does not increase the aerobic response more than is seen with BSO alone. Cells treated simultaneously with BSO + DEM show little increase in the hypoxic radiation response, compared to DEM alone, but are more sensitive under aerobic conditions. Decreased cell survival for aerobically irradiated log phase A549 cells occurs within minutes after addition of a mixture of BSO + DEM. The decreased cell survival following aerobic irradiation, after prolonged treatment with BSO or acute exposure to BSO + DEM, may be in part due to inhibition of glutathione peroxidases. For example, glutathione-S-transferase, known to have glutathione peroxidase activity (non-selenium), is nearly completely inhibited by the BSO treatments. In addition, cellular capacity to react with peroxide (glutathione peroxidase, selenium containing) was also inhibited. We suggest that the enhanced aerobic radiation response is related to an inability of GSH depleted cells to inactivate either peroxy radicals or hydroperoxides that may be produced during irradiation of BSO treated cells. Furthermore, enhancement of the aerobic radiation response may be useful in vivo if normal tissue responses are not also increased.

Depletion of intracellular GSH and NPSH by buthionine sulfoximine and diethyl maleate: factors that influence enhancement of aerobic radiation response.

Many investigators have observed aerobic sensitization of V79, CHO and A549 (human lung carcinoma) cells upon depletion of GSH using buthionine sulfoximine (BSO). Recently we discovered that this aerobic sensitization can be reversed if WR-2721 or N-acetylcysteine is added to the cells just prior to irradiation. Reversal requires that the exogenous thiols be present during the time of irradiation. One possible explanation was that these thiols entered the cells and either increased the pool of cellular nonprotein thiols or reversed the thiol-depleted state by stimulation of GSH synthesis. Cells treated with BSO do not readily regenerate intracellular GSH because this agent irreversibly inhibits gamma-glutamyl synthetase. For A549 monolayer cultures, there is approximately 50% regeneration 6 hr after removal of 0.01 mM BSO, 20% 6 hr after 0.1 mM BSO, and only 5% 6 hr after 0.5 mM BSO. We found that addition of WR-2721 or N-acetylcysteine to BSO-treated cells did not affect the rate of regeneration of intracellular GSH. Thus, reversal of the aerobic sensitization of A549 cells by BSO cannot be explained on the basis of intracellular thiol levels alone, or by rapid reversal of BSO inhibition. In addition, diethylmaleate (DEM)-treated cells are considerably different from BSO-treated cells with respect to the ability to regenerate GSH. After removal of DEM, A549 cells immediately begin GSH resynthesis, and return to control levels occurs within 2 hr. Exogenous 5 mM GSH increases the rate of resynthesis of GSH in DEM-treated cells, but not in BSO-treated cells.

Sensitivity to chemical oxidants and radiation in CHO cell lines deficient in oxidative pentose cycle activity.

In this paper we examine the susceptibility of a series of G6PD- CHO cell lines to a variety of chemical oxidants. Addition of these drugs to K1D, the parental cell line, results in as much as a 20-fold increase in pentose cycle (PC) activity over control values. In two of our mutant lines, E16 and E48, little or no stimulation of PC activity is seen. These lines are shown to be much more susceptible to the toxic effects of the chemical oxidants t-butyl hydroperoxide and diamide. PC activity is also stimulated by ionizing radiation in K1D cells. One of the G6PD- cell lines has an increased aerobic radiation response compared to the parental line. However, since this is not the case with the other G6PD- cell lines, it is unclear whether this represents a difference in the absolute value of PC activity or some additional variable that may be influencing the results.

A biochemical basis for the radiosensitizing and cytotoxic effects of chlorpromazine hydrochloride in vitro and in vivo.

There have been many investigations on the radiosensitizing and cytotoxic effects of chlorpromazine, both in vitro and in vivo. We have extended the studies to include the chemotherapeutic effect of chlorpromazine on lymphosarcoma and on a mammary adenocarcinoma arising spontaneously in breeding female CBA mice. Chlorpromazine was ineffective in the former tumor but enhanced the longevity of tumor-bearing mice in the latter case. The drug was also observed to potentiate the hyperthermic killing of L5178Y cells and inhibit cellular oxygen utilization by cultured mammalian cells. The possible mechanisms involved are discussed.

Enhanced cytotoxicity of melphalan by prolonged exposure to nitroimidazoles: the role of endogenous thiols.

It was first demonstrated that prolonged exposure of hypoxic V-79 cells to misonidazole prior to irradiation produced an increased radiosensitization in 1977; it was postulated that the reduction of misonidazole resulted in intermediates capable of depleting cells of endogenous thiols, substances known to play a role in the hydrogen repair of target radicals produced by ionizing radiation. The present study shows that a prolonged exposure of V-79 cells to a variety of nitroimidazoles (misonidazole, Ro-05-9963, SR-2508, and MTR1-80) results in an enhanced cytotoxicity when these cells are subsequently exposed to melphalan. This process of enhanced melphalan toxicity occurred only when cells were pretreated with misonidazole under hypoxic conditions, suggesting that nitroreduction is necessary for chemosensitization as it is for increased radiosensitization. Different nitroimidazoles tested vary in the extent to which they sensitize cells to the subsequent action of melphalan. Repair from a misonidazole pretreatment is essentially complete by six hours. This study demonstrated that cysteamine could reduce the cytotoxicity of misonidazole and the enhancement of melphalan toxicity. This was an effect reversible with time and one implying similar mechanisms for the preincubation effect observed in vitro for radiation and chemotherapy agents.

Enhancement in the aerobic toxicity of misonidazole and SR-2508 by buthionine sulfoximine and 4-hydroxypyrazole: the role of hydrogen peroxide.

Chronic aerobic exposure of A549 human lung carcinoma cell cultures to 0.1 mM L-buthionine-S,R-sulfoximine and 1 mM misonidazole, or 1 mM SR-2508 results in inhibition of cell growth and decreased clonogenic survival. These patterns are not apparent with the individual drug treatments. Both parameters demonstrate maximum toxicity after 72 hr in culture, which parallels the time required to deplete A549 cells of glutathione with 0.1 mM L-BSO under these growth conditions. Toxicity appears to be related to hydrogen peroxide-produced during 1 electron reduction of the nitro compounds in the presence of oxygen. A549 cells have a lowered capacity to reduce peroxide due to the effect of thiol depletion on the enzymes GSH-peroxidase and GSH-S-transferase, which require the tripeptide as a substrate. The addition of catalase, another important enzyme involved in peroxide reduction, partially reverses the observed toxicity. 4-Hydroxypyrazole, which inhibits endogenous catalase activity, causes an increase in the observed cytotoxicity. Similar effects of L-BSO and 4-hydroxypyrazole are seen for toxicity due to hydrogen peroxide being added directly to cell cultures.

The effect of L-buthionine sulfoximine on the aerobic radiation response of A549 human lung carcinoma cells.

Our data show that A549 cells are increasingly radiosensitive with prolonged exposure to L-BSO. The resulting glutathione and protein thiol depleted cells show both loss of shoulder and slope modification. Furthermore, there is an increase in single strand DNA breaks and irrepairable cross-linking. The aerobic radiation damage in the thiol depleted state appears to be different from that obtained with hypoxic cells. Any postulated role for GSH in reducing or preventing peroxidative radiation damage must also include protection against single strand DNA breaks as well as involvement in repairing DNA-protein cross-links. The latter effect may be related to decreased protein thiol content as reflected in a decreased enzyme capacity to repair DNA damage.

Role of glutathione in the aerobic radiation response.

We will review the relationships between glutathione (GSH), protein thiols, and cellular responses to radiation, peroxides, and peroxide-producing drugs. Our primary interest involves the behavior of sulfhydryls as electron and hydrogen carriers, and their capacity to protect various target molecules against radiation and peroxidative damage. We used reagents such as L-buthionine sulfoximine (LBSO), alone and in combination with N-ethyl maleimide (NEM), diamide, and dimethylfumarate, to decrease GSH so that it could no longer participate in the electron transfer reactions. Our results indicate that aerobic sensitization produced by GSH depletion can be further enhanced if electron-accepting agents, such as tertiary butyl hydroperoxide (t-BOOH), are present during irradiation. Hydroperoxide is a substrate for glutathione peroxidase and diverts electrons and hydrogen away from target molecules during its reduction. Sensitivity to radiation seems to be due to the inhibition of the mitochondria's capacity to reduce hydroperoxide. We will also report the mitochondria's ability to reduce the oxygen radicals produced by radiation and drugs. Data also indicate that t-BOOH oxidizes protein thiols which are enzymatically involved in repair of DNA damage.

Inhibition of glycolysis of mammalian cells by misonidazole and other radiosensitizing drugs. prevention by thiols.

Prolonged anaerobic incubation of Ehrlich ascites tumor cells and Chinese hamster V79-379A cells with misonidazole, desmethylmisonidazole, or niridazole led to inhibition of both glucose consumption and lactate formation. This effect was measured in cells washed free of the nitro compounds and resuspended in fresh buffer or medium. The degree of inhibition of glucose utilization was related to drug concentration, and to the rate of metabolic reduction, as measured under aerobic conditions by KCN-insensitive oxygen consumption. Misonidazole-induced inhibition of glycolysis developed concurrently with depletion of intracellular non-protein thiol (NPSH) and was protected against by the presence of cysteamine, cysteine and, to some extent, GSH in the cell incubate. These findings suggest diethyl maleate was used to deplete 90% of the endogenous NPSH, but this depletion did not alter the effects of misonidazole on glycolysis.

Nitroheterocycle metabolism in mammalian cells. Stimulation of the hexose monophosphate shunt.

Misonidazole, SR-2508, nitrofurazone and other nitroheterocycles stimulated release of 14CO2 from [1-14C]glucose but not from [6-14C]glucose when incubated with mouse Ehrlich ascites cells or human A549 lung carcinoma cells in vitro. This demonstrated that the nitro compounds activated the hexose monophosphate shunt and is evidence that an important pathway of nitro reduction in these cell lines is electron transfer from NADPH-dependent cytochrome c reductase to the nitro group. Shunt activity was stimulated under both aerobic and anaerobic conditions. For catalase-free Ehrlich cells, aerobic effects were greater than anaerobic, indicating that NADPH was used for reduction of H2O2, via GSH peroxidase and reductase, as well as for one-electron nitro reduction, under aerobic conditions. Several of the compounds tested stimulated 14CO2 release from [2-14C]glucose as well as from [1-14C]-glucose. This shows that the cellular requirement for NADPH, in the presence of nitro drug, was great enough to cause recycling of pentose phosphates. Recycling could decrease the availability of ribose-5-P needed for nucleic acid synthesis, which could partly explain the inhibition of DNA synthesis observed upon prolonged aerobic incubation of cells with nitro compounds. Comparison of the rate of disappearance of nitrofurazone from anaerobic A549 cell suspensions with the rate of 14CO2 release suggests that the drug reduction in this cell line was catalyzed almost entirely by NADPH-requiring enzymes.

Combined depletion of O6-alkylguanine-DNA alkyltransferase and glutathione to modulate nitrosourea resistance in breast cancer.

MCF-7 human breast cancer cells possess high levels of O6-alkylguanine-DNA alkyltransferase and moderate levels of glutathione, and are more resistant to chloroethylnitrosoureas (CNUs) than cells with low levels of either molecule. The role of each as a component of CNU resistance was assessed using O6-benzylguanine (O6-bG) or O6-methylguanine (O6-mG) to deplete the alkyltransferase and L-buthionine sulfoxamine (L-BSO) to deplete glutathione. O6-bG and O6-mG potentiated 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity, resulting in a dose modification factor of 5.4 and 2.3, respectively, which reflected the more potent inhibitory effect of O6-bG. L-BSO alone had little effect on the survival of MCF-7 cells following BCNU exposure, but when combined with O6-mG, BCNU cytotoxicity was additive, yielding a dose modification factor of 3.2. O6-bG or O6-mG and L-BSO acted independently, as neither class of inhibitor affected the other's mechanism of CNU resistance. Furthermore, MCF-7 cells overexpressing GST mu were not more resistant to BCNU than the parent cell line in either the presence or absence of O6-bG or L-BSO. These results indicate that on a relative basis in MCF-7 cells, the alkyltransferase is the cell's first line of defense against CNUs. This suggests that therapeutic trials based on O6-bG-induced biochemical modulation of CNU resistance may increase the efficacy of these chemotherapeutic agents against human malignant cells and that L-BSO may have little additive effect when used with these agents.

Biochemistry of reduction of nitro heterocycles.

Misonidazole is a metabolically active drug. Its addition to cells causes an immediate alteration in cellular electron transfer pathways. Under aerobic conditions the metabolic alterations can result in futile cycling with electron transfer to oxygen and production of peroxide. Thiol levels are extremely important in protecting the cell against the peroxide formation and potentially hazardous conditions for hydroxyl radical production. Nevertheless such electron shunting out of cellular metabolism will result in alterations in pentose cycle, glycolysis and cellular capacity to reduce metabolites to essential intermediates needed in DNA metabolism (i.e. deoxyribonucleotides). Glutathione must be depleted to very low levels before toxic effects of misonidazole and other nitro compounds are manifested in cell death via peroxidative damage. Under hypoxic conditions misonidazole also diverts the pentose cycle via its own reduction; however, unlike the aerobic conditions, there are a number of reductive intermediates produced that react with non-protein thiols such as GSH as well as protein thiols. The reaction with protein thiols results in the inhibition of glycolysis and other as yet undetermined enzyme systems. The consequences of the hypoxic pretreatment of cells with nitro compounds are increased vulnerability to radiation and chemotherapeutic drugs such as L-PAM, cis-platinum and bleomycin. The role that altered enzyme activity has in the cellular response to misonidazole and chemotherapeutic agents remains to be determined. It is also clear that the GSH depleted state not only makes cells more vulnerable to oxidative stress but also to hypoxic intermediates produced by the reduction of misonidazole beyond the one electron stage. The relevancy of the present work to the proposed use of thiol depletion in vivo to enhance the radiation or chemotherapeutic response of tumor tissue lies with the following considerations. Apparently, spontaneous peroxidative damage to normal tissue such as liver can occur with GSH depletion to 10-20% of control and with other normal tissue when GSH reaches 50% of control. This situation can obviously become more critical if peroxide producing drugs are administered. The only advantage to such combined drug treatments would lie in the possibility that tumors vary in their catalase and peroxidase activity and consequently may be more vulnerable to oxidative stress (cf. review by Meister. Our tumor model, the A549 human lung carcinoma cell in vitro, appears to be an exception because it has catalase, peroxidase and a high content of GSH.(ABSTRACT TRUNCATED AT 400 WORDS)

pH-dependent effects of the ionophore nigericin on response of mammalian cells to radiation and heat treatment.

The extracellular pH (pHe) in many solid tumors is often lower than the pH of normal tissues. The K+/H+ ionophore nigericin is toxic to CHO cells when pHe is below but not above 6.5, and thus it has potential for selective killing of tumor cells in an acidic environment. This study examines the pH-dependent effects of nigericin on the response of CHO cells to radiation and heat treatment. Cells held for 4 h in Hank's balanced salt solution, after 9 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is maximal at pHe 6.7-6.8. Addition of nigericin, postirradiation, not only inhibits PLDR when pHe is below 6.8, but interacts synergistically with radiation to reduce survival below that of cells plated immediately after irradiation when pHe is 6.4 or lower. Nigericin enhances heat killing of CHO cells perferentially under acidic conditions, and where neither heat nor drug treatment alone is significantly toxic. Survival of cells held for 30 min at 42.1 degrees C in the presence of 1.0 microgram/ml nigericin is 0.6, 0.08, 0.003, and 0.00003 at pHe 7.4, 6.8, 6.6, and 6.4, respectively, relative to survival of 1.0 in untreated cultures. The biochemical effects of nigericin at pHe 7.4 vs pHe 6.4 have been investigated. Nigericin inhibits respiration, stimulates glucose consumption, and causes dramatic changes in intracellular concentrations of Na+ and K+ at pHe 7.4 as well as 6.4. The drug reduces intracellular levels of ATP, GTP, and ADP but has more pronounced effects under acidic incubation conditions. Others have shown that nigericin equilibrates pHe and intracellular pH (pHi) only when pHe is 6.5 or lower. Our observations and those of others have led us to conclude that lowering of pHi by nigericin is either the direct or indirect cause of enhancement of radiation and heat killing of cells in an acidic environment.