Characterization of 2,3-bis(chloromethyl)-1,4-naphthoquinone induced mitochondrial swelling

Chem.-Biol. Interactions, 35 (1981) 241--253 © Elsevier/North-Holland Scientific Publishers Ltd.

241

CHARACTERIZATION OF 2,3-BIS(CHLOROMETHYL)-I,4-NAPHTHOQUINONE INDUCED MITOCHONDRIAL SWELLING

RONALD S. PARDINI, MARGARET A. TILKA, CHRIS A. PRITSOS, A.J. LIN and ALAN C. SARTORELLI

Allie M. Lee Laboratory for Cancer Research, Division o f Biochemistry, University of Nevada, Reno, N V 89557 and Department o f Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT 06510 (U.S.A.) (Received June 14th, 1980) (Revision received November 8th, 1980) (Accepted December 23rd, 1980)

SUMMARY

Mitochondrial swelling induced by 2,3-bis(chloromethyl)-l,4-naphthoquinone (CMNQ) was found to be a non-energy linked, oxygen and sulfhydryl-dependent, substrate-independent, osmotic process, that lacks cation specificity. Swelling was inhibited by cysteine and DTNB, and the CMNQ induced swelling resulted in a decrease in mitochondrial reactive sulfhydryl groups~ thus, mitochondrial sulfhydryl interaction was mandatory in the CMNQ swelling process. The non-enzymatic reaction of CMNQ with cysteine but not cystine resulted in the consumption of oxygen, implicating sulfhydryl redox activity in the swelling process. High levels of tocopherol and histidine depressed the CMNQ induced swelling, suggesting that free radicals and singlet oxygen are important in the CMNQ induced swelling process. These findings support the proposition that CMNQ interacts with mitochondrial reductase systems and sulfhydryl groups in such a way as to generate superoxide radical which subsequently may dismute to H202 and produce -OH and possibly singlet oxygen. These toxic oxygen species may be responsible for the CMNQ-promoted sulfhydryl depletion and mitochondrial swelling.

INTRODUCTION

A series of benzo- and naphthoquinone's capable of functioning as alkyAbbreviations: CMNQ, 2,3-bis(chloromethyl)-l,4-naphthoquinone; DTNB, 5,5'-dithiobis( 2-nitrobenzoic acid)

242 lating agents after bioreductive activation were found to possess antitumor activity against Sarcoma 180 and Adenocarcinoma 755 in vivo [1--5], to be inhibitors of the synthesis of DNA and R N A [1,2,6] and to inhibit mitochondrial respiratory enzyme systems, including NADH-oxidase and succinoxidase [2,4]. A p r o t o t y p e of this class of bioreductive alkylating agents, CMNQ was shown to be capable of undergoing reductive activation via a mitochondrial respiratory chain induced oxidation of NADH, occurring on the substrate side of the rotenone site of inhibition [6]. The primary mitochondrial action of CMNQ appeared to be the release of respiratory control, and higher doses of CMNQ induced a measurable burst of oxygen consumption followed b y inhibition of respiration [6,7]. CMNQ was also found to bind to mitochondrial proteins in vitro [7], an observation consistent with the bioreductive alkylation mechanism proposed for its antineoplastic activity [8]. Sarcoma 180 mitochondrial ATPase was found to be stimulated b y the addition of CMNQ to mitochondria in vitro [7], and CMNQ-induced large amplitude swelling of rat liver mitochondria in vitro [6]; thus, CMNQ has the capability of affecting a wide variety of mitochondrial functions in vitro. In order to further characterize the interaction between CMNQ and mitochondrial membrane systems, studies were conducted on the cation specificity, energy requirement, sulfhydryl involvem e n t and oxygen dependence of CMNQ-induced large amplitude mitochondrial swelling and these findings are described in this report. MATERIALS A N D METHODS

Tightly coupled liver mitochondria (av. RCR = 4.8) were isolated from rats fasted for 48 h b y differential centrifugation according to the procedures of Lardy and Johnson [9]. Swelling was monitored as the decrease in turbidity of dilute suspensions of mitochondria at 520 nm in 1-cm cuvettes on a Cary 14 or Aminco DW-2 UV-visible spectrophotometer in the split beam mode. Mitochondrial suspensions (20--70 ~l) containing approx. 0.5 mg o f protein were diluted to 3 ml with suspending buffer (150 mM KC1, 20 mM Tris--HC1, pH 7.4) to give an initial absorbance of 0.7--1.0 at 520 nm. Mitochondrial thiol groups were estimated in the incubation mixture by the procedure of Ellman [10], employing 5,5'
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Fig. 1. The e f f e c t o f various a m o u n t s o f C M N Q o n rat liver m i t o c h o n d r i a l swelling. M i t o c h o n d r i a ( 0 . 5 mg protein) were s u s p e n d e d in 3 ~ 1 o f 1 5 0 ~ KCI c o n t a i n i n g 20 l ~ l Tris--HCI ( p H 7 . 4 ) at r o o m temperature. C M N Q was added at the c o n c e n t r a t i o n s s h o w n in the s p e c t r o p h o t o m e t r i c tracings f o l l o w i n g 1 min o f incubation. Swelling induced b y p h o s p h a t e ( 2 . 0 m M ) and t h y r o x i n e ( 0 . 0 1 2 raM) initiated f o l l o w i n g 1 m i n i n c u b a t i o n with 5.0 mM succinate is s h o w n for comparison. The c o n t r o l tracing d o e s n o t have any additions. The arrow indicates the p o i n t o f a d d i t i o n o f CMNQ. 0.4 NH4CI

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Fig. 2. T h e e f f e c t o f different m o n o v a l e n t c a t i o n s o n C M N Q - i n d u c e d swelling o f rat liver m i t o c h o n d r i a . T h e c o n d i t i o n s o f this e x p e r i m e n t were as described in the legend to Fig. 1, e x c e p t that 3 7 5 n m o l o f C M N Q were added to the reaction m e d i u m containing various chloride salts ( 1 5 0 mM). T h e arrow indicates the p o i n t o f a d d i t i o n o f CMNQ.

244 of swelling was independent of substrate, i t appears that CMNQ-induced swelling is a non-energy linked process. To assess the cation specificity for CMNQ-induced mitochondrial swelling, the process was measured in a buffer containing NH4 ÷, Li ÷ or Na ÷ in place of K ÷. The findings, shown in Fig. 2, indicate that neither the rate nor the extent o f the CMNQ-induced swelling were significantly affected b y varying the permeant cation; thus, CMNQ-induced mitochondrial swelling lacks monovalent cation specificity. In order to ascertain whether the mitochondrial swelling induced by CMNQ was an osmotic process, swelling was measured in the presence of sucrose, which does n o t impermeate the mitochondrial inner membrane and thereby establishes an opposing osmotic force to the swelling process [12]. The results, shown in Fig. 3, demonstrate that increasing concentrations of sucrose in the swelling medium caused a concomitant decrease in the extent of CMNQ-induced swelling. In addition, the observation that deoxycholate, a well known detergent that disrupts the membrane structure of mitochondria, caused a decrease in absorbance in the presence of 0.3 M sucrose (Fig. 3) suggests that CMNQ-induced swelling is an osmotic process and n o t simply a dbtergent.like disruption of the mitochondrial membrane which would result in decreased turbidity in the assay system

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MINUTES Fig. 3. T h e e f f e c t of sucrose o n C M N Q - i n d u c e d swelling o f r a t liver m i t o c h o n d r i a . T h e c o n d i t i o n s o f t h i s e x p e r i m e n t w e r e as d e s c r i b e d in t h e legend t o Fig. 1, e x c e p t t h a t 3 7 5 n m o l o f C M N Q were a d d e d to t h e r e a c t i o n m e d i u m c o n t a i n i n g v a r y i n g c o n c e n t r a t i o n s o f sucrose. Swelling i n d u c e d b y d e o x y c h o l a t e (0.8 rag) in 0.3 M sucrose r e a c t i o n m e d i u m is s h o w n for c o m p a r i s o n . T h e a r r o w i n d i c a t e s t h e p o i n t o f a d d i t i o n of t h e swelling a g e n t s CMNQ a n d d e o x y c h o l a t e .

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Fig. 4. The effects of 2,4-dinitrophenol (DNP), electron transport inhibitors and cysteine on CMNQ-induced swelling of rat liver mitochondria. The conditions of the experiment were as described in the legend to Fig. 1, except that 375 nmol of CMNQ were added to the reaction medium containing either 0.12 mM DNP, electron transport inhibitors (RAKO- 12.6 ~M rotenone, 0.15 /~M antimycin A, 0.66 mM KCN and 2 ~g oligomycin), or varying amounts of cysteine as indicated on the tracings. The arrow indicates the point of addition of CMNQ. Fig. 5. The effects of anaerobiosis and varying substrate on CMNQ-mediated mitochondrial swelling. The conditions were as described in the legend to Fig. 1 except that 375 nmol of CMNQ were added to the reaction medium containing varying concentrations of succinate. Anaerobic conditions were obtained by flushing the buffer for 15 rain with nitrogen. The Aminco DW-2 spectrophotometer was equipped with the anaerobic cell adapter-plunger for the anaerobic experiment and CMNQ was added with the plungeradapter at zero time.

employed. Furthermore, inhibitors of the mitochondrial electron transport system (RAKO-rotenone, antimycin, KCN and oligomycin) and dinitrophenol, an uncoupler of oxidative phosphorylation, do not prevent CMNQinduced mitochondrial swelling, whereas increasing concentrations of cy~ teine proportionally inhibits the CMNQ-induced swelling (Fig. 4). The data in Fig. 5 show that CMNQ-modulated swelling was oxygen dependent as anaerobic conditions prevented swelling. Furthermore, these data also show that the drug-induced swelling process was not effected by electron transport substrate as the swelling process was similar in the presence and absence of succinate. These findings establish that CMNQ-induced swelling is not an energy linked process, is oxygen-dependent, substrate-independent and they show that CMNQ interacts with the sulfhydryl groups of cysteine in such a way as to protect the mitochondria from this agent. To further assess the involvement of mitochondrial sulfhydryl groups in CMNQ-pro-

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Fig. 6. T h e e f f e c t s o f D T N B o n C M N Q - i n d u c e d smelling o f r a t liver m i t o c h o n d r i a . T h e c o n d i t i o n s o f t h e e x p e r i m e n t were as d e s c r i b e d in t h e legend t o Fig. 1, e x c e p t t h a t 3 7 5 n m o l o f C M N Q were a d d e d t o t h e r e a c t i o n m e d i u m c o n t a i n i n g varying c o n c e n t r a t i o n s o f D T N B . T h e a r r o w i n d i c a t e s t h e p o i n t o f a d d i t i o n o f CMNQ.

moted swelling, mitochondria were preincubated with DTNB, a disulfide reagent that reacts with sulfhydryl groups. The data in Fig. 6 demonstrate that preincubation of rnitochondria with 334 mM DTNB significantly depressed the CMNQ-induced mitochondrial swelling in a dose-dependent fashion; thus, CMNQ-promoted mitochondrial swelling was dependent on the availability of mitochondrial sulfhydryl groups. However, this exTABLE

I •

THE CONTENT OF SULFHYDRYL FOLLOWING EXPOSURE TO CMNQ

GROUPS

CMNQ nmol added

S u l f h y d r y l c o n t e n t aJ~ (%)

0 187 375

100 13.2 0

IN R A T

LIVER

MITOCHONDRIA

a R a t liver m i t o c h o n d r i a (5 m g p r o t e i n p e r 3 ml were i n c u b a t e d in 20 m M Tris--HCi ( p H 7.4) at r o o m t e m p e r a t u r e in t h e p r e s e n c e ~n~l a b s e n c e o f CMNQ. A f t e r 2 min, t h e s u l f h y d r y l c o n t e n t was e s t i m a t e d d i r e c t l y in t h e i n c u b a t i o n m i x t u r e b y t h e proc e d u r e o f E l l m a n [ 1 0 ] using 0 . 0 2 ml o f D T N B ( 7 9 . 2 ~g) a n d m o n i t o r i n g t h e r e a c t i o n s p e c t r o p h o t o m e t r i c a l l y at 4 1 2 n m for 5 min. T h e r e f e r e n c e c u v e t t e h a d n o D T N B a d d e d . T h e r e s u l t i n g a b s o r b a n c e was used t o c a l c u l a t e t h e c o n c e n t r a t i o n of s u l f h y d r y l groups. bAverage of 3 determinations.

247 periment did n o t eliminate the possibility that unreacted DTNB from the pre-incubation mixture reacted directly with CMNQ. To assess further the effects o f CMNQ on the sulfhydiT1 c o n t e n t of mitochondria, sulfl~ydryl groups were measured after CMNQ treatment. The addition of 187 nmol of CMNQ decreased the concentration o f free mitochondrial sulfhydryl groups b y a b o u t 80%, and 375 nmol of CMNQ virtually eliminated all of the reactive mitochondrial membrane snlfhydryl groups (Table I). To further probe the role of substrate and sulfhydryl groups in the action of CMNQ on mitochondrial systems, we measured the effect of substrate and cysteine on the CMNQ induced burst of oxygen consumption [6,7]. The findings in Fig. 7 show that the mitochondrial burst of oxygen consumption is dependent on the concentration of substrate. In Fig. 8, a dose dependent oxygen consumption was also observed when cysteine and CMNQ were mixed non-enzymatically, whereas cystine and CMNQ produced no oxygen burst. These findings demonstrate that CMNQ has the ability to undergo a redox reaction with either a mitochondria] reductase (Fig. 7) system or with sulfhydryl groups (Fig. 8). Since DTNB blocks the mitochondrial swetling caused b y CMNQ, it appears that the swelling process in d e p e n d e n t on reducing equivalents in the form of sulfhydryl groups. Since in the absence of succinate, the addition of CMNQ to mitochondrial suspensions produces a small burst of oxygen consumption (Fig. 7) it follows .49@ *,~'~'-.--'. .

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that the reducing equivalents for the oxygen consumption are the sulfhydryl groups, a reaction that apparently causes membrane alteration and subsequent mitochondrial swelling. The data in Fig. 7 show that only a small burst of oxygen consumption is required to induce mitochondrial swelling, which suggests that only a few key sulfhydryl groups are involved. What isn't clear from these investigations, is the role in the swelling process of the cyanide insensitive burst of oxygen consumption caused by CMNQ. It plays an essential role in the swelling process since swelling is oxygen-dependent. It is well known that membrane lipid peroxidation can induce mitochondrial swelling [13--15] and Lynch and Fridovich [16] have demonstrated that the enzymatic generation of superoxide anion results in the peroxidation of erythrocyte membrane lipids and subsequent membrane disruption. Since we have previously reported a cyanide-insensitive burst of oxygen consumption in mitochondrial preparations following the addition of CMNQ [6,7; Fig. 7], which suggests that O2- is produced, we have conducted preliminary studies to assess the role of free radicals in the CMNQinduced swelling of rat liver mitochondria. Adriamycin [17], streptonigrin [18], ~-lapachone [19], paraquat [20] and a variety of other compounds [21] have all been reported to participate in enzyme-catalyzed redox cycling in the presence of oxygen, with subsequent generation of superoxide and hydrogen peroxide and possibly singlet oxygen and hydroxyl radical via the Haber-Weiss or Fenton reactions. Since CMNQ has the potential to function through such a mechanism, we measured the effects of free radical and singlet oxygen scavengers on CMNQ-induced swelling; these data are shown in Fig. 9 and 10. Tocopherol, a general free radical scavenger (Fig. 9) and histidine, a scavenger of singlet oxygen (A~'O:), decreased the rate of

249 0.4 OpM

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MINUTES Fig. 9. The effect of e-tocopherol on CMNQ-induced swelling of rat liver mitochondria. The conditions of the experiment are as described in the legend to Fig. 1, except that 94 nmol of CMNQ were added to the reaction medium containing varying concentrations of e-toeopherol. The arrow indicates the point of addition of CMNQ.

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MINUTES Fig. 10. T h e e f f e c t of h i s t i d i n e o n C M N Q - i n d u c e d swelling o f rat liver m i t o c h o n d r i a . T h e c o n d i t i o n s o f t h e e x p e r i m e n t are as d e s c r i b e d in t h e legend t o Fig. 1, e x c e p t t h a t 94 n m o l o f C M N Q were a d d e d to t h e r e a c t i o n m e d i u m c o n t a i n i n g varying c o n c e n t r a t i o n s o f histidine. T h e a r r o w i n d i c a t e s t h e p o i n t of a d d i t i o n o f CMNQ.

250

CMNQ-induced mitochondrial swelling (Fig. 10). These findings suggested that free radicals and Ag'O2 may be in part responsible for CMNQ-induced mitochondrial swelling; however, they do not exclude the possibility that CMNQ reacts directly with either a-tocopherol or histidine. The observation that 50 mM histidine (Fig. 10) depresses swelling to the same extent as 150 mM sucrose (Fig. 3) indicates that the histidine effect is not due solely to an osmolarity effect. DISCUSSION

Olenev et al. [22] classified the types of mitochondrial swelling as deoxycholate-induced mitochondrial membrane alteration, valinomycin-induced active uptake of potassium, active calcium accumulation and non-energy linked lipid peroxidation. Our results demonstrate that CMNQ-.induced mitochondrial swelling is a cation non-specific, oxygen-dependent, nonenergy linked, osmotic process which excludes the ionophore induced and active cation uptake mechanisms of swelling as classified by Olenev et al. [22]. The CMNQ-induced mitochondrial swelling process has characteristics similar to those described for ascorbate-induced [13], glutathione-(GSHGSSG) [14] and ferrous ion--induced [15] mitochondrial swelling. These redox active compounds induce mitochondrial swelling in proportion to the amount of mitochondrial lipid peroxides produced; thus, CMNQ-induced swelling appears to possess all of the characteristics of the membrane lipid peroxidation mechanism. That the addition of a-tocopherol blocks CMNQinduced swelling is consistent with a peroxidative mechanism, although we did not attempt to measure directly lipid peroxide formation as a result of CMNQ treatment. The noted protection against CMNQ-induced swelling by sulfhydrylcontaining amino acids, the observed decrease in mitochondrial sulfhydryl groups produced by treatment of mitochondria with this agent and the dependence on sulfhydryl groups for swelling is consistent with the observations of Riley and Lehninger [23] that lipid peroxide formation is accompanied by a decrease in mitochondrial sulfhydryl groups during GSH-GSSG induced mitochondrial swelling. These authors reported that 12% of the sulfhydryl groups that rapidly react with Ag÷ were depleted during GSHGSSG induced mitochondrial swelling. The inhibition of CMNQ-induced mitochondrial swelling by the addition of DTNB supports the view that membrane sulfhydryl groups are essential for the CMNQ-induced swelling process. Consistent with this proposed mechanism are the findings of Bindoli et al. [ 24] who reported that ascorbate-induced generation of lipid peroxides in mitochondrial preparations in vitro was dependent upon a decrease of approx. 15% of the membrane sulfhydryl groups. Thus, a relationship between oxidation of mitochondrial membrane sulfhydryl groups, lipid peroxide formation and mitochondrial swelling exists. The findings herein reported are consistent with the mechanism proposed in Fig. 11 that CMNQ undergoes a redox reaction with NADH in the mito-

251 NADH

NAD +

mem-S

mem-SH

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CMNQH=

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H+ + O z-

~

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OH Fig. 11. Proposed mechanism for interaction of CMNQ with mitochondrial membranes.

chondrial membrane via a mitochondrial reductase forming NAD ÷ and CMNQH2. Alternatively or coincidentally, CMNQ may react with mitochondrial sulfhydryl groups forming disulfide and CMNQH2. In either case, the resulting CMNQH2 then can react with oxygen in a univalent fashion at the membrane surface, as described by Misra and Fridovich [25], to form 0 2 - and CMNQH-. The 0 2 - may then dismutate spontaneously or enzymatically to form H202 (reaction 1), and the 02- and H202 may react via the Fe 2+ catalyzed Haber-Weiss reaction (superoxide-mediated Fenton chemistry) (reaction 2) to form OH. [26] which can induce lipid peroxidation and mitochondrial swelling. Furthermore, our data support the interaction of CMNQ with key mitochondrial sulfhychT1 groups to render them unreactive towards DTNB. This reaction could also be involved in the induction of mitochondria] swelling since swelling was found to be substrate independent, and sulfhydryl and oxygen dependent.

252 Reaction 1 Reaction 2

0 2 - + 0 2 - + 2 H ÷ ~ O2 + H 2 0 2 O : - + F e 3÷ ~ F e 2÷ + O2 F e 2+ + H 2 0 2 -~ F e 3÷ + O H - + - O H

Although CMNQ was designed as a bioreductive alkylating agent which generated a quinone methide intermediate following bioreduction in the oxygen
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253 13 E.F. Hunter, A. Scott, P.E. Hoffstein, F. Guerra, J. Weinstein, A. Schneider, B. Schultz, J. Fink, L. Ford and E. Smith, J. Biol. Chem., 239 (1964) 604. 14 E.F. Hunter, A. Scott, J. Weinstein and A. Schneider, J. Biol. Chem., 239 (1964) 622. 15 E.F. Hunter, J.M. Gebicki, P.E. Hoffstein, J. Weinstein and A. Scott, J. Biol. Chem., 238 (1963) 828. 16 R.E. Lynch and I. Fridovich, J. Biol. Chem., 253 (1978) 1838. 17 J. G o o d m a n and P. Hochstein, Biochcm. Biophys. Res. Commun., 77 (1977) 797. 18 R. Docampo, F.S. Cruz, A. Boveris, R.P.A. Muniz and D.M.S. Esquivel, Biochem. Pharmacol., 28 (1979) 723. 19 F.S. Cruz, R. Docampo and A. Boveris, Antimicrob. Agents Chemother., 14 (1978) 630. 20 H.M. Hassan and I. Fridovich, J. Biol. Chem., 253 (1978) 8143. 21 H.M. Hassan and I. Fridovich, Arch. Biochem. Biophys., 196 (1979) 385. 22 V.I. Olenev, T.B. Suslova and Y.A. Vladimirov, Studia Biophysica, Berlin, 58 (1976) 147. 23 M.V. Riley and A.L. Lehninger, J. Biol. Chem., 239 (1964) 2083. 24 A. Bindoli, L. Cavallini and N. Siliprandi, Chem.-Biol. Interact., 19 (1977) 383. 25 H.P. Misra and I. Fridovich, J. Biol. Chem., 247 (1972) 188. 26 E.W. Kellog and I. Fridovich, J. Biol. Chem., 250 (1975) 8812. 27 L.W. Oberley and G.R. Bucttner, Cancer Res., 39 (1979) 1141.