Chem.-BioL Interactions, 72 (1989) 335-345 335 Eisevler Scientific Publishers Ireland Ltd.

D I F F E R E N T I A L E F F E C T S OF G L U T A T H I O N E D E P L E T I O N AND M E T A L L O T H I O N E I N INDUCTION ON T H E INDUCTION OF DNA SINGLE-STRAND B R E A K S AND CYTOTOXICITY BY tert-BUTYL H Y D R O P E R O X I D E IN C U L T U R E D M A M M A L I A N CELLS

TAKAFUMI OCHI" and PETER A. CERUTTI b

aDepartmont of Environmental Tozwology, Faculty of Pharmaceutwal Sciences, Te~kyo Universzty, Sagamiko, Kanagawa 199-01 fJapan~ and bDepartment of Carcmogenesia, Sur~ss Institute for Experimental Cancer Research, CH-1066 Epahnges/Lausanne (Su~tzerland)

(Received December 12th, 19881 (Revision received May 11th, 1989) (Accepted May 12th, 1989)

SUMMARY

Induction of DNA single-strand breaks (ssb), their resealing and cytotoxicity by tert-butyl hydroperoxide (t-BuOOH) were investigated in cul- tured Chinese hamster V79 cells. The effect of the depletion of cellular gluta- thione (GSH), iron chelation and induction of metaUothionein (MT) on these parameters was studied, t-BuOOH in a concentration range of 0.02--0.5 mM induced DNA ssb in a dose-dependent fashion. Strand breakage increased as a function of time up to 1 h. Divalent iron chelator o-phenanthroline sup- pressed markedly the induction of DNA ssb while the trivalent iron chelator desferrioxamine had no effect. GSH-depletion increased cytotoxicity of t- BuOOH. In contrast, the depletion of GSH did not affect the efficiency of formation of DNA ssb by t -Bu00H and the rate of resealing of the DNA damage. The induction of MT did not influence the efficiency of formation of DNA ssb by t-Bu00H. In summary, while GSH depletion and MT induction affected the formation of DNA ssb and cytotoxicity differently divalent iron was required for both. Therefore, appears likely that DNA breakage and cytotoxicity by t-BuOOH are caused by independent mechanisms.

Key words: Hydroperoxide -- DNA strand breaks -- Iron chelation -- Glutathione depletion -- Metallothioneins

INTRODUCTION

Active oxygen species (AO) and hydroperoxides cause a variety of struc- tural alterations of cellular macromolecules in vitro, cultured cells and ani- mals in vivo [1--3]. AO is known to induce DNA damage such as strand

0009-2797/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published m Ireland

336

breaks [3--6] and base damage including thymine glycols [7,8] and 8-hydroxy- deoxyguanosine [9]. It has been shown to participate in radiation- and chemi- cal-induced carcinogenesis, in particular in tumor promotion and progression [10-13]. However, the role of oxidant-induced DNA damage in carcinogene- sis remains unclear. The effects of oxidants on living cells are at tenuated by an elaborate defense system consisting of superoxide dismutase, catalase, glutathione (GSH) and GSH-related enzymes such as GSH-peroxidase or GSSG-reductase as well as antioxidant vitamins [3,10].

In earlier studies [14,15], we at tempted to relate the cytotoxicity of the organic hydroperoxide tert-butyl hydroperoxide (t-BuOOH) on V79 cells to the protective capacity of their antioxidant system. We found that divalent iron-catalysed radical reactions were responsible for the cytotoxicity of t- Bu0OH. Cytotoxicity was markedly enhanced by GSH depletion.

In the present work we studied the mechanism of the induction of DNA ssb by t-BuOOH, repair of the damage and the relationship between DNA ssb, cytotoxicity, GSH concentration and metallothionein (MT) levels. Inductin of MT had been demonstrated previously to protect cells from cytotoxicity by the hydroperoxide [14].

MATERIALS AND METHODS

Chemicals L-Buthionine-SR-sulfoximine (BSO), reduced form GSH and t-BuOOH were

purchased from Sigma Chemical Co. (St. Louis, MO). O-Phenanthroline and zinc acetate were obtained from Wako Pure Chemical Co. (Osaka, Japan); tetra-n-propylammoniumhydroxide from Tokyo Kasei Kogyo Co. (Tokyo, Japan); desferrioxamine (desferal mesylate) from Ciba Geigy (Basel, Switzer- land). [2-14C]Thymidine (58 mCi/mmol) was obtained from ICN Radiochemicals, (California, U.S.A.).

Cell culture V79 cells from lung fibroblasts of male Chinese hamster were grown in a

monolayer in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 pg/ ml). The cells were cultured in a CO 2 incubator with 50/0 CO s in humidified air. HEPES (10 raM, pH 7.4}-buffered MEM medium without FBS was used for the t reatment with hydroperoxide.

Cell survival Cells in triplicate (3 wells) were plated into Linbro 12-well plates at a cell

density of 1.2 × 105 cells/ml/well. After a 20-h incubation, cells were incu- bated in the medium with or without 0.2 mM BSO for 6 h prior to challenge with t-BuOOH. The challenge was performed for 1 h at 37°C in HEPES-buff- ered MEM without FBS in the presence or absence of BSO. Following the treatment, 200 or 2000 cells were plated on 6-cm dishes containing control medium. Colonies formed after one week of culture were fixed and stained with crystal violet in methanol and counted the number.

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DNA strand breaks DNA single-strand breaks were measured by alkaline elution method of

Kobn et al. [16]. V79 cells were seeded at a cell density of 1 × 106 cells in 3.5-cm petri dishes. After a 20-h incubation, [14C]thymidine at 0.05/~Ci/ml was added to the cultures and DNA was labelled by a 24-h incubation. After removal of the radioactive medium the cells were grown in fresh medium with or without 0.2 mM BSO for 6 h or 10 -4 M zinc acetate for 10 h. Before t reatment with t-BuOOH, medium was placed to HEPES-buffered MEM ( -FBS) . Following t reatment with t-BuOOH for 1 h in the presence or absence of iron chelators, the medium was removed, cells were scraped into 2 ml PBS without Ca 2÷, Mg 2÷ and collected on polycarbonate filters for lysis with 5 ml 2% SDS/50 mM Tris (pH 9.6) for 1 h at room temperature. Filters were washed thrice with 5 ml 20 mM EDTA pH 10.0 and DNA was eluted with 3.2% tetram-propylammoniumhydroxide/0.1% SDS/40 mM EDTA (pH 12.0) at a flow rate of 0.05 ml/min. Ten fractions of 4.5 ml were collected directly in scintillation vials and counted after the addition of 9 ml acidified ACS II (Amersham, U.S.A.). The radioactivity remaining on the filter was eluted by treatment with 0.4 ml 1 N HC1 for 1 h at 70 °C.

RESULTS

Induction of DNA single-strand breaks by t-BuOOH and effects of iron chela- tors on the induction

The induction of DNA single-strand breaks (ssb) and/or alkali labile sites (pH 12.0) by t-BuOOH in V79 cells was investigated at toxic and non-toxic concentrations, respectively. Figure 1 shows DNA elution curves of a typical experiment. A dose dependent increase of DNA elution was observed after 1-h incubation of ceils with t-BuOOH, t-BuOOH induced DNA ssb even at concentrations below 0.05 mM which were not toxic to the cells. In an earlier study [14,15] we had shown that intracellular free iron was required for t- BuOOH cytotoxicity. Figure 2 shows that the DNA ssb by 0.5 mM t-BuOOH were markedly suppressed by the presence of 100 pM o-phenanthroline. In contrast, the trivalent iron chelator desferrioxamine which does not readily diffuse into cells did not suppress the formation of DNA ssb by the hydrope- roxide although it has been shown earlier to diminish cytotoxicity.

Effects of GSH depletion on the cytotoxicity of t-BuOOH and on the induction of DNA ssb

As demonstrated in an earlier s tudy [17] cellular GSH was depleted to approximately 5% of controls upon treatment of V79 cells with 0.2 mM BS0 for 6--7 h. BSO is a selective inhibitor of r-glutamylcysteine synthetase [18] which did not affect cell growth [17].

Susceptibility to t-BuOOH-induced cytotoxicity and DNA ssb was compared between controls and GSH depleted cells. As shown in Fig. 3, GSH depletion (GSH-) strongly increased the sensitivity to toxicity by t-BuOOH. In contrast, DNA ssb by 0.1 mM t-BuOOH increased with duration of treat- ment up to 1 h with similar kinetics in GSH- and GSH* cells. Indeed, the

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extent of DNA ssb was somewhat lower in GSH- cells (Fig. 4). Figure 5 shows dose response curves for DNA ssb in both GSH ÷ and GSH- cells upon treatment with t-BuOOH for I h. At all concentrations of t-BuOOH, strand breaks were somewhat lower in GSH- cells than in GSH ÷ cells.

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repair of DNA ssb induced by 0.5 mM t-BuOOH was very h s t during the first 10 rain and then diminished. Most of the DNA ssb were repaired within 60 min. DNA ssb induced by 0.1 mM t-BuOOH were repaired to control lev- els within 10 rain. Little differences in the rates of DNA repair were observed for controls and GSH-depleted cells.

Effects of induction of metaUothionein fMT) synthesis on the induction o/ DNA ssb by t-BuOOH

As demonstrated previously [14,17], metallothionein (MT) is induced upon t reatment of V79 cells with 10 -4 M zinc acetate• The amount of MT attained a maximum within 1 0 - 1 2 h following addition of the metal ion and in part protected cells from the toxicity of t-BuOOH. Figure 7 shows the effect of a maximal induction of MT synthesis on the induction of DNA ssb by t- BuOOH. It is evident that preinduction of MT did not significantly suppress the formation of DNA ssb by 0•02--0•1 mM t-BuOOH.

DISCUSSIONS

We studied the effect of iron chelators, GSH-depletion and MT induction on the formation of DNA ssb and cytotoxicity of t-BuOOH in V79 cells. Only

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the divalent iron chelator o-phenanthroline but not desferrioxamine sup- pressed DNA breakage and cytotoxicity. This may be due to the fact that only o-phenanthroline readily penetrates the cellular and nuclear mem- branes. Efficient suppression of t-BuOOH-indeuced DNA strand breakage by o-phenanthroline was also observed for mouse epidermal cells JB6 [19]. Again desferrioxamine was much less effective in this regard.

GSH occupies a central role in many fundamental cellular processes and, in particular, it contributes in a major way to the defence against extra- and intracellular stress by oxidants and other toxic agents [20]. Indeed, GSH- depletion has been demonstrated to increase the sensitivity of cells to heavy metal ions [17], alkylating agents [21], X-rays [22], heat [23], UV-light [24] and AO generated by inflammatory cells [25]. We demonstrated in the present

343

work that GSH-depletion also sensitized V79 cells towards the cytotoxicity of t-BuOOH. However, unexpectedly it had no major effect on the efficiency of the induction of DNA ssb. Similarly, GSH-depletion did not affect the rate of resealing of t-BuOOH induced ssb. These negative results may be due to the failure of BSO to efficiently deplete nuclear relative to cytoplasmic levels of GSH which has been reported in the literature [26]. Our results suggest that t-BuOOH induced cytotoxicity to V79 cells and the formation of DNA ssb are caused by different mechanisms. In support of this interpretation Ward et al. [27] had observed a large difference in the efficiencies for the formation of DNA ssb and killing of V79-171 cells by H202 and concluded that DNA ssb were ineffectual in causing cell death.

Meanwhile, oxidants are known to cause DNA base damages such as thy- mine glycols [7,8] or 8-OH-deoxyguanosine [9]. It is also known that plasma membrane blebbing and cytoskeletal alterations are caused by oxidative stress generated during the metabolism of menadione [28]. It is therefore conceivable that the cytotoxic effect of hydroperoxides is the consequence of damage to membranes rather than DNA or of DNA damages other than ssb.

MT is a low molecular weight, cysteine rich, metal binding protein [29]. Its induction is correlated with a decrease in the sensitivity of cells to the toxic effects of heavy metal ions [30--32]. MT is also known to protect cells from the radiation damage [33], which is in part caused by radical species such as hydroxyl radicals generated by the radiolysis of water.

In an earlier s tudy [14] we demonstrated that maximal induction of MT protected cells from the cytotoxicity of t-BuOOH. Our data suggested that MT plays a protective role as a scavenger of radical species which are gener- ated in reactions of divalent iron with hydroperoxides. Therefore, we investi- gated the effect of MT induction on the formation of DNA ssb by t-Bu0OH. As shown in Fig. 7, maximal induction of MT did not suppress DNA ssb. In analogy to the modulation of GSH levels by BS0 induction of MT may mostly affect cytoplasmic rather than nuclear concentration of the protein. Nevertheless, it would be expected that cytoplasmic GSH and MT concentra- tions affect the amounts of t-BuOOH which reach the nucleus.

Hydroperoxides may mostly play a role in late steps in carcinogenesis. Evidence has been presented that they act as promoters and progressors [34,35]. In particular the clastogenic action of oxidants could cause the inactivation of suppressor genes or the rearrangement and amplification of protooncogenes.

ACKNOWLEDGEMENTS

This work was supported in part by an International Cancer Research Technology Transfer (ICRETT) award.

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