Effects of glutathione depletion and induction of metallothioneins on the cytotoxicity of an organic hydroperoxide in cultured mammalian cells

Toxicology, 50 (1988) 257--268 Elsevier Scientific Publishers Ireland Ltd.

EFFECTS OF GLUTATHIONE DEPLETION AND INDUCTION OF METALLOTHIONEINS ON THE CYTOTOXICITY OF AN ORGANIC HYDROPEROXIDE IN CULTURED MAMMALIAN CELLS

TAKAFUMI 0CHI

Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 {Japan) (Received October 20th, 1987) (Accepted November 24th, 1987)

SUMMARY

The effects of glutathione depletion and induction of metallothioneins (MTs) on the cytotoxicity of t-butyl hydroperoxide (t-BHP) were investigated in cultured Chinese hamster V79 cells. The cytotoxicity of t-BHP was enhanced with increasing duration of the pretreatment with L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of y-glutamylcysteine synthetase, and was correlated with the decrease of cell glutathione, indicating that glutathione constitutes a cellular defense against toxicity by t-BHP. Desferrioxamine, a specific iron chelator, suppressed partly inhibition of cell growth induced by t-BHP and suppressed completely the increase of the cytotoxicity caused by glutathione depletion. Butylated hydroxytoluene, a diffusible radical scavenger, showed almost the same suppressive effect as desferrioxamine. These results suggest that the cytotoxicity of t-BHP enhanced by the depletion of glutathione is attributable to an action of ironmediated reactive radical species. Pretreatment with zinc (10-4 M) suppressed the cytotoxicity of t-BHP that was enhanced by depletion of glutathione and the extent of suppression was paralleled with increasing duration of zinc pretreatment that correlated with increased synthesis of metallothioneins (MTs). Maximum induction of MTs also suppressed the t-BHP-induced inhibition of cell growth at 4°C in glutathione-depleted cells. These results suggest that MTs act as a scavenger for the reactive radical species which are formed in an ironmediated manner.

Key words: Glutathione; Metallothioneins; Hydroperoxide toxicity; Iron chelator; Radical scavenger 0300-483X/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

257

INTRODUCTION

Organic hydroperoxides are known to generate free radicals [ 1 - 3 ] and to cause a variety of cellular injuries either directly or indirectly [4-11]. Recent biochemical studies have shown that t-butyl hydroperoxide impaired the ability of liver mitochondria to retain Ca 2÷ [12-14], caused Ca 2÷ release from perfused liver [15], increased the formation of membane lipid hydroperoxides in red blood cells [16--18], and that site-specific DNA damages in vitro were induced by the hydroperoxides of linoleic acid [19] and by autoxidation products of methyl linoleate [20]. However, the relationship between hydroperoxide-induced molecular changes and resulting biological effects still remains unclear. Therefore, the specific target molecule which is responsible for the biological alterations caused by organic hydroperoxides needs to be determined. On the other hand, some hydroperoxides have been known to possess tumor promoting activity [21,22]. Arachidonic acid hydroperoxides which can be released from human monocytes stimulated by a skin tumor promotor, phorbol-12-myristate-13-acetate (PMA), stimulated DNA synthesis, induced ornithine decarboxylase (ODC) in rat colon [23] and were clastogenic to cultured mammalian cells [24]. Thus, recent study hypothesizes the involvement of hydroperoxides in chemical carcinogenesis, particularly in the process of tumor promotion. Meanwhile, living cells have an elaborate defense system against reactive oxygen species and hydroperoxides consisting of antioxidant enzymes such as superoxide dismutase {SOD), catalase and glutathione peroxidase or endogenous antioxidants such as the reduced form of glutathione [25]. They are as a whole playing a homeostatic or protecting role against reactive species. However, a high and rapid production of reactive oxygen species can overcome the cellular defense and lead to cellular injuries that might be related to tumor promotion. In this context, it is necessary to investigate in detail the cellular defense system against hydroperoxides either for evaluating action mechanism and extent of each factors or for a better understanding of the variations in sensitivity of cells or organs to the reactive species. In the present study, we focused on cell glutathione as an intrinsic protector against organic hydroperoxides and on metallothioneins as an induced cellular defense. In practice, as a first-step approach to the molecular analysis of the specific target of hydroperoxides, we evaluated at the cellular level the cytotoxicity and the mechanism of a model organic hydroperoxide, t-butyl hydroperoxide (t-BHP), under the depletion of cell glutathione and the induction of metallothioneins. MATERIALS AND METHODS

Chemicals Acrylamide,

258

N,N'-methylenebisacrylamide,

N,N,N',N'-tetramethylethyl-

enediamine and ammonium persulfate were purchased from Bio-Rad Laboratories, Richmond (U.S.A.). L-Buthionine-SR-sulfoximine (BSO), t-butyl hydroperoxide (t-BHP), glutathione (GSH) and glutathione reductase (GSSGR, 220 U/mg protein) were obtained from Sigma Chemical Co., St. Louis (U.S.A.), butylated hydroxytoluene (BHT) from Tokyo Kasei Kogyo Co., Tokyo (Japan); Coomassie brilliant blue G-250, sodium thiosulfate, silver nitrate, iodoacetic acid, and zinc acetate from Wako Pure Chemical Co., Osaka (Japan); desferrioxamine (desferal mesylate) from Ciba Geigy, Basel (Switzerland); 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) from Nakarai Chemical Co., Kyoto (Japan); NADPH from Oriental Yeast Co., Tokyo (Japan).

Cell line and medium 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 U/ml) and streptomycin (100 ~g/ml). The cells were cultured in a CO2 incubator with 5o/o CO2 in humidified air. In experiments where the challenge treatment with t-BHP was carried out at 4°C, Hepes-buffered (pH 7.4) medium was used.

Cytotoxicity of hydroperoxide to sulfoximine (BSO) and~or zinc acetate

cells

pretreated

with

buthionine

Cells in triplicate were plated in Linbro 12-well plates at a cell density of 1.2 × 105 cells/well. After a 20-h incubation, cultures were pretreated with 0.2 mM BSO for up to 6 h and then were challenged with t-BHP for 1 h at 37 o or 4 °C in MEM medium including 10% FBS. For the combined treatment with BSO and zinc, cells cultured in control medium for 20 h were incubated with 1 × 10 -4 M zinc acetate for 6 or 12 h and with 0.2 mM BSO for 6 h prior to challenge with t-BHP. When used, desferrioxamine or BHT were added 30 min before the t r e a t m e n t with t-BHP in MEM medium without FBS. Stock solution of BHT was in dimethyl sulfoxide (DMSO) and the final concentration of the DMSO in cultures presence or absence of BHT was 0.1%. After challenge with the hydroperoxide for 1 h at 37 ° or 4°C, the cells were washed twice with Hanks' balanced salt solution (BSS) and plated in control medium for further incubation. After a 20-h incubation, the cells were washed, trypsinized and counted with hemocytometer.

Assay of glutathione and metallothioneins (MTs) For the assay of glutathione, 5 × 106 cells were harvested, washed 4 times with Ca 2÷, Mg2÷-free PBS and lysed with 0.25 ml of 0.2% Triton X-100. Then 50% sulfosalicylic acid was added to the lysates to give a final concentration of 2.5%. The proteins precipitated were removed by centrifugation at 5000 g for 10 min and the supernatants were used for the total glutathione assay. Glutathione was measured according to the same DTNB-GSSG reductase recycling assay as used in the previous study [26]. For the assay Of MTs, cells treated with 1 × 10 -4 M zinc acetate for up to 12 h in the presence or absence of 0.2 mM BSO were washed 4 times with

259

Ca 2÷, Mg2÷-free PBS and 5 x 106 cells were lysed with 100 /zl of the lysis buffer (10 mM T r i s - H C 1 (pH 7.5), 0.15 M NaC1, 3 mM MgCl2, 0.50/o Nonidet P-40 and 5 mM 2-mercaptoethanol). After being kept on ice for 5 min, the lysates were agitated and centrifuged at 12 000 g for 5 rain. The supernatants were used for the assay of MTs as described before [27,28]. RESULTS

Effect of glutathione depletion on the cytotoxicity of t-BHP Cellular glutathione was depleted by t r e a t m e n t of cells with buthionine sulfoximine (BSO), a selective inhibitor of y-glutamylcysteine synthetase. The details for the depletion of glutathione by BSO are shown in our previous study [28]. The level of cell glutathione was decreased to 78% of the initial level 1 h after addition of 0.2 mM BSO, 32% after 2 h, 18% after 3 h, 15% after 4 h, 10% after 5 h and 6% after 6 h. The susceptibility of cells to t-BHP toxicity was evaluated in relation to the extent of glutathione depletion. Cells pretreated with 0.2 mM BSO for a certain time up to 6 h were challenged with 0.5 mM t-BHP for 1 h at 37 °C. As shown in Fig. 1, the depletion of cell glutathione by BSO caused increase Time a f t e r a d d i t i o n of BSO (h) 0 I00

50-

1

2

3

4

5

6

,

,

i

,

i

i

O

o

~3

o

c~

10m

r-" {D

5-

Fig. 1. Increased sensitivity of cultured V79 cells to t-butyl hydroperoxide (t-BHP) with the depletion of cellular glutathione. Cells pretreated with 0.2 mM butbionine sulfoximine(BSO) for a certain time by 6 h were challenged with 0.5 mM t-BHP for 1 h at 37°C and subsequent cell growth was evaluated after 20 h.

260

Concn. of t-BHP (mM) 0 lO0 !.

O.l 0.2 0.3 0.4 0.5

50-

8 u

0

Fig. 2. Effect of glutathione depletion on the inhibition of cell growth by t-BHP. Cells were incubated with (e) or without (O} 0.2 mM BSO for 6 h prior to challenge with t-BHP for 1 h.

of t-BHP cytotoxicity. The susceptibility to t-BHP of the cells which were depleted of glutathione to 6°/o of the control value by 6-h t r e a t m e n t with BSO was also investigated as functions of concentrations of the hydroperoxide. As shown in Fig. 2, t r e a t m e n t of control cells with t-BHP for 1 h at 37 °C inhibited cell growth to 84O/o of the control growth at 0.1 mM of the hydroperoxide, 75% at 0.2 mM and 56% at 0.5 mM. In contrast, growth of the cells depleted of glutathione was decreased to 52% at 0.1 mM, 2 7 e at 0.2 mM and 7.2% at 0.5 mM t-BHP.

Effects of desferrioxamine or butylated hydroxytoluene inhibition of cell growth by t-BHP

(BHT) on the

As an approach at the cellular level for a b e t t e r understanding of the mechanism by which t-BHP causes inhibition of cell growth, the effects of a specific iron chelator, desferrioxamine, and of a diffusible radical scavenger, butylated hydroxytoluene (BHT), were investigated in cells depleted or not of glutathione. As shown in Fig. 3a, a 30-rain preincubation with desferrioxamine suppressed partly the inhibition of cell growth induced by tBHP on V79 cells in MEM medium without FBS and suppressed completely

261

Concn. of t-BHP (mM)

,oo!. "#

O.l

0.2 0.3

0.4

0.5

0

O.l

0.2

0.3

0.4 0.5

50-

"s

I0-

(a)

(b)

Fig. 3. Effects of desferrioxamine (a) and butylated hydroxytoluene (b) on the cytotoxicity of tBHP in the cells depleted (GSH-) or not (GSH*) of glutathione. Cells preincubated with or without 0.2 mM BSO for 6 h were challenged with t-BHP for 1 h in the presence or absence of 1 mM desferrioxamine or 50 ~M butylated hydroxytoluene (BHT) added 30 min prior to the challenge treatment, o, GSH÷ cells treated with t-BHP; O, GSH÷cells treated with t-BHP in the presence of desferrioxamine or B H T ; . , GSH- cells treated with t-BHP; [~, GSH- cells treated with t-BHP in the presence of desferrioxamine or BHT.

the increase of the c y t o t o x i c i t y caused by glutathione depletion. showed almost the same effects as d e s f e r r i o x a m i n e (Fig. 3b).

BHT

Protection of t-BHP-induced cytotoxicity by metaUothioneins (MTs) V79 cells w e r e e x p r e s s i n g t r a c e a m o u n t s of MTs in absence of inducer. H o w e v e r , as s h o w n in Fig. 4, e x p o s u r e of cells to 1 x 10 -4 M zinc a c e t a t e induced MT s y n t h e s i s a f t e r a lag time of 2 h. The induction was c o r r e l a t e d with the time of e x p o s u r e and r e a c h e d a m a x i m u m 1 0 - - 1 2 h after addition of zinc. P r e s e n c e of 0.2 mM BSO did not influence the stimulation of MT s y n t h e s i s by zinc (data not shown). The effect of MT induction on t-BHP-induced c y t o t o x i c i t y was e v a l u a t e d with r e s p e c t to the depletion of cellular glutathione. Cells w e r e p r e t r e a t e d simultaneously with 1 x 10 -4 M zinc for 6 or 12 h and with 0.2 mM BSO for 6 h and t h e n challenged with t-BHP for 1 h at 37 °C or 4°C. As s h o w n in Fig. 5, glutathione-depleted cells w e r e highly susceptible to t-BHP t r e a t m e n t at 37 °C. In contrast, the c y t o t o x i c i t y of t-BHP on glutathione-depleted cells was r e d u c e d by the p r e t r e a t m e n t with zinc. This s u p p r e s s i v e effect was c o r r e l a t e d with the d u r a t i o n of zinc p r e t r e a t m e n t , and hence with the stimulation of MT synthesis. On glutathione-depleted cells, a 12-h

262

I0

8

6

==

>~

4

2

I

!

2

4

I

I

6 8 Hours a f t e r a d d i t i o n of zinc

I

I

I0

12

Fig. 4. Time course of the induction of metallothioneins (MTs) after application of zinc acetate in cultured V79 cells. After incubation of cells with 1 x 10-4 M zinc acetate for various time by 12 h, the amount of induced MTs was determined as described in Materials and Methods.

0

0. I

Concn. of t-BHP 0.2 0.3 0.4

0.5 (mM)

.tJ 0

10-

Fig. 5. Effect of pretreatment with zinc on the cytotoxicity of t-BHP in glutathione-depleted cells. Cells were pretreated with 0.2 mM BSO for 6 h and with 1 x 10-4 M zinc acetate for 6 and 12 h prior to challenge treatment with t-BHP for 1 h at 37°C. O, cells with normal level of glutathione; o , cells depleted of glutathione; A, cells depleted of glutathione and pretreated with zinc for 6 h; V, cells depleted of glutathione and pretreated with zinc for 12 h.

263

Concn. of t-BHP 0

I0

20

30 (mM)

I00

5O

0

I0

Fig. 6. Effect of p r e t r e a t m e n t with zinc on the cytotoxicity of t-BHP at 4°C in glutathionedepleted cells. Cells were preincubated with 0.2 mM BSO for 6 h and 1 × 10-~ M zinc acetate for 12 h prior to challenge with t-BHP for 1 h at 4°C. O, cells with normal level of glutathione; o , glutathione-depleted cells; A, cells depleted of glutathione and pretreated with zinc for 12 h.

pretreatment with zinc that allows a 8-fold stimulation of MT synthesis restored a level of t-BHP-induced inhibition of cell growth comparable to which was observed in cells with normal level of glutathione. A suppressive effect of MTs on t-BHP-induced inhibition of cell growth was also observed when the cells were challenged with the hydroperoxide at 4°C. As shown in Fig. 6, a concentration of t-BHP greater than 30 mM was needed to induce cytotoxicity at 4°C on cells with normal level of glutathione. Glutathione-depleted cells were highly susceptible to t-BHP even at 4°C. A 12-h p r e t r e a t m e n t with zinc that induced a maximum stimulation of MT synthesis suppressed significantly the t-BHP-induced inhibition of cell growth at 4°C in glutathione-depleted cells. DISCUSSION

Glutathione peroxidase can catalyse the reduction of a wide variety of hydroperoxides. With the glutathione as electron donor the enzyme reduces the hydroperoxides to corresponding alcohols. Oxidized glutathione (GSSG) generated during this reaction is reduced by the GSSG reductase, NADPH acting as cofactor. Thus, homeostatic and protective role of glutathione peroxidase (GSH-Px) cycle have recently been documented. However, biological significance of the GSH-Px cycle against hydroperoxides added exogenously in the toxicological aspect and also the relative importance of the glutathione redox cycle compared to other protective substances such as

264

antioxidant vitamins which can directly interact with radical species are still debated. The present study was undertaken to evaluate the mechanism of the cytotoxicity of a model hydroperoxide, t-BHP, at the cellular level by comparing the sensitivity of cells depleted or not depleted of glutathione. The treatment of cells with 0.1--0.5 mM t-BHP caused an inhibition of cell growth up to 55% of untreated control (Fig. 2). This inhibition of cell growth by t-BHP may be due to an overloading of the capacity of the GSH-Px to detoxify the hydroperoxide or to an inhibition of GSH-Px by reactive radical species. On the other hand, as shown in Figs. 1 and 2, depletion of glutathione by BSO markedly enhanced the cytotoxicity of t-BHP and the extent of this enhancement was correlated with the depletion of cellular glutathione. These results apparently show the protective role of cellular glutathione against the cytotoxicity of t-BHP and also suggest the other role of glutathione than as electron donor in GSH-Px reaction. For a better understanding of the mechanism by which glutathione depletion sensitizes cells to t-BHP, the mechanism for the inhibition of cell growth by the hydroperoxide must be evaluated. Organic hydroperoxides, in vitro, are known to cause the formation of the reactive radical species such as alkoxy or peroxy radicals in the presence of trace elements such as iron [1-3]. Dysfunction by the reactive radical species of the molecules which are critical for the expression of normal cellular function may be a cause for the growth inhibition by t-BHP. In this sense, we tried to use a specific iron chelator, desferrioxamine, to suppress the formation of reactive radical species from t-BHP. Butylated hydroxytoluene (BHT), a diffusable radical scavenger, was also used to scavenge the reactive species formed intracellularly. As shown in Fig. 3a, pretreatment with desferrioxamine suppressed partly t-BHP-induced inhibition of cell growth in cells with normal level of glutathione. In contrast, the cytotoxicity of t-BHP that was enhanced by the depletion of glutathione was completely suppressed by desferrioxamine. Almost the same results were obtained with BHT (Fig. 3b). These results suggest strongly that the increased cytotoxicity of t-BHP that depends on the depletion of glutathione is attributable to an action of iron-mediated reactive radical species. Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins and their protection against heavy metal toxicity is well documented [ 2 9 31]. On the other hand, recent publications have shown that high amount of MTs could protect cells from radiation damage [32-34]. Accordingly, taking into consideration the radical mechanism in radiation effects, it is natural to consider that MTs play a protective role against cellular damage by hydroperoxides which can form reactive radical species in the presence of trace elements. Previous study [28] have shown that treatment of cultured V79 cells with 1 × 10 -4 M zinc induced MT synthesis after a lag time of 2 h and that depletion of glutathione by BSO did not have a great influence on MT synthesis. Therefore, protective role of MT induction on the t-BHPinduced cytotoxicity was evaluated in the present study with respect to the depletion of cell glutathione that sensitizes cells to hydroperoxide toxicity.

265

As shown in Fig. 5, pretreatment with zinc markedly suppressed the cytotoxicity of t-BHP that was enhanced by depletion of glutathione and the protective effect of zinc was dependent upon duration of pretreatment, being parallel with the increased accumulation of MTs. As shown in Fig. 3, the cytotoxicity of t-BHP that is enhanced by depletion of glutathione is attributed to the action of the reactive radical species. Also, MTs can be considered to be a major cellular factor induced by treatment with zinc in conjunction with the suppression of t-BHP toxicity, because the metal did not influence the level of cell glutathione [28]. Furthermore, the suppression of t-BHP-induced cytotoxicity by MT induction was observed even when challenged with the hydroperoxide at 4°C at which temperature radical reactions are mostly operative in the cells. Therefore, the suppression of t-BHP toxicity by MTs strongly suggest the possibility that MTs act as scavenger for the reactive radical species that are formed in an iron-mediated manner. Little is known about the specific target molecules responsible for the cytotoxicity of hydroperoxides. It is known that t-BHP causes membrane lipid hydroperoxidation in red blood cells and that this action can be markedly suppressed by a lipid soluble antioxidant, BHT. But, in V79 cells used in the present study, no formation of lipid hydroperoxides which could be evaluated by the formation of thiobarbituric acid (TBA)-reactive substances was observed by treatment with the concentration of t-BHP that was effective for the inhibition of cell growth (data not shown). Therefore, lipid peroxidation may not be implicated in the inhibition of cell growth by tBHP in the case of cultured V79 cells. On the other hand, unpublished data (Ochi and Cerutti) have shown that DNA strand breaks were induced by t-BHP in the concentration range effective for the growth inhibition of cells and that the DNA lesions were also induced even in the treatment at 4°C. Moreover, DNA breaks by t-BHP were observed on free DNA in the presence of trace elements. These data suggest that t-BHP induces DNA damage via formation of reactive radical species. Poly ADP-ribosylation that can be considered to be closely related to DNA strand breaks was also stimulated by t-BHP (unpublished). Accordingly, use of desferrioxamine or BHT on the induction of DNA breaks by t-BHP in cultured V79 cells may provide an insight to clarify a causeeffect relationship between t-BHP-induced inhibition of cell growth and DNA strand breaks. This kind of approach for the DNA strand breaks is under investigation.

ACKNOWLEDGEMENTS The author thanks Dr. Motohisa Kaneko and Mr. Domonique Mfihlmatter for helpful discussion. The author also wishes to thank Dr. Motoyasu 0hsawa for encouragement of this study.

266

REFERENCES 1

2 3 4

5 6 7 8 9 10 11 12

13

14 15

16

17

18

19 20 21

P.J. Thornalley, R.J. Trotta and A. Stern, Free radical production from the reaction of tbutylhydroperoxide with iron complex, in B. Wolf, S. Manfred and T. David (Eds.), Oxygen Radicals in Chemistry and Biology, Walter de Gruyter and Co., Berlin, New York, 1984, p. 215. B. Halliwell and J.M.C. Gutteridge, Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J., 219 (1984) 1. S.D. Aust, L.A. Morehouse and C.E. Thomas, Role of metals in oxygen radical reactions. J. Free Radicals Biol. Med., 1 (1985) 3. V.J. Horgan, J.S.L. Phflpot, B.W. Porter and D.B. Roodyn, Toxicity of autoxidized squalene and linoleic acid and of simpler peroxides in relation to toxicity of radiation. Biochem. J., 67 (1957} 551. S.E. Lewis and E.D. Wills, The destruction of - S H groups of proteins and amino acids by peroxides of unsaturated fatty acids. Biochem. Pharmacol., 11 (1962) 901. R.S. Chio and A.L. Tappel, Inactivation of ribonuclease and other enzymes by peroxidizing lipids and by malonaldehydes. Biochemistry, 8 (1969) 2827. R.C. Green, C. Little and P.J. O'Brien, The inactivation of isocitrate dehydrogenase by a lipid peroxide. Arch. Biochem. Biophys., 142 (1971) 598. A. Benedetti, A.F. Cassini, M. Ferrali and M. Comporte, Effects of diffusible products of peroxidation of rat liver microsomal lipids. Biochem. J., 180 {1979} 303. Y.A. Vladimirov, V.I. Olenev, T.B. Suslova and Z.P. Cheremisina, Lipid peroxidation in mitochondrial membrane. Adv. Lipid Res., 17 (1980) 173. M.K. Logani and R.E. Davies, Lipid oxidation: Biological effects and antioxidants, A review. Lipids, 15 (1980) 485. H. Nielson, Covalent binding of peroxidized phospholipid to protein: III. Reaction of individual phospholipids with different proteins. Lipids, 16 (1981) 215. H.R. Lotscher, K.W. Winterhalter, E. Carafoli and C. Richter, Hydroperoxides can modulate the redox state of pyridine nucleotides and the calcium balance in rat liver mitochondria. Proc. Natl. Acad. Sci., U.S.A., 76 (1979) 4340. V. Prpic and F.L. Bygrave, On the inter-relationship between glucagon action, the oxidation-reduction state of pyridine nucleotides and calcium retention by rat liver mitochondria. J. Biol. Chem., 255 (1980) 6193. G. Bellomo, S.A. Jewell and S. Orrenius, The metabolism of menadione impairs the ability of rat liver mitochondria to take up and retain calcium. J. Biol. Chem., 257 {1982) 11558. H. Sies, P. Graf and J.M. Estrela, Hepatic calcium efflux during cytochrome P-450dependent drug oxidations at the endoplasmic reticulum in intact liver. Proc. Natl. Acad. Sci. U.S.A., 78 {1981} 3358. R.J. Trotta, S.G. Sullivan and A. Stern, Lipid peroxidation and hemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide: Dependence on glucose metabolism and hemoglobin status. Biochim. Biophys. Acta, 679 (1981) 230. R.J. Trotta, S.G. Sullivan and A. Stern, Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide: Effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate. Biochem. J., 204 {1982} 405. R.J. Trotta, S.G. Sullivan and A. Stern, Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide: The relative role of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis. Biochem. J., 212 (1983) 759. S. Iooue, Site-specific cleavage of double-strand DNA by hydroperoxide of linoleic acid. FEBS Lett., 172 (1984) 231. K. Ueda, S. Kobayashi, J. Morita and T. Komano, Site-specific DNA damage caused by lipid peroxidation products. Biochem. Biophys. Acta, 824 (1985) 341. T.J. Slaga, A.J.P. Klein-Szanto, L.L. Triplett, L.P. Yotti and J.E. Trosko, Skin tumorpromoting activity of benzoyl peroxide, a widely used free radical generating compound. Science, 213 (1981} 1023.

267

22 23

24 25 26

27 28

29 30 31 32 33

34

268

T.J. Slaga, Overview of tumor promotion in animals. Environ. Health Perspect., 50 (1983) 3. A.W. Bull, N.D. Nigro, W.A. Golembieski, J.D. Crissman and L.J. Marnett, In vivo stimulation of DNA synthesis and induction of ornithine decarboxylase in rat colon by fatty acid hydroperoxides, autooxidation products of unsaturated fatty acids. Cancer Res., 44 (1984) 4924. T. Ochi and P.A. Cerutti, Clastogenic action of hydroperoxy-5,8,11,13-icosatetraenoic acids on the mouse embryo fibroblasts C3H10T1/2. Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 990. P.A. Cerutti, Prooxidant states and tumor promotion. Science, 227 (1985) 375. T. Ochi, K. Takahashi and M. Ohsawa, Indirect evidence for the induction of a prooxidant state by cadmium chloride in cultured mammalian cells and a possible mechanism for the induction. Mutat. Res., 180 (1987) 257. F. Otsuka, S. Koizumi, M. Kimura and M. Ohsawa, Silver staining for carboxymethylated metallothioneins in polyacrylamide gels. Anal. Biochem., 168 (1988) 184. T. 0chi, F. 0tsuka, K. Takahashi and M. Ohsawa, Glutathione and metallothioneins as cellular defense against cadmium toxicity in cultured Chinese hamster cells. Chem.-Biol. Interact., 65 (1988) 1. J.H.R. Kaji and M. Nordberg (Eds.), Metallothionein, Birkhauser-Verlag, Basel, 1979. M. Webb, The metallothioneins, in M. Webb (Ed.), The chemistry, biochemistry and biology of cadmium. Elsevier/North-Holland Biomedical Press, Amsterdam, 1979, p. 195. E.C. Foulkes (Ed.), Biological roles of metaUothioneins, Elsevier, New York, 1982. A. Bakka, A.S. Johnsen, L. Endresen and H.E. Rugstad, Radioresistance in cells with high content of metallothionein. Experientia, 38 (1982) 381. P.L. Thornalley and M. Vasak, Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochem. Biophys. Acta, 827 (1985) 36. J. Matsubara, Y. Tajima and M. Karasawa, Promotion of radioresistance by metallothionein induction prior to irradiation. Environ. Res., 43 (1987) 66.