Lack of effect of glutathione depletion on cytotoxicity, mutagenicity and DNA damage produced by doxorubicin in cultured cells

Chem.-Biol. Interactions, 57 (1986) 189--201 Elsevier Scientific Publishers Ireland Ltd.

189

LACK OF E F F E C T OF GLUTATHIONE DEPLETION ON CYTOTOXICITY, MUTAGENICITY AND DNA DAMAGE PRODUCED BY DOXORUBICIN IN CULTURED CELLS

GIOVANNI CAPRANICOa, NORA BABUDRIb, GINO CASCIARRIc, LUCILLA DOLZANIb, ROMOLO A. GAMBETTAa, ESTER LONGONIc, BIANCA PANIb, CARLA SORANZOa and FRANCO ZUNINOa,* aIstituto Nazionale per lo Studio e la Cura dei Tumori, Milan, bIstituto di Microbiologia, Universitd di Trieste, Trieste and CBoehringer Biochemiea Robin, Milan (Italy)

(Received July 15th, 1985) (Revision received November 12th, 1985) (Accepted November 28th, 1985)

SUMMARY Since endogenous glutathione (GSH), the main non-protein intracellular thiol compound, is k n o w n to provide protection against reactive radical species, its depletion by diethylmaleate (DEM) was used to assess the role of free radical formation mediated by doxorubicin in DNA damage, cytotoxicity and mutagenicity of the anthracycline. Subtoxic concentrations of DEM that produced up to 75% depletion of GSH did not increase doxorubicin c y t o t o x i c i t y in a variety o f cell lines, including Chinese hamster ovary (CHO) and lung (V-79) cells, L o V o human carcinoma cells and P388 murine leukemia cells. Similarly, the number of doxorubicin-induced DNA single strand breaks in CHO cells and the mutation frequency in V-79 cells were not affected by GSH depletion. The results obtained suggest that mechanisms other than free radical formation are responsible for DNA damage, c y t o t o x i c i t y and mutagenicity of anthracyclines. K e y w o r d s : Doxorubicin -- DNA damage -- Cytotoxicity

INTRODUCTION Most evidence presently favors the theory that DNA binding of anthracyclines is directly responsible for the cytotoxic activity of these antitumor agents. Evidence for this mechanism comes from studies of the interaction *To whom correspondence should be sent. Abbreviations: CHO, Chinese hamster ovary; DEM, diethylmaleate; FCS, fetal calf serum; GSH, glutathione; SDS, sodium dodecyl sulfate; 6-TG, 6-thioguanine. 0009-2797/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

190 of the drugs with nucleic acids [ 1 ] , from observations of their effects on DNA synthesis and structure [2,3] and from detailed structure-activity relationship studies [4--6]. However, since these drugs have a number of biochemical actions [7], the evidence for DNA as the primary target of drug action is not yet conclusive. One of the proposed alternative mechanisms for the cytotoxic effects of anthracyclines involves formation of intraceUular oxygen radicals (superoxide, hydroxyl radicals) generated b y drug metabolic activation [8,9] with consequent damage to DNA [ 1 0 , 1 1 ] , membranes and other biologically important macromolecules [12]. In this study, modulation of intracellular GSH, a known detoxifying agent towards toxic oxygen radical species (i.e., antioxidant) [13,14], was used to assess the role of free oxygen radical production by anthracyclines in the cytotoxicity and induction of DNA breaks in in vitro cultured cell lines. Since free radicals have also been implicated in the genotoxic effect of anthracyclines [ 1 5 ] , we also studied the influence of GSH levels on mutagenicity of doxorubicin in mammalian cells. MATERIAL AND METHODS Cell cultures CHO cells and Lo Vo human colon carcinoma cells were grown in monolayer culture in F-12 medium {Flow Laboratories Inc., McLean, VA, U.S.A.) supplemented with 10% and 20% fetal calf serum (FCS) {Flow Laboratories Inc.), respectively, and antibiotics. P388 murine leukemia cells were collected from a tumor-bearing mouse 3 days after transplant and maintained in suspension culture in RPMI 1640 medium (Grand Island Biological Co., Grand Island, NY, U.S.A.) supplemented with 15% FCS, 10 pM 2-mercaptoethanol and antibiotics up to 2 months and transplanted 3 times a week. The V-79 cell line, clone G5 [ 1 6 ] , was cultured in Dulbecco's modified Eagles' minimum essential medium with 10% FCS and antibiotics. Incubations were carried o u t at 37°C in a humidified atmosphere with 5% CO2. Cells in exponential growth phase were used in all experiments. For alkaline elution experiments, CHO cell DNA was labelled with 0.05--0.1 pCi/ml [2-14C] thymidine (56 mCi/mmol, Amersham, U.K.) for 20 h. Drug exposure Drug solutions were prepared immediately before use. Doxorubicin (Farmitalia-Carlo Erba, Milan, Italy) and DEM (Fluka, Switzerland) were dissolved in distilled water and in culture medium, respectively. All cells were treated with DEM at subtoxic doses (determined in preliminary experiments), for 1 h at 37°C. After washing twice, fresh medium and doxorubicin were added and incubated for another hour. After doxorubicin treatment, cells were washed, trypsinized (CHO, LoVo and V-79) or pelleted (P388), and resuspended in appropriate medium for further processing. In the case of V-79 cells, a simultaneous treatment schedule with doxorubicin and DEM (for 1 h) was also used.

191

Assays o f cytotoxic and mutagenic activities In CHO, LoVo, and V-79 cells lines, doxorubicin cytotoxicity was determined by the colony-forming inhibition test. After drug exposure, a fixed number of cells were plated and allowed to grow in fresh medium for 8--10 days. Cell colonies were then stained and counted. In the P388 cell line, drug c y t o t o x i c i t y was determined b y the growth inhibition test. After drug treatment, cells were cultured in drug-free medium for 72 h and then counted. In alkaline elution experiments, CHO cell viability was also assessed b y the trypan blue exclusion test. Mutagenic activity of doxorubicin on V-79 cells was determined as previously described [16]. Alkaline elution procedures The alkaline elution analyses used to determine DNA single-strand breaks (DNA-SSB) were essentially the same as described b y Kohn et al. [17,18] with minor modifications. '4C-Labelled CHO cells (7 × l 0 s) were diluted in ice-cold buffered saline (0.14 M NaCl, 1.47 mM KH2PO4, 2.7 mM KC1, 8.1 mM Na~HPO4, 0.53 mM Na:EDTA, pH 7.3), layered on 25-mm diameter, 2-pm pore-size polyvinylchloride filters (Millipore Corp., Bedford, MA U.S.A.), and washed several times. In each experiment, control untreated cells and 300-rad x-ray-irradiated cells as external standard were included. Cells were irradiated at 0°C with Stabilipan, Siemens, 200 kV, 0.5 mm Cu, 30 DFP, 221 rad/min and kept at 0°C until lysis. Cells on the filters were lysed at room temperature by addition of 5 ml of 2% sodium dodecyl sulfate (SDS), 25 mM Na2EDTA, 0.1 M glycine (pH 10), then incubated with 4 ml of the same lysing solution containing 0.5 mg/ml proteinase K (Merk, F.R.G.) for 50 min at room temperature. Filters were then rinsed once with 5 ml of 20 mM Na~EDTA ( p n 10). The DNAs on the filters were eluted in the dark with 20 mM EDTA (acid form), 0.1% SDS and tetrapropylammonium hydroxide (10% in water; Eastman, Kodak, Rochester, NY, U.S.A.), added in the amount to give pH 12.2, at a constant flow rate of 0.04--0.06 ml/min. Fractions were collected at l l 0 - m i n intervals. One ml of each fraction was mixed with 10 ml of Emulsifier (Packard Instruments, Downers Grove, IL, U.S.A.) and counted. Filters were treated with 0.4 ml of 1 M HC1 at 70--80°C for 60 min; after cooling at room temperature, 0.6 ml of 1 M NaOH was added for 45 min. They were finally neutralized and counted in 10 ml of Emulsifier. The fraction of ['4C] DNA retained on the filter was plotted as a function of elution time. From retention values of untreated control cells (r0), 300rad-irradiated cells (R0), and drug-treated cells (rn ) of the same experiment at a fixed elution time, the DNA-SSB frequency was calculated according to Kohn et al. [ 1 7 ] , using the following formula: Pn = [l°g(rn/ro)/log(Ro/ro)] × 300 rad where DNA-SSB frequency was expressed as rad-equivalents (Pn)"

192

Determination of cellular GSH Cellular GSH was determined by the procedure of Ellman [19] and expressed as nmol/mg of cellular proteins. Proteins were determined by the method of Lowry et al. [20]. RESULTS GSH, the main endogenous non-protein thiol compound, is known to provide protection against toxic radical species [13,14]. A 1-h exposure to DEM, a thiol conjugating reagent, produced depletion of intracellular GSH. GSH content of cell lines used in this work following treatment with various subtoxic doses of DEM is shown in Table I. In these cell systems, GSH depletion increased with DEM concentration and reached about 75% at 1 mM. Higher doses of the depleting agent were found to be cytotoxic. GSH content in DEM-treated CHO cells did not change after an additional hour of incubation in the absence of DEM and in the presence of cytotoxic concentrations (<2 #M) of doxorubicin (Table I); thus, doxorubicin did not increase GSH depletion, which was maintained at the same level during doxorubicin treatment. Doxorubicin itself, at pharmacological (i.e., cytotoxic) concentrations, did not appreciably affect GSH concentration, after 1 h exposure. However, a marginal depletion of GSH was seen at higher concentrations of the anthracycline. Cell lines used in this work (CHO, V-79, LoVo) showed somewhat different sensitivities to doxorubicin (Fig. 1). In all the cell lines tested, drug cytotoxicity was not appreciably influenced by pretreatment or simultaneous treatment (V-79 cells} with DEM at any dose used of the depleting agent; the observed differences were not statistically significant (Student's t-test). In another set of experiments, the sensitivity of V-79 cells was not affected by pretreatment with DEM (not shown). The effect of GSH depletion was also examined in the murine leukemia P388 cells (Table II). Again, a reduction of intracellular GSH level by subtoxic DEM concentrations (1 mM) did not modify the sensitivity to doxorubicin. It is well known that X-ray irradiation produces free oxygen radicals [21] and, like oxidizing agents, induces DNA breakage [22]. Under our experimental (i.e., aerated) conditions, 300 tad X-ray irradiation of DEM-pretreated CHO cells caused a higher elution rate of DNA from the filter than that of non-pretreated cells (Fig. 2). This finding is consistent with the involvement of GSH .as an intracellular protective agent against DNAdamaging reactive free radicals [ 23]. In contrast to X-ray irradiation, GSH depletion had no effect on DNA damage produced by doxorubicin in exponentially growing CHO cells (Fig. 3). At drug levels higher than 3.4 × 10 -7 M, the number of DNA-SSB increased with doxorubicin concentration and, as observed in different cell lines [24], reached a plateau around 3.4 × 10 -6 M. In this range of drug concentration, depletion of GSH by 0.2 mM DEM only marginally modified DNA fragmentation produced by doxorubicin.

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DOXORUBICIN(M) Fig, 1. E f f e c t o f g l u t a t h i o n e d e p l e t i o n o n C H O a n d L o V o cells, b e f o r e 1 h e x p o s u r e m e d i u m w i t h o u t DEM (e), w i t h 0.05 m M 1 m M DEM (D). V-79 cells were e x p o s e d DEM ( * ) or in t h e p r e s e n c e o f 0.5 m M DEM t i o n s (S.D. n o t m o r e t h a n 25%). T h e lines doxorubicin.

cell survival a f t e r e x p o s u r e t o d o x o r u b i c i n . t o d o x o r u b i c i n , were i n c u b a t e d for 1 h in DEM (o), w i t h 0.2 m M DEM (*), a n d w i t h t o d o x o r u b i c i n for 1 h in t h e a b s e n c e o f (A). T h e p o i n t s are m e a n s o f 2--5 d e t e r m i n a refer t o survival o f cells t r e a t e d o n l y w i t h

In these experiments, the observed differences were not statistically significant. In addition, the extent of DNA-SSB produced by doxorubicin was not appreciably influenced by DEM concentration, since further GSH depletion (obtained by treatment with 1 mM DEM, the highest tolerated dose) did not increase DNA damage (Fig. 3B). In all these experiments, cell viability, evaluated by the dye exclusion test, was always higher than 95%. Again, mutagenic effects of doxorubicin, tested in V-79 cells, were not affected by incubation with subtoxic doses of DEM (Fig. 4). At concentrations lower than 0.5 pM, doxorubicin was found to be weakly mutagenic on V79 cells, as already observed [16]. The mutagenic effect of the drug could T A B L E II E F F E C T O F DEM ON IDso O F D O X O R U B I C I N IN P388 L E U K E M I A C E L L S DEM cone. (raM)

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195

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not be studied above this concentration due to marked cytotoxicity. Under these conditions a mean GSH depletion of a b o u t 70%, produced by 0.5 mM DEM (Table I), was ineffective in increasing the number of 6-thioguanine (6TG)-resistant clones. At the present time, we cannot explain the difference observed between DEM-treated and untreated cells in the absence of doxorubicin. Although in a dose-response curve of separate experiments (in the range of subtoxic doses of DEM, i.e., < 1 mM) a trend towards an increase in the mutation frequency b y increasing DEM concentration was observed, the effect of DEM itself should be considered negligible in the experiments of Fig. 4. DISCUSSION

The presence of the quinone group in the anthracycline structure has been implicated in a variety of biochemical actions that lead to cytotoxic effects [8] and to organ~specific toxicity [25] of these antitumor agents. Indeed, the quinone group is able to undergo oxidation-reduction reactions which may generate free radical intermediates and reactive oxygen species [26].

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197 In particular, since anthracyclines, like a variety of DNA intercalating agents [27], induce damage in DNA, it has also been proposed that DNA strand breaks can be generated b y a free radical mechanism [10,11]. DNA break production b y anthracycline-mediated free radicals has been suggested as a mechanism contributing to drug cytotoxicity [7,8 ]. In this work, reduction of intracellular GSH levels has been used to assess the role of oxygen-based radicals in the doxorubicin-induced DNA breaks in CHO cells. The finding that depletion of GSH increased the number of DNA strand breaks b y ionizing radiation is consistent with the free radical scavenger activity of this thiol c o m p o u n d inside the cell [13,23]. In contrast, DNA break production b y doxorubicin was not affected b y pretreatment with subtoxic concentrations of DEM. The lack of a synergistic interaction between doxorubicin and DEM was also accompanied b y a lack of modification in the mutagenic activity of doxorubicin in V-79 cells following depletion of GSH. Although there is evidence that GSH is compartmentalized inside the cells [ 2 8 ] , the protective effect of nonprotein sulfhydryl compounds against DNA damage produced b y X-rays (Fig. 2) and, in contrast, the lack of potentiation of DNA alterations in doxorubicintreated cells following GSH depletion suggest that, wherever the thiols are compartmentalized, a different mechanism is responsible for the observed DNA alterations. Thus, the results obtained suggest that DNA lesions and mutagenicity of anthracyclines are not related to free radical formation. Indeed, in agreement with the results of other authors [ 2 4 ] , at pharmacological drug levels, a free radical mechanism may make a marginal (if any) contribution to DNA damage. These findings are also consistent with the results obtained by the use of exogenous free radical scavengers [ 2 9 ] . Other indirect evidence [30] supports our conclusions. Indeed, it is unlikely that a free radical mechanism would explain the breaks in cellular DNA produced by other intercalators lacking the quinone group [27]. Recent findings suggest that an enzymatic process is involved in the intercalator-induced break production [ 31]. Due to low cytotoxic levels of doxorubicin (<2 pM) in our cell systems, we did not attempt to determine the extent of oxygen radicals produced b y drug treatment. Depletion of cellular GSH was reported at very high doxorubicin concentration (/>100 #M) in different cell lines [ 3 2 , 3 3 ] , probably as a consequence of oxygen radical formation. At cytotoxic drug concentrations (<2/~M), the relevance of possible formation of free radicals remains uncertain. The results presented in this work provide indirect evidence that free radicals are not likely to be involved in the cytotoxic action of doxorubicin in these cell lines, which are highly sensitive to the drug. The observation that the drug sensitivity of a variety of cell lines of different origin was not affected b y subtoxic concentrations of DEM argues against a cytotoxic mechanism involving formation of reaction oxygen radical species. The finding that exogenous free radical scavengers slightly decreased lethal effects of doxorubicin only at relatively high drug concentration [29] is consistent with the lack of effect of GSH depletion. Since DEM was found

198 to reduce only the cytoplasmic GSH [28], the present results do not exclude the possibility that mitochondrial toxicity was unaffected by the depleting agent. However, the results obtained by other authors [28,33] on rat hepatocytes after exposure to high drug concentrations (>100/~M) could not be extrapolated to our situation, in which sensitive cells were exposed to pharmacological drug levels. In contrast to cell lines used in the present work, rat hepatocytes appeared relatively insensitive to lethal effects of anthracyclines [33], thus requiring the use of higher doses. In addition, at therapeutic doses, doxorubicin is known to localize essentially in nuclei [34,35]. These observations suggest that different effector mechanisms resulting in cytotoxicity may be operative at different drug levels. Similarly, an oxygen-dependent degradation of DNA (presumably by free radicals) in L1210 cells had been detected only at high drug concentration [24,36]. The observation by Kennedy et al. [37] that doxorubicin is preferentially toxic to hypoxic tumor cells suggests the existence of oxygen-independent lethal effects. In addition, the acquired resistance of P388 cells was not related to increased ability to detoxify reactive oxygen species [38]. Taken together with this indirect evidence, the results reported in this work suggest that, at pharmacological drug concentrations [24], production of free radicals plays a marginal, if any, role in drug cytotoxicity. Although mechanisms other than drug-mediated free radical generation appear to be operational in the lethal effects of doxorubicin in the cell lines studied in this work, these results could not be generalized since different cells may be characterized by different enzymatic abilities to reduce doxorubicin to intermediate free radicals [39], and by different defenses against reactive oxygen species [40--42]. This may be the case of the cardiac tissue characterized by limited antioxidant defenses [43]. Thus, the drug-induced free oxygen radicals may play a critical role in damaging myocardial cells. It is likely that the GSH system may be involved in the modulation of doxorubicin-induced toxicity, since GSH depletion by DEM markedly potentiated doxorubicin lethal toxicity [44] and sulfhydryl-containing compounds protected against acute lethality and cardiac lesions produced by doxorubicin [45,46]. However, treatment with thiols did not interfere with the antitumor activity of the anthracycline [45,46]. Although our findings do not eliminate the possibility that, in some cases, free radicals may still contribute to antineoplastic action of anthracyclines, there is no clear evidence to link this mechanism with cytotoxic effects in tumor cells. Thus, the results of this work indirectly support the original hypothesis that DNA intercalation plays a major role in the antitumor effect of these agents [4]. ACKNOWLEDGEMENTS This research was supported by a grant from the Consiglio Nazionale deUe Ricerche, Rome, Italy (Progetto Finalizzato Oncologia). The authors thank Ms. B. Johnston for editing and preparing the manuscript.

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