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1,4-reductive addition of glutathione to quinone epoxides
Free Radical Biology & Medicine, Vol. 6. pp. 149-165, 1989 Printed in the USA. All rights reserved.
0891-5849/89 $3.00 + .00 © 1989 Pergamon Press plc
Original Contribution 1,4-REDUCTIVE ADDITION OF GLUTATHIONE TO QUINONE EPOXIDES Mechanistic studies with h.p.l.c, with electrochemical detection under anaerobic and aerobic conditions and Evaluation of chemical reactivity in terms of autoxidation reactions ANDERS BRUNMARK
and ENRIQUE C A D E N A S
Department of Pathology II, University of Linkfping, S-581 85 Linkfping, Sweden
(Received 4 February 1988; Accepted 6 April 1988)
Abstract--The nucleophilic addition of GSH to quinonoid compounds, characterized as a 1,4-reductive addition of the Michael type, was studied with p-benzoquinone- and 1,4-naphthoquinone epoxides with different degree of methyl substitution. Identification and evaluation of molecular products from the above reaction were assessed by h.p.l.c, with either reductive or oxidative electrochemical detection, based on the redox properties retained in the molecular products formed. It was found that the degree of methyl substitution of the quinone epoxide, from either the 1,4naphthoquinone- or p-benzoquinone epoxide series, determined their rate of reaction with GSH. The reductive addition implied the rearrangement of the quinone structure with opening of the epoxide ring yielding as the primary product a hydroxy-glutathionyl substituted adduct of either p-benzohydroquinone or 1,4naphthohydroquinone. The primary product undergoes elimination reactions and redox transitions which bring about a number of secondary molecular products. The distribution pattern of the latter depends on the degree of methyl substitution of the quinone epoxide studied and on the concentration of 02 in the solution. The occurrence of the hydroxy-substituent in position a, adjacent to the carbonyl group, enhances the autoxidation properties of the compound resulting in an augmented 02 consumption and H202 production. Therefore, it could be expected that the chemical reactivity of the products originating from the thiol-mediated nucleophilic addition to quinone epoxides would be of toxicological interest. Keywords--Quinone epoxides, Glutathione, Free radicals, H.p.l.c. with electrochemical detection, 1,4-Naphthoquinones, p-Benzoquinones, Quinone-glutathione conjugates, Autoxidation, H20~
of the redox chemistry of quinones are of particular interest, for they affect the likelihood and efficiency of [a] their reduction by different flavoproteins ~'6 as well as [b] their participation in autoxidation reactions. The redox properties of quinones were reported in some instances to sustain the toxicological effects observed in quinone-induced cell and tissue injury. 9-~ The latter feature, the addition chemistry of quinones, involves, among others, their reaction with 02 and sulphur nucleophiles. The nature of the new substituent in the quinone molecule will determine, in large measure, its subsequent chemical reactivity. 4 Addition reactions with 02 nucleophiles can be exemplified by the H202 (HOO-)-mediated oxidation of quinones to yield quinone epoxide products. This p r o cess is based on the known property that the - C = C -
INTRODUCTION
The cellular toxicity of quinones is partially based on two interrelated properties inherent to these compounds, their redox chemistry ~-3 and their addition chemistry.4 The former feature, in biological terms, implies, on the one hand, the cellular redox activation of quinones by either one- or two-electron transfer flavoproteins yielding semiquinone or hydroquinone derivatives, respectively t'3'~,6 and, on the other, subsequent autoxidation reactions with formation of O2 ~ , H202, and, in some cases, a triplet electronically-excited state of the quinone. 7'8 The substitution pattern of the quinone determines both the redox potential and the electron distribution in the quinonoid structure. These properties 149
150
A. BRUNMARKand E. CADENAS
double bonds of the quinonoid nuclei can be epoxidized. 4 At variance with the p-benzoquinone series, the adjacent benzene ring in the 1,4-naphthoquinone compounds precludes other possible sites for the H202mediated epoxidation. Moreover, the oxidation products of naphthoquinone series are less prone to fragmentation reactions than those of the p-benzoquinone series. 12 We have studied previously 8'~3 some aspects of the O2 addition reactions to quinonoid compounds, namely the oxidation by H202 ofp-benzoquinones and 1,4-naphthoquinones which yields the corresponding quinone epoxide products: 2,3-epoxy-p-benzoquinone and 2,3-epoxy-l,4-naphthoquinone derivatives, respectively. 13 The second order rate constants for the epoxidation reaction is similar for both unsubstituted quinone series, whereas it decreases with the increasing degree of methyl substitution of the quinones.~3 Addition reactions to quinones with sulphur nucleophiles could be characterized as 1,4-reductive additions of the Michael type 4 with formation of, for the case of GSH, the corresponding hydroquinone-glutathionyl conjugate. Recent mechanistic studies carried out with 1,4-naphthoquinone or menadione and GSH under anaerobic conditions suggested that the hydroquinone-thioether adduct underwent oxidation with formation of GSH-conjugated semiquinones intermediates. 14-16 Another recent finding indicates that the redox potential of the menadione-GSH conjugate (around - 0 . 1 9 2 volts) 17does not differ substantially from that of the parent quinone ( - 0.203 volts). ~8Therefore, the products resulting from reductive addition of GSH to quinones may hypothetically undergo autoxidation reactions, thus posing the question whether this feature of 1,4-reductive addition chemistry to quinones--involving thiol compounds--could be a potential source of 02 free radical species, with further deleterious effects on the cell. It was recently pointed that this conjugation reaction might promote cell injury in a fashion which is not linked to oxidative stress, 19,2°that is, without involving an autoxidation component. Thus, the 1,4-reductive addition of GSH to quinones provides new implications to quinone metabolism which might bear an antioxidant and/orprooxidant character. This research is concerned with the 1,4-reductive addition of GSH to quinone epoxides and mechanistic studies point to the reductive cleavage of the epoxide ring to yield as primary molecular product a 2-OHglutathionyl-hydroquinone conjugate. The hydroxy group in position e~, adjacent to the carbonyl group, introduces in the molecule a particular chemical reactivity, distinct from that originating from the GSHmediated reductive addition of the parent quinone compounds lacking the epoxide ring. This feature might broaden the biological relevance--in terms of the tox-
icological aspects--bound to the 1,4-reductive addition of GSH to quinone epoxides. These mechanistic studies were extended to 1,4-naphthoquinone epoxides with different degree of methyl substitution as well as p-benzoquinone epoxides. However, in the latter instances, identification of molecular products was complicated due to the lability of the intermediate compounds and their rapid decomposition to a variety of subproducts. The molecular products originating from the nucleophilic addition of GSH to quinone epoxides were assessed under anaerobic and aerobic conditions. The former approach does not involve autoxidation reactions and allows the evaluation of redox interactions within molecular products. The latter approach permits to determine the involvement of the different molecular products--identified under anaerobic conditions--in autoxidation reactions, that is, one- or two-electron transfer to 02 with formation of O2~ and H202, respectively. This research was carried out mainly by h.p.l.c. with electrochemical detection, taking advantage of the redox properties of all products formed under either anaerobic or aerobic situations. Further, this technique can measure minute amounts of quinones or hydroquinones present in reaction mixtures 8,21,22 and it provides--by means of the corresponding hydrodynamic voltamograms--the half-wave potential of the quinone/hydroquinone couples, a parameter which permits to predict the likelihood of redox interactions between different molecules. MATERIALS AND METHODS
Chemicals, biochemicals, and synthesis of quinone epoxides 1,4-naphthoquinone, 2,3-epoxy-, 1,4-naphthoquinone and 2-OH-l,4-naphthoquinone were obtained from Aldrich Chemical Co. (Steinheim, FRG). Scopoletin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals were from Merck (Darmstadt, FRG) and biochemicals from Boehringer (Mannheim, FRG). 2,3-Dimethyl-2,3-epoxy1,4-naphthoquinone was a gift from Dr. M. D. Threadgill (Pharmaceutical Sciences Institute, Aston University, Birmingham, UK). 2,3-epoxy-p-benzoquinone and 6-methyl-2,3-epoxyp-benzoquinone were prepared as described previously. 23 2-Methyl-2,3-epoxy-l,4-naphthoquinone (menadione epoxide) and 2-methyl-3-OH-1,4-naphthoquinone (phthiocol) were prepared according to the method described by Fieser 24and 2-methyl-3-glutathionyl- 1,4naphthoquinone (thiodione) by the procedure described by Nickerson et al. 25
Quinone epoxides
2-OH-3-glutathionyl-l,4-naphthoquinone was prepared by mixing 0.70-g 1,4-naphthoquinone epoxide dissolved in 14-ml ethanol with 1.23-g GSH dissolved in 70-ml distilled H20 and incubating at 37°C for 5 days. The reaction mixture was extracted with chloroform, lyophilized, and chromatographed on an 5/~m Spherisorb ODS-2 250 mm x 8 mm semi-preparative h.p.l.c, column (Jones Chromatography Ltd, Llanbradach, Mid Glamorgan, UK) with 25% methanol/75% H20 as the eluent. Fractions containing the desired product were lyophilized. The elemental analysis of the synthesised compound was performed by Mikro Kemi AB, Uppsala, Sweden; found: C, 48.8; H, 4.4; N, 8.7; S, 6.0, in agreement with that calculated for C20H22N309S: C, 50.0; H, 4.6; N. 8.7; S, 6.7. 3-Glutathionyl-l,4-naphthoquinone was prepared by the method designed by Nickerson et a1.25for thiodione and the product was purified from its corresponding diconjugate on the preparative chromatographic system described above, but with 40% methanol/60% H20 as the mobile phase. The elemental analysis data: found: C, 50.3; H, 4.8; N, 8.8; O, 27.3; S, 7.5, in agreement with that calculated for C20H22N308S: C, 51.7; H, 4.8; N, 9.0; O, 27.6; S, 6.9. Assay conditions
All reactions were carried out in 0.1 M potassium phosphate buffer, pH 7.65, containing 100--or 500 aM p-benzoquinone--or naphthoquinone epoxides, respectively, and equimolar amounts of GSH. Assay temperature was 37°C. Anaerobic conditions were obtained by purging the system with helium for 10 min and the reaction mixture was kept under helium atmosphere during the reaction period.
151
spectromonitor D (LDC, Riviera Beach, Florida, USA), operated at h = 220 nm, was connected in series with the electrochemical detector. Injection volume was 20 /tl. The mobile phases were selected in order to obtain optimal separation of the quinonoid derivatives studied and were as follows: (a) A mobile phase consisting of 35% 2-propanol/ 65% H20, buffered to pH 6.5 with 50 mM sodium phosphate, was used for the evaluation of 2,3dimethyl-2,3-epoxy- ! ,4-naphthoquinone, 2,3-epoxy-pbenzoquinone, and 6-methyl-2,3-epoxy-p-benzoquinone. Flow rate was 0.6 ml x min-~. (b) A modification of the mobile phase described by Hernandez et al. z6 was used. A buffer solution was made by diluting 5 ml of phosphoric acid to 1 litre with H20 and the pH was adjusted to 7.0 by addition of Tris-base. 10% of this buffer, 25% methanol, and 65% H20 containing 50 mM sodium sulphate was used as mobile phase for the characterization of 2,3-epoxy1,4-naphthoquinone,2-OH-3-glutathionyl- 1,4-naphthoquinone, 3-glutathionyl-1,4-naphthoquinone, and 2OH-1,4-naphthoquinone. Flow rate was 1.0 ml x min- ~. (c) A mobile phase similar to that described above but with a methanol content increased to 37% at expense of H20 was used for the evaluation of 2-methyl2,3-epoxy- 1,4-naphthoquinone, 2-methyl-3-OH- 1,4naphthoquinone, and 2-methyl-3-glutathionyl-l,4naphthoquinone. Flow rate was 1.0 ml x min -~. All the mobile phases were carefully degassed in vacuo, followed by at least 1 h of helium purging prior to analysis in order to eliminate oxidation by 02 of reduced products during h.p.l.c, runs.
H.p.l.c. with electrochemical detection
The concentration of quinones, quinone epoxides, and hydroxy- and glutathionyl quinone derivatives was measured by h.p.l.c, with either reductive or oxidative electrochemical detection. A 150 mm x 3.9 mm Novapak 4 / t m C18 reverse-phase column (Waters-Millipore AB, V. Fr61unda, Sweden) with an Uptight 20 mm × 2 mm C- 18 precolumn (Skandinaviska Genetec, Kungsbacka, Sweden) was connected to a Constametric III pump (LDC, Riviera Beach, Florida, USA). The eluent was monitored by an electrochemical detector unit consisting of a LC-4 potentiostat with a TL5A three electrode glassy carbon-flow cell and an Ag/ AgCI reference electrode (BAS, West Lafayette, IN, USA) operated at either - 0 . 9 volts (reductive mode) or +0.9 volts (oxidative mode). In experiments with 2,3-dimethyl-2,3-epoxy-l,4-naphthoquinone a LDC
02 uptake
02 consumption was measured in a thermostated cuvette filled with a Clark-type 02 electrode (Hansatech Ltd., Norfolk, UK).
H202 measurements H202 was measured with the scopoletin method27 as reduction of fluorescence intensity (hexcitation ---- 360 nm; hemission ---- 460 nm) on a Shimadzu 540 spectrofluorimeter (Shimadzu Corporation, Kyoto, Japan). Total GSH and GSSG measurements
Total GSH, expressed as glutathione equivalents, was measured as described in z8 with a Varian DMS-
152
A. BRUNMARKand E. CADENAS
100 spectrophotometer (Varian AB, Solna, Sweden). Samples were either diluted five-fold with distilled H20 and stored for a short time on ice or analyzed directly after withdrawal from the reaction mixture. GSSG was measured following NADPH oxidation ( E . 3 4 0 a m : 6.22 mM-Jcm-]). 28 RESULTS AND DISCUSSION
I. H.p.l.c. with electrochemical detection properties of 1,4-naphthoquinone- and menadione epoxide and hydroxy- and glutathionyl-substituted 1,4-naphthoquinones H.p.l.c. with electrochemical detection was used for the evaluation of products arising from the 1,4-nucleophilic addition of GSH to un- and methyl-substituted 1,4-naphthoquinone epoxides. This method is based on the redox properties retained in the molecular products originating from the above reaction and allows detection of minute amounts of products. 8'2~'22 Figure IA shows a chromatogram of the standards (see Materials and Methods section) used for the evaluation of the reaction between 2,3-epoxy-l,4naphthoquinone and GSH: 2-OH-3-glutathionyl-l,4naphthoquinone (peak a) was eluted first, followed by
the 2-OH- 1,4-naphthoquinone (lawsone) (peak b). The 3-glutathionyl-l,4-naphthoquinone adduct (peak c) was retained more strongly despite the hydrophilic character of the glutathionyl moiety; this may be due to the formation of ionic pairs between the compound and certain components in the mobile phase. Peak d corresponds to 2,3-epoxy- 1,4-naphthoquinone. Retention times (R,) and half-wave potentials (El/2) of the above quinonoid derivatives are listed in Table 1. Figure IB shows a chromatogram of the hydroxyand glutathionyl-adducts of the monomethyl-substituted 1,4-naphthoquinone (menadione) and of menadione epoxide. 2-Methyl-3-OH-1,4-naphthoquinone was eluted first followed by 2-methyl-3-glutathionyl-l,4naphthoquinone and finally--with the longest retention time--2-methyl-2,3-epoxy-1,4-naphthoquinone or menadione epoxide. Retention times (Rr) and half-wave potentials (Ej/2) are summarized in Table 1. It should be noted that a decreasing half-wave potential was observed in both series (unsubstituted and methyl-substituted naphthoquinones) with the following order: glutathionyl-substituted derivatives > hydroxy-substituted derivatives > quinone epoxides. This is illustrated in Figure 2 with the hydrodynamic voltamograms of 3-glutathionyl-l,4-naphthoquinone
O
(2@ °
SG
O [2 nn'l~s] O
O
O
0 o
I
2 1 ~ nA
o
SG 0 14 amok~]
B O
0 [4 nmalcsl
I~miami
;
O
o
O
Time (rain) Fig. 1. H.p.l.c. with reductive electrochemical detection chromatograms of 1,4-naphthoquinone- and menadione epoxides and hydroxy- and glutathionyl-substituted naphthoquinones. Assay conditions and synthesis of standard compounds as described in the Materials and Methods section. (A). Chromatogram of 1,4-naphthoquinone derivatives: (a) 2-OH-3-glutathionyl-1,4-naphthoquinone; (b) 2-OH-1,4-naphthoquinone; (c) 3-glutathionyl-1,4-naphthoquinone; (d) 2,3-epoxy-1,4-naphthoquinone. (B). Chromatogram of menadione derivatives: (a) 2-methyl-3-OH-1,4-naphthoquinone; (b) 2-methyl-3-glutathionyl-1,4-naphthoquinone; (c) 2-methyl-2,3-epoxy- 1,4-naphthoquinone. Applied potential - 0.9 volts.
Quinone epoxides
(1) Studies under aerobic conditions. Figure 3A shows the chromatogram corresponding to a reaction mixture containing 2,3-epoxy- 1,4-naphthoquinone obtained with h.p.l.c, with reductive electrochemical detection. The chromatogram obtained after 10-min incubation of the quinone epoxide with equimolar amounts of GSH (Fig. 3B) showed a large decrease in the intensity of the peak d corresponding to 2,3-epoxy-l,4-naphthoquinone and formation of three main products: peaks a, b, and c co-chromatographed with the standards shown in Figure 1A and possessed the same El/2 values listed in Table 1 and were identified as 2-OH-3-glutathionyl1,4-naphthoquinone, 2-OH-l,4-naphthoquinone, and 3-glutathionyl-l,4-naphthoquinone, respectively. The peak between peaks a and b in Figure 3B was not identified but it would correspond probably to a glutathione-diconjugate of 1,4-naphthoquinone. Analysis of the spent reaction mixture with oxidative applied potential (Fig. 3C) did not show significant amount of reduced products, except for a peak with a short retention time. This peak remained unaffected upon oxygenation and, therefore, we cannot ascribe it to a reduced form of any quinone. Figure 4 shows the time courses of GSH and 2,3epoxy-l,4-naphthoquinone consumption as well as product formation. Both reactants, GSH and 2,3-epoxy1,4-naphthoquinone were consumed at initial rates of about 240 I~M x min- ~, equivalent to a second-order rate constant of about 16 M-%-~. The total amount of GSH consumed was slightly lower than the amount of 2,3-epoxy-l,4-naphthoquinone consumed during the reaction ([GSH] . . . . . . . d / [quinone epoxide] ..... n~ = 0.93).
Table 1. H.p.l.c. with reductive electrochemical detection characteristics of un- and methyl-substituted 1,4-naphthoquinone derivatives Retention Time Rt(min)
Half-Wave Potential Etn(-volts)
1.8
0.68
4.6 10.1 17.8
0.55 0.22 0.72
2.3 4.1
0.50 0.30
13.3
0.79
Unsubstituted naphthoquinone derivatives 2-OH-3-glutathionyl-
1,4-naphthoquinone 2-OH-1,4-naphthoquinone 3-glutathionyl-1,4-naphthoquinone 2,3-epoxy-1,4-naphthoquinone Menadione derivatives 2-methyl-3-OH-1,4-naphthoquinone 2-methyl-3-glutathionyl1,4-naphthoquinone 2-methyl-2,3-epoxy1,4-naphthoquinone
153
Note. H.p.l.c. mobile phases used are specified in the Materials and Methods section.
(E,/2 0.22 volts), 2-OH-l,4-naphthoquinone (Ej/2 = - 0 . 5 5 volts), and 2,3-epoxy-l,4-naphthoquinone (E1/2 = - 0 . 7 2 volts).
H. GSH-mediated 1,4-reductive addition to 2,3-epoxy- 1,4-naphthoquinone The GSH-mediated 1,4-reductive addition to 2,3-epoxy-l,4-naphthoquinone was studied under aerobic- and anaerobic conditions in order to assess the importance of autoxidation reactions and potential interaction of secondary molecular products.
1.0
O
O
O °
$O o
0.5
0.2
0.4
0.6
01~
,i0
Appli~l volt~e (- ,,,olu)
Fig. 2. Hydrodynamic voltamograms of 3-glutathionyl-l,4-naphthoquinone, 2-OH-1,4-naphthoquinone, and 2,3-epoxy-l,4naphthoquinone. Assay conditions and synthesis of standards as described in the Materials and methods section.
154
A. BRUNMARK and E. CADENAS
ISOOnA
A
GSH, 85% (425/zM) quinone epoxide was consumed and the following quantitative distribution of molecular products was found: 51% (216 ktM) 2-OH-3-glutathionyl- 1,4-naphthoquinone, 20% (86/~M) 2-OH- 1,4naphthoquinone, and 22% (95/zM) 3-glutathionyl-1,4naphthoquinone (Table 2). The GSH-mediated reductive addition to 2,3-epoxy1,4-naphthoquinone was associated with 02 consumption, which was accounted for in terms of H202 formation (Fig. 5). The initial rates of 02 consumption and H202 formation were about 94- and 98 ltM × min -1, respectively (( - d[Oz]/dt)/(+ d[H202]/dt) 1). The distribution of molecular products found with h.p.l.c, with electrochemical detection (Fig. 3B) as well as the associated autoxidation reactions can be accounted for in terms of (a) a main reaction consistent with the reductive nucleophilic addition of GSH to the naphthoquinone epoxide and (b) secondary pathways involving elimination reactions and redox transitions.
-'V" 0
10
20
i
B
c SOOnA
I
SOOmA
b
1
0
10
20
0
5
10
(a) Main reaction: 1,4-reductive nucleophilic addition of GSH to quinone epoxide. The 1,4-reductive addition of GSH to 2,3-epoxy-l,4-naphthoquinone results in epoxide ring opening and formation of the primary molecular product 2-OH-3-glutathionyl-l,4-naphthohydroquinone (reaction 1).
Time (rain)
Fig. 3. H.p.l.c. with electrochemical detection analysis of products from the reaction of 2,3-epoxy-1,4-naphthoquinone with GSH under aerobic conditions. Assay conditions as described in the Materials and methods section. (A) H.p.l.c. with reductive electrochemical detection (applied potential - 0 . 9 volts) chromatogram of 500/tM 2,3-epoxy-l,4-naphthoquinone. (B) H.p.l.c. with reductive electrochemical detection chromatogram of 500 pM 2,3-epoxy- 1,4-naphthoquinone after 10-min incubation with equimolar amount of GSH. (C) H.p.l.c. with oxidative electrochemical detection (applied potential + 0.9 volts) chromatogram of the spent reaction mixture described in (B). Labelled peaks were identified as: (a) 2-OH-3-glutathionyl-1,4naphthoquinone; (b) 2-OH-1,4-naphthoquinone; (c) 3-glutathionyl1,4-naphthoquinone; (d) 2,3-epoxy-1,4-naphthoquinone. Peak (a) in Figure 3C could not be ascribed to a reduced form of any quinone; see explanations in the text.
The spent reaction mixture contained about 30 /tM GSSG, probably arising from the oxidation of GSH by H202, a reaction that has been reported29 in order to account for GSSG production during the interaction of GSH with menadione. The time courses of molecular product formation are illustrated in Figure 4B, C, and D for 2-OH-3glutathionyl-1,4-naphthoquinone, 2-OH- 1,4-naphthoquinone, and 3-glutathionyl-l,4-naphthoquinone, respectively. After 10-min incubation of 500 ~M 2,3epoxy- 1,4-naphthoquinone with equimolar amounts of
c+° O
+ GS-
--
O O2H +
(1)
O (b) Secondary reactions: elimination reactions and redox transitions. The secondary reactions give rise to the molecular products identified by h.p.l.c, with reductive electrochemical detection shown in Figure 3B. These involve elimination reactions and redox transitions. Elimination reactions. Peak c in Figure 3B co-chromatographed with the 3-glutathionyl-1,4-naphthoquinone standard (Fig. 1A). Its formation can be rationalized in terms of an elimination reaction as illustrated in reaction 2 and as previously proposed for the inter-
155
Quinone epoxides Table 2. Relative contribution of molecular products during the aerobic- and anaerobic GSH-mediated reductive addition to 2,3epoxy- 1,4-naphthoquinone
0
O
0
o Aerobiosis Anaerobiosis
C so
0
0
O
51 43
20 29
22 --
-29
O°-
+ 2H+ SG O 0
+ OHSG
(2)
0
Redox transitions. The primary molecular product, 2-OH-3-glutathionyl- 1,4-naphthohydroquinone (as shown in reaction 1) can transfer electrons to either unreacted 2,3-epoxy- 1,4-naphthoquinone (reaction 3) or 02 (reaction 4). The former reaction is accompanied by the opening of the epoxide ring and is similar to the two-electron enzymatic reduction catalyzed by DT-diaphorase. 3~ The molecular products are 2-OH-3-glutathionyl-l,4naphthoquinone (identified as peak a in the chromatogram in Fig. 3B) and 2-OH-naphthohydroquinone (reduced lawsone).
the bulk of quinone epoxide is consumed via reaction 1, whereas a part of it is consumed in redox transitions within the frame of reaction 3. This is in agreement with the observed slight excess of quinone epoxide consumed over GSH consumed (Fig. 4A, B). The latter reaction, 02 consumption and consequent H 2 0 2 formation (Fig. 5), is accounted for by further redox transitions represented by electron transfer to 02 from either the 2-OH-3-glutathionyl-l,4-naphthoquinone (reaction 4) or 2-OH-l,4-naphthohydroquinone (reaction 5) (the latter compound being a product of reaction 3). 2-OH-1,4-naphthoquinone, a product of reaction 5, is identified as peak b in the chromatogram of Figure 3B. O+
02
+2H +
O-
~
O O- + H202 SG
(4)
+ H202
(5)
O O"
O
°+ O2+2H +
O SG O-
SG
0
action of l-phenyl-lH-tetrazole-5-thiol with substituted 2,3-epoxy- 1,4-naphthoquinones. 3°
O
o
O-
O
0 0
0-
+ SG O
+ H+ (3)
O
O-
Reactions 1 and 3 represent two different pathways for quinone epoxide consumption. It is assumed that
It is suggested that reactions 4 and 5 represent the main autoxidation reactions associated with the GSHmediated nucleophilic addition to 2,3-epoxy-l,4-
156
A. BRUNMARKand E. CADENAS
A
B 50~
!N)"
500
Y
|
==
125
2so ;/3
!
[ |
o
,.4
Time(rain)
Time(min)
0 150
150
o
100 ,3
¢
..=
f~
0
Time(mini
Time(rain)
Fig. 4. Time courses for reactant consumption and product formation during the reaction of 2,3-epoxy-1,4-naphthoquinone with GSH under aerobic and anaerobic conditions. Assay conditions as described in the Materials and methods section. Open and closed symbols correspond to assays carried out under anaerobic and aerobic conditions, respectively. (A) Consumption of total glutathione equivalents; (B) Consumption of 2,3-epoxy-1,4-naphthoquinone and formation of 2-OH-3-glutathionyl-1,4-naphthoquinone product. (C) Formation of 2-OH-1,4-naphthoquinone product. (D) Formation of 3-glutathionyl-1,4-naphthoquinone and its reduced counterpart.
naphthoquinone. Most likely, electrons are transfered directly to O2 from 2-OH-3-glutathionyl-1,4-naphthohydroquinone and 2-OH- 1,4-naphthohydroquinone. This is based on the observation that the addition of 100 /iM 2-OH- 1,4-naphthoquinone or 3-glutathionyl- 1,4naphthoquinone to a reaction mixture consisting of GSH plus 2,3-epoxy-l,4-naphthoquinone did not increase significantly the rate of 02 uptake by the system. This could be the expected if the above compounds were required intermediates for electron transfer to 02. Reactions 4 and 5 are written as a two-electron transfer to O2 with direct formation of H202. It is likely though, that production of HzO2 (Fig. 5) proceeds in two one-electron transfer steps with formation of glutathione-semiquinone intermediate ~4-16and 02 ~. However, we cannot provide ESR evidence for the occurrence of a glutathione-semiquinone intermediate and
reactions 4 and 5 reflect solely the results summarized in Figure 5. The relative contribution of the autoxidation reactions (reactions 4 and 5) to H202 formation could be analyzed on the basis of the quantitative data presented in Figs. 4 and 5 and summarized in Table 2. About 190/IM H202 is formed during the reductive addition of GSH to 2,3-epoxy-l,4-naphthoquinone; approximately 86 ~M 2-OH-1,4-naphthoquinone is detected in the spent reaction mixture and this would correspond (according to reaction 5) to an equimolar amount of H202. The remaining H202 should be formed out of the autoxidation of 2-OH-3-glutathionyl-1,4-naphthohydroquinone (reaction 4), that is, about 104 ,uM. Because about 216 /tM 2-OH-3-glutathionyl-l,4naphthoquinone is detected in the reaction mixture, the remaining product should originate from the redox
Quinone epoxides
.~:~:ZZ ............................................................................. 200
v
loo
J
0
10
T
20
Time (min) Fig. 5. Time courses of 02 consumption and H,_O2 formation upon supplementation of 2,3-epoxy-l,4-naphthoquinone epoxides with GSH. Assay conditions as described in the Materials and methods section. --., ---, and - - indicate O: consumption for 2,3-dimethyl2,3-epoxy-l,4-naphthoquinone, 2-methyl-2,3-epoxy-l,4-naphthoquinone, and 2,3-epoxy-l,4-naphthoquinone, respectively....e..., --e--, and - e - indicate H20: production for 2,3-dimethyl-2,3-epoxy1,4-naphthoquinone, 2-methyl-2,3-epoxy-l,4-naphthoquinone, and 2,3-epoxy- 1,4-naphthoquinone, respectively.
157
to 3-glutathionyl-l,4-naphthoquinone was also observed. Analysis of the spent reaction mixture with oxidative electrochemical detection (Fig. 6B) showed a main peak (peak c) corresponding to 3-glutathionyl-1,4-naphthohydroquinone, the compound with the highest redox potential in the system (Table 1). The intensity of this peak decreased instantly and was abolished completely following oxygenation of the reaction mixture. Similarly to what commented for peak a in Figure 3C, we cannot ascribe the analogous peak in Figure 6B to a reduced form of any quinone and, accordingly, this peak remained unaffected upon oxygenation. The time courses of GSH and 2,3-epoxy-l,4naphthoquinone consumption as well as product formation under anaerobic conditions are summarized in Figure 4. The total amount of GSH consumed during the reductive addition to 2,3-epoxy-l,4-naphthoquinone did not change appreciably (Fig. 4A) in comparison with experiments carried out under aerobiosis, whereas the total amount of quinone epoxide consumed exceeded that observed in the presence of 0 2 (A[quinone epoxide] ...... bio~iJA[quinone epoxide]acrobio~ = 1.15) (Fig. 4B). Moreover, under anaerobic conditions, 2,3epoxy- 1,4-naphthoquinone was consumed in large ex-
I
5OO n A
transition shown in reaction 3. In summary, the 216 ~uM2-OH-3-glutathionyl- 1,4-naphthoquinone detected in the reaction mixture should be formed with similar efficiency from [a] redox transition with unreacted epoxide (providing about 112 pM; reaction 3) and [b] autoxidation reactions (providing about 104 pM; reaction 4). (2) Studies under anaerobic conditions. The study of the GSH-mediated nucleophilic addition to 2,3-epoxy1,4-naphthoquinone under anaerobic conditions brings about the following advantages: it precludes electron transfer to 02 and, thereby, it facilitates the evaluation of possible interactions within the molecular products. H. p. l.c. with reductive electrochemical detection of the spent reaction mixture carried out under anaerobic conditions showed two peaks (Fig. 6A): peak a and b co-chromatographed with the standards shown in Figure 1A and corresponded to 2-OH-3-glutathionyl-1,4naphthoquinone and 2-OH-1,4-naphthoquinone, respectively. A very small peak (peak c) corresponding
_._3,_ 6
~
5OOnA
A
~ 1'o
2~
o
5
10
Time (min) Fig. 6. H.p.l.c. with electrochemical detection chromatograms of the reaction between 2,3-epoxy-1,4-naphthoquinone and GSH under anaerobic conditions. Assay conditions as described in the Materials and methods section. Figure shows chromatograms obtained after 10-min incubation of 500 pM 2,3-epoxy-l,4-naphthoquinone and equimolar amounts of GSH under anaerobic conditions analyzed with reductive (A) and oxidative (B) applied potential ( - 0 . 9 volts and + 0.9 volts, respectively). Labelled peaks [a-d] are the same as in Figure 3 and co-chromatograph with the standards in Figure IA.
158
A. ]3RUNMARKand E. CADENAS
cess over GSH: (A[quinone epoxide] - A[GSH] = 108/~M). Under anaerobic conditions 97% (486 ItM) of 2,3epoxy-1,4-naphthoquinone was consumed, whereas 76% (378/zM) GSH was consumed. This uneven molar ratio for consumption of reactants suggested the interaction of derived molecular products with 2,3-epoxy-l,4naphthoquinone which had not reacted with the thiol. The quantitative distribution pattern of products under anaerobiosis was as follows: 43% (211 /~M) 2-OH-3glutathionyl-l,4-naphthoquinone, 29% (145 /tM) 2OH-1,4-naphthoquinone, and 29% (142 ltM) 3-glutathionyl- 1,4-naphthohydroquinone. The differences between the distribution of molecular products under anaerobiosis and aerobiosis during the thiol-mediated reductive addition of 2,3-epoxy-l,4naphthoquinone are listed in Table 2. The excess of 2,3-epoxy-l,4-naphthoquinone consumed over that of GSH under anaerobic conditions could be interpreted as reaction 3 prevailing over reaction 4, for autoxidation reactions are negligible under these conditions. The molecular mechanism for the formation of the products identified during the GSH-mediated reductive addition to 2,3-epoxy-l,4-naphthoquinone under anaerobiosis, that is, 2-OH-1,4-naphthoquinone and 2OH-3-glutathionyl-l,4-naphthoquinone, is implied in reaction 1 followed by redox transitions as that of reaction 3.3-Glutathionyt- 1,4-naphthoquinone would be formed through an elimination reaction similar to reaction 2. This compound would display a main oxidant activity, substituting for 02 (as in reactions 4 and 5)
in the oxidation of reduced lawsone (reaction 6) or 2OH-3-glutathionyl-l,4-naphthohydroquinone (Fig. 6B). O-
O
O-
O
SG
O
O(6)
O
O-
111. GSH-mediated nuclephilic addition to methyl-substituted naphthoquinone epoxides The h.p.l.c, with reductive electrochemical detection chromatogram of 500 /~M 2-methyl-2,3-epoxy1,4-naphthoquinone is shown in Figure 7A (peak a) and that of the spent reaction mixture (after 20-min incubation of menadione epoxide with equimolar amounts of GSH) in Figure 7B. The peak corresponding to 2-methyl-2,3-epoxy-l,4-naphthoquinone decreased substantially upon incubation of the methylsubstituted quinone epoxide with GSH resulting in the formation of a compound with a shorter retention time and more hydrophilic character (peak b; Fig. 7B). Peak b was identified as 2-methyl-3-glutathionyl-1,4naphthoquinone (thiodione), which was the sole molecular product in this reaction. H.p.l.c. with oxidative
A
B
1000 nA
5
10
15
0
5
1'0
1'5
Time (min) Fig. 7. H.p.l.c. with electrochemical detection analysis of products originating from the 1,4-nucleophilic addition of GSH to 2-methyl-2,3-epoxy-l,4-naphthoquinone or menadione epoxide under anaerobic conditions. Assay conditions as described in the Materials and methods section. (A) Chromatogram obtained with reductive applied potential (-0.9 volts) of a solution containing 500/tM 2,3-epoxy-2-methyl-l,4-naphthoquinone(peak a). (B) Chromatogramobtained with reductive applied potential of a solution of 500/tM 2-methyl-2,3-epoxy-l,4-naphthoquinoneafter 20-min incubation with equimolar amounts of GSH: peak a decreases at expense of product formation (= peak b, identified as 2-methyl-3-glutathionyl-l,4-naphthoquinone (thiodione) after comparison with standards in Fig. 1B).
Quinone epoxides
electrochemical detection did not reveal any reduced quinonoid compound even under anaerobic conditions (not shown). Figure 8 illustrates the time course for menadione epoxide and GSH consumption and concomitant formation of the molecular product 2-methyl-3-glutathionyl-l,4-naphthoquinone (thiodione), which was produced in equimolar amounts. 2-Methyl-2,3-epoxy1,4-naphthoquinone or menadione epoxide and GSH react with an initial rate of about 123 ,uM x min -l, that is, at half the rate observed with the unsubstituted 1,4-naphthoquinone epoxide (Fig. 4A,B). Menadione epoxide was consumed at identical rates regardless whether the assay was carried out under aerobic- or anaerobic conditions. Reaction 7 intends to rationalize the findings observed during the reductive addition of GSH to menadione epoxide over an intermediate which undergoes a rapid elimination reaction to yield 2-methyl-3-glutathionyl-l,4-naphthoquinone. The latter compound corresponds to peak b in Figure 7B and represents the main or sole molecular product. The addition of nbutanethiol to menadione epoxide gave a similar product, that is, 2,n-butylthio-3-methyl-l,4-naphthoquinone or menadione-thioether. 32 O
OH
0
0
~
O CH3 +
OH-
(7)
SG O
In accordance with the mechanism illustrated in reaction 7, autoxidation reactions with formation of H202 (reactions 4 and 5 above) would be of little significance. Consequently, the amount of 02 consumed and H202 produced during the GSH-mediated nucleophilic addition to 2-methyl-2,3-epoxy-1,4-naphthoquinone was negligible in comparison with that observed with the unsubstituted naphthoquinone epoxide (Fig. 5). At the moment we cannot provide a satisfactory explanation for the formation of H202; it is clear that mechanisms other than that comtemplated in reaction 7 may operate as a source of H202 during the above reaction. A small amount of GSSG (about 10/~M) was found in the reaction mixture. It is likely that redox transitions involving glutathionyl radicals, as those proposed by Ross et a1.,29 would be operative in this system. Of
159
| E,h e~
0
110
210
Time(min) Fig. 8. Time courses for reactant consumption and product formation during the l-4-reductive addition of GSH to 2-methyl-2,3epoxy- 1,4-naphthoquinone or menadione epoxide. Assay conditions as described in Fig. 7 and in the Materials and methods sections. Points correspond to samples withdrawn at different intervals from the ongoing reaction mixture.
note, however, is that these reactions should be considered secondary to the primary thiol-mediated reductive addition to menadione epoxide described in Figures 7 and 8 and rationalized in reaction 7. 2,3-Dimethyl-2,3-epoxy-l,4-naphthoquinone reacted with GSH at a rate that was far slower (18 ItM x min- l) than that of other epoxides in the naphthoquinone series. The rate of consumption of GSH was higher than that of quinone epoxide (not shown). The system did not interact with 02 as could be seen from the total absence of O2 consumption and H202 production (Fig. 5). A UV detector, connected in series with the electrochemical detector, showed a signal which increased in intensity with time during the reaction (not shown). This signal appeared close to the void volume, which suggests the presence of a hydrophilic molecule, probably containing the glutathionyl rest. No products, however, were detected by h.p.l.c, with electrochemical analysis, indicating the formation of a compound which did not possess the redox properties required for its evaluation with this method, most likely due to loss of the quinonoid structure of the product. The loss of the quinonoid structure can be understood as a consequence of a change in the electron configuration in the molecule produced upon GSH conjugation. To regain a quinonoid structure two substituents on the quinone ring would be required to leave, that is, hydroxyl
160
A. BRUNMARK and E. CADENAS
anion (HO-) and glutathionyl anion ( G S ) , which would be equivalent to a four-electron oxidation. The hypothetical molecule possesses no reducing equivalents and, thus, it will not undergo rearrangement into a quinonoid structure. A speculative mechanism is described in reaction 8, where the product should be considered as an intermediate compound which may undergo further rearrangements involving scission between C2 and C3. 0
0 +
v
GS-
(8)
"1]" CH3 O
~k~cHSG O
O" H 3 C ~ O" ~+ G S "
IV. Reaction of un- and methyl-substituted p-benzoquinone epoxides with GSH The reductive addition of GSH to p-benzoquinone epoxides was studied with 2,3-epoxy-p-benzoquinone and 6-methyl-2,3-epoxy-p-benzoquinone. Figure 9A, B shows the time courses for GSH and p-benzoquinone epoxide consumption. 2,3-Epoxy-pbenzoquinone was consumed at a rate 10-fold higher than 6-methyl-2,3-epoxy-p-benzoquinone (117- and 1 ! /~M × min- ~, respectively). This large difference may be due to the electron-donating properties of the methyl substituent in 6-methyl-2,3-epoxy-p-benzoquinone, which would decrease considerably its rate of reaction with GSH. Reaction 9 exemplifies the mechanism for the 1,4-reductive addition of GSH to p-benzoquinone epoxides. O H3C~o +
GS-
O O-
H3C ~
two additions each followed by oxidation. 4 The excess of GSH consumed cannot be accounted for in terms of GSSG formation. The latter observation may indicate secondary reactions (secondary to the initial 1,4-reductive addition of GSH to p-benzoquinone epoxide; reaction 9), probably comprising a redox transition between the hydroxy-p-benzohydroquinone-thioether and p-benzoquinone epoxide (reaction 10). This reaction (similar to reaction 3) would lead to epoxide ring opening yielding an unconjugated 2-OH-p-benzohydroquinone, a compound shown to possess strong autoxidation properties? ~ O H 3 C ~
O"
O
O O
O-
GS - "-~ O
O-
Consequently, the overall addition reaction of GSH to p-benzoquinone epoxides was associated with a substantial consumption of 02, which was largely accounted for as H202 production (Fig. 9C). A lag phase was observed to precede the maximal rate of 02 uptake and H202 production, thus suggesting that electrons were mainly transferred to 02 by an intermediate accumulating in the reactioo mixture. The hydroxy-hydroquinone derivatives resulting from secondary interactions, as that illustrated in reaction 10, seem to support the autoxidation features of this system, unlike the stable p-benzohydroquinone conjugate 8,~9 formed during the reductive addition of GSH to p-benzoquinone lacking an epoxide ring.
O'+ 2H+
(9)
GS" " ( / O-
The time courses in Figure 9A, B also indicate that (a) the amount of GSH consumed is higher than that of either p-benzoquinone epoxide tested and (b) the consumption of the unsubstituted p-benzoquinone epoxide still proceeds after GSH has been totally consumed. The former observation may indicate a second site of conjugation, thus involving the usual sequence of
CONCLUSIONS AND COMMENTS The 1,4-reductive addition of GSH to the unsubstituted quinone epoxides (from either the p-benzo- or 1,4-naphthoquinone epoxide series) studied here implies predominantly epoxide ring opening and leads primarily to the formation of a hydroxy-glutathionylhydroquinone derivative. Further reactions following the primary formation of a 2-OH-3-glutathionyl-hydroquinone derivative are determined by (a) the degree of methyl substitution of the quinone epoxide and (b) the 02 concentration in the reaction mixture, parameters which are by no means
Quinone epoxides A
161 C
B
100,
100
]
[,,0
,.v
l
0
150L
10
20
0
10
20
0
0
10
20
Tlrlae (rain)
Fig. 9. Time courses of reactant consumption, 02 consumption, and HzO2 formation during reaction between GSH and un- or methyl-substituted p-benzoquinone epoxide. Assay conditions as described in the Materials and methods section. (A) Consumption of 2,3-epoxy-p-benzoquinone ( 0 ) or 6-methyl-2,3-epoxy-p-benzoquinone ( • ) during its reaction with GSH~ (B) Consumption of GSH during its reaction with 2,3-epoxy-p-benzoquinone ( 0 ) or 6-methyl-2,3-epoxy-p-benzoquinone ( 0 ) . (C) O2 uptake (traces) and H20: (symbols) production during the nucleophilic addition of GSH to 2,3-epoxy-p-benzoquinone ( - - and 0 ) and 6-methyl-2,3-epoxy-p-benzoquinone (--- and • ) .
independent of each other. They will both influence to different extent the interaction of secondary products leading to an ultimate pattern of molecular product distribution, which is characteristic for every quinone epoxide studied.
(a) Influence of the degree of methyl substitution on the 1,4-reductive addition of GSH to quinone epoxides The rate of the 1,4-reductive addition of GSH to pbenzoquinone- and 1,4-naphthoquinone epoxides decreases with increasing degree of methyl substitution of the quinone epoxide (Table 3). Parent quinones (lacking epoxide groups) react more rapidly with GSH than those containing an epoxide ring. Similar considerations apply to the differences found between the 1,4-naphthoquinone- and p-benzoquinone epoxide seTable 3. Initial rates of quinone epoxide consumption upon nucleophilic attack by GSH. Dependence on degree of methyl substitution in the quinone epoxide molecule -d[Quinone epoxide]/dt ,uM x rain -t 1,4-Naphthoquinone series 2,3-epoxy- 1,4-naphthoquinone 2-methyl-2,3-epoxy! ,4-naphthoquinone 2,3-dimethyl-2,3-epoxy1,4-naphthoquinone p-Benzoquinone series 2,3-epoxy-p-benzoquinone 6-methyl-2,3-epoxy-p-benzoquinone
240 123 18 117 11
Note. Assay conditions as described in the Materials and Methods section.
ries, for the p-benzoquinone offer a - C ~ C - double bond opposite to the site possessing the epoxy group. The former (with a lower electron density) will be a more favorable site for the nucleophilic addition than the latter (with a higher electron density). The decreased rate of 1,4-reductive addition of GSH to quinone epoxides with increasing degree of methyl substitution can be interpreted on the basis of the substituent in C2: the electron-donating properties of the - C H 3 groups will decrease accordingly the electrophilicity in C3 and, hence, its ability to react with GSH. A hydroxy substituent in C:, with a high electron density, such as that in lawsone or 2-OH-1,4-naphthoquinone, will prevent definitely the nucleophilic addition of GSH. 25 We have previously shown that the increasing degree of methyl-substitution of quinone epoxides was related to a decrease in the half-wave potential value. ~3 It could be expected that the redox potential of a quinone epoxide would influence the rate of reaction of the latter with GSH, assuming the requirement of an intermediate electron transfer complex within the reaction. However, it is difficult at the moment to draw a conclusive relationship between, on the one hand, the rate of GSH-mediated 1,4-reductive addition to naphthoquinone epoxides with different degree of methyl substitution and, on the other, their corresponding redox potential (as reflected by the half-wave potential13). Preliminary approaches, though, indicate that the rate of 1,4-reductive addition decreases exponentially with the decrease of the redox potential of the quinone epoxide. In summary, it is proposed that the rate of addition
162
A. BRUNMARKand E. CADENAS
of a quinone or quinone derivative with a sulphur nucleophile (GS-) will decrease in following order: quinone > methyl-substituted quinone > quinone epoxide > methyl-substituted quinone epoxide.
and 5). These relationships are summarized in the scheme in Figure 10. The absence of 02 in the system will prevent autoxidation of the primary molecular product (2-OH-3glutathionyl-l,4-naphthohydroquinone). This will be reflected in the prevailance of elimination reactions (reaction 2) and redox transitions with unreacted naphthoquinone epoxide (reaction 3) with respective formation of 3-glutathionyl-l,4-naphthoquinone and 2-OH-1,4-naphthohydroquinone (reduced lawsone). Under these circumstances, 3-glutathionyl- 1,4-naphthoquinone will substitute for 02 in reactions 4 and 5 as the ultimate oxidizing species, because it possesses the highest redox potential among the quinonoid compounds present in the system. Its reaction with both 2OH-3-glutathionyl- 1,4-naphthohydroquinone and 2-OH1,4-naphthohydroquinone will bring about the distribution of molecular products observed during the 1,4reductive addition of GSH to 2,3-epoxy-1,4-naphthoquinone under anaerobic conditions. These mechanistic relationships are summarized in the scheme in Figure 11. Qualitative alterations in the pathways drawn in the scheme in Figure 10 (under aerobic conditions) are expected to be determined by the increasing degree of
(b) Interaction with 02 subsequent to the 1,4-reductive addition of GSH to quinone epoxides The primary product resulting from the GSH nucleophilic addition to 2,3-epoxy-l,4-naphthoquinone, namely, 2-OH-3-glutathionyl-l,4-naphthohydroquinone, is endowed with a high reducing power and it can be oxidized by either (a) unreacted quinone epoxide (reaction 3) or (b) 02 with formation of H202 (reaction 4). Another possibility is an elimination reaction (reaction 2). The presence of 02 in the reaction mixture favors the autoxidation of the primary product, thereby decreasing the contribution to the formation of final molecular products through both elimination reactions and quinone/hydroquinone derivative redox transitions. Autoxidation reactions are relevant under aerobic conditions and the main sources of H20: (Fig. 5) would involve electron transfer to 02 from 2-OH-3glutathionyl- 1,4-naphthohydroquinone and 2-OH- 1,4naphthohydroquinone (reduced lawsone) (reactions 4
I'IO"
0-
GS-
0
SG
o-
~
O
o
o
°
o-
o
o
o O2
H202
SG
02
~H202 o
o
Fig. 10. Scheme of reactions for the GSH-mediated 1,4-reductive addition to 2,3-epoxy-l,4-naphthoquinoneunder aerobic conditions. Boxes below the chemical structures indicate the peak identified in the spent reaction mixture shown in Figurc 3B and identified after comparison with retention times and half-wavepotentials in Figure 1A and Table 1.
Quinone epoxides o
O"
GS"
O
O
o-
0
163 I-K)-
SG
O
O
SG
o-
/ ½so O
O
O
o
Fig. 11. Scheme of reactions for the GSH-mediated 1,4-reductive addition to 2,3-epoxy-l,4-naphthoquinone under anaerobic conditions. Boxes below the chemical structures indicate the peak identified in the spent reaction mixture shown in Figure 6 and identified after comparison with retention times and half-wave potentials in Figure IA and Table 1.
methyl-substitution of 2,3-epoxy-l,4-naphthoquinone. The GSH nucleophilic addition to the unsubstituted naphthoquinone epoxide yielded substantial amounts of H202, whereas the addition to menadione epoxide (2-methyl-2,3-epoxy-l,4-naphthoqui. none) showed significantly lower H202 production, and that to the dimethyl-substituted 1,4-naphthoquinone epoxide showed neither 02 consumption nor H202 production. In terms of their relative contribution to autoxidation associated with the nucleophilic addition to 1,4-naphthoquinone epoxides, following order could be observed: 2,3-epoxy-l,4-naphthoquinone > 2-methyl-2,3-epoxy-1,4-naphthoquinone > 2,3-dimethyl-2,3-epoxy- 1,4-naphthoquinone. The primary product formed upon GSH addition to menadione epoxide is disubstituted in C3 and the compound is not to be considered a hydroquinone. To regain a quinonoid structure a H O - has to be eliminated from the intermediate compound within an oxidation reaction (reaction 7). Consequently, the formation
of 2-methyl-3-glutathionyl-l,4-naphthoquinone occurs mainly through loss of a H O - rather than a redox transition with O2 with formation of H202. A mechanism as that proposed by Nickerson et al. 25 for H202 production is not applicable within the context of these experiments, for their experimental model consisted of [menadione]/[GSH] molar ratio of 2, and the ultimate autoxidizing species was reported to be the reduced form of menadione. 25 The mechanistic implications related to the 1,4-reductive addition of GSH to 2,3-dimethyl-2,3-epoxy1,4-naphthoquinone are difficult to interpret. The structure drawn in reaction 8 is merely hypothetical and should be considered as an intermediate compound prone to undergo scission reactions. The decreased participation in redox reactions of the products originating from the GSH nucleophilic addition to monomethyl- (reaction 7) and dimethyl(reaction 8) substituted 1,4-naphthoquinone can be understood in terms of: (a) the occurrence of inter-
164
A. BRUNMARKand E. CADENAS
m e d i a t e p r o d u c t s w i t h d i s u b s t i t u t e d c a r b o n a t o m s , (b) the c o n s e q u e n t p r e v a i l a n c e o f e l i m i n a t i o n or f r a g m e n tation r e a c t i o n s , and, t h e r e b y (c) the i m p o s s i b i l i t y o f r e a r r a n g e m e n t into a r e d u c e d q u i n o n o i d structure. A l i k e the u n s u b s t i t u t e d 1 , 4 - n a p h t h o q u i n o n e e p o x ide, the G S H - m e d i a t e d r e d u c t i v e a d d i t i o n to p - b e n z o q u i n o n e e p o x i d e s s t u d i e d h e r e w o u l d l e a d indisting u i s h a b l y to the f o r m a t i o n o f a H O - s u b s t i t u t e d pb e n z o h y d r o q u i n o n e . S u c h a c o m p o u n d , as s h o w n in F i g u r e 9, is p r o n e to u n d e r g o r e d o x r e a c t i o n s , in s o m e instances implying autoxidation. F i n a l l y , it can be c o n c l u d e d that the r e a c t i o n o f q u i n o n e e p o x i d e s with G S H u n d e r a e r o b i c c o n d i t i o n s p r o d u c e s h y d r o x y - and g l u t a t h i o n y l - q u i n o n e d e r i v a tives, c o m p o u n d s m o r e prone to autoxidation than those r e s u l t i n g f r o m the n u c l e o p h i l i c a d d i t i o n to q u i n o n e s l a c k i n g an e p o x i d e g r o u p . T h e m a i n feature o f this r e a c t i o n is the o c c u r r e n c e o f a h y d r o x y substituent in p o s i t i o n e~, a d j a c e n t to the c a r b o n y l g r o u p , w h i c h appears to w i t h s t a n d the e n h a n c e d a u t o x i d a t i o n o b s e r v e d , T h i s h i g h e r c h e m i c a l r e a c t i v i t y m a y be o f t o x i c o l o g i c a l interest. H o w e v e r , it s h o u l d be b e a r e d in m i n d , as d e s c r i b e d a b o v e , that the m e t h y l s u b s t i t u t i o n o f quin o n e e p o x i d e s l o w e r e d the rate o f t h e i r c h e m i c a l addition w i t h s u l p h u r n u c l e o p h i l e s , t h e r e b y p r o d u c i n g less r e a c t i v e m o l e c u l a r p r o d u c t s , at least in t e r m s o f autoxidation reactions. This lower chemical reactivity m a y not translate d i r e c t l y into a low t o x i c i t y , for a less r e a c t i v e c o m p o u n d is, b e c a u s e o f its l o n g e r l i f e t i m e , m o r e l i k e l y to be t r a n s p o r t e d a w a y f r o m its g e n e r a t i o n loci to an a d e q u a t e m o l e c u l a r target. Acknowledgements--Supported by grants 7679 and 4481 from the
Swedish Medical Research Council. REFERENCES
1. Yamazaki, I. Free Radicals in enzyme-substrate reactions. In: Pryor, W. A., ed. Free radicals in biology. New York: Academic Press; 1977:183-218. 2. Chambers, J. Q. Electrochemistry of quinones. In: Patai, S., ed. The chemistry of quinonoid compounds. London: John Wiley & Sons; 1974:737-791. 3. Mason, R. P. Free radical intermediates in the metabolism of toxic chemicals. In: Pryor, W. A., ed. Free radicals in biology. Vol. V. New York: Academic Press; 1982:183-218. 4. Finley, K. T. The addition and substitution chemistry of quinones. In: Patai, S., ed. The chemistry of the quinonoid compounds. London: John Wiley & Sons; 1974:877-1144. 5. Ernster, L. DT-Diaphorase: a historical review. Chemica Scripta 27A:1-13; 1987. 6. Powis, G.; Appel, P. L. Relationship of the single-electron reduction potential of quinones to their reduction by flavoproteins. Biochem. Pharmacol. 29:2567-2572; 1980. 7. Cadenas, E. Generation of electronically-excited states during electron transfer to quinones. Chemica Scripta 27A:113-115; 1987. 8. Brunmark, A.; Cadenas, E. Electronically-excited states generation during the reaction of p-benzoquinone with H202. Relation to product formation: 2-OH- and 2,3-epoxy-p-benzoquinone. The effect of glutathione. Free Radic. Biol. Med. 3:169180; 1987.
9. Smith, M. T.; Evans, C, G.; Thor, H.; Orrenius, S. Quinoneinduced oxidative injury to cells and tissues. In: Sies, H., ed. Oxidative stress. London: Academic Press; 1985:91-113. 10. Miller, M. G.; Rodgers, A.; Cohen, G. M. Mechanisms of toxicity of naphthoquinones to isolated hepatocytes. Biochem. Pharmacol. 35:1177-1184; 1985. 11. Thornalley, P. J.; d'Arcy Doherty, M. ; Smith, M. T.; Bannister, V.; Cohen, G. M. The formation of active oxygen species following activation of l-naphthol, 1,2- and 1,4-naphthoquinone by rat liver microsomes. Chem.-Biol. Interactions 48:195-206; 1984. 12. Hodge, P. Fragmentation reactions of quinones. In: Patai, S., ed. The chemistry of the quinonoid compounds. London: John Wiley & Sons; 1974:559-616. 13. Brunmark, A.; Cadenas, E.; Segura-Aguilar, J.; Lind, C.: Ernster, L. DT-diaphorase-catalysed two-electron reduction of various p-benzoquinone and 1,4-naphthoquinone epoxides. Free Radic. Biol. Med. 5:133-143; 1988. 14. Takahashi, N.; Schreiber, J.; Fischer, V.; Mason, R. P. Formation of glutathione-conjugated semiquinones by the reaction of quinones with glutathione: an ESR study. Arch. Biochem. Biophys. 252:41-48; 1987. 15. Gant, T. W.; d'Arcy Doherty, M.; Odowole, D.; Sales, K. D.; Cohen, G. M. Semiquinone anion radicals formed by the reaction of quinones with glutathione or amino acids. FEBS Lett. 201:296-300; 1986. 16. Gant, T. W.: Cohen, G. M. Reactions of glutathione or amino acids with quinones forming semiquinone radicals. In: RiceEvans, C., ed. Free radicals, oxidant stress and drug action. London: Richelieu Press; 1987:377-397. 17. Wilson, I.; Wardman, P.; Lin, T.-S.; Sartorelli, A. C. Reactivity of thiols towards derivatives of 2- and 6-methyl-1,4-naphthoquinone bioreductive alkylating agents. J. Med. Chem. 29:13811384; 1986. 18. Butler, J.; Hoey, B. M. The apparent inhibition of superoxide dismutase activity by quinones. J. Free Radio. Biol. Med. 2:7781; 1986. 19. Rossi, L.; Moore, G. A.; Orrenius, S.; O'Brien, P. J. Quinone toxicity in hepatocytes without oxidative stress. Arch. Biochem. Biophys. 251:25-35; 1986. 20. Bellomo, G.; Mirabelli, F.; DiMonte, D.; Richelmi, P.; Thor, H.; Orrenius, C.; Orrenius, S. Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl- 1,4-naphthoquinone). Biochem. Pharmacol. 36:1313-1320; 1987. 21. Rappaport, S. M.; Jin, Z. L.; Xu, X. B. High-performance liquid chromatography with reductive electrochemical detection of mutagenic nitro-substituted polynuclear aromatic hydrocarbons in diesel exhausts. J. Chromatogr. 240:145-154; 1982. 22. Fluck, D. S.; Rappapport, S. M.; Eastmond, D. A.; Smith, M. T. Conversion of 1-naphthol to naphthoquinone metabolites by rat liver microsomes: demonstration by high-performance liquid chromatography with reductive electrochemical detection. Arch. Biochem. Biophys. 235:351-358; 1984. 23. Rashid, A.; Read, G. Quinone epoxides. Part 11. Synthesis of (_+)-terreic acid and some related epoxides. J. Chem. Soc.(C):1323-1325; 1967. 24. Fieser, L. F. Convenient procedures for the preparation of antihemorrhagic compounds. J.Biol. Chem. 133:391-396; 1940. 25. Nickerson, J. W.; Falcone, G.; Strauss, G. Studies on quinonethioethers. I. Mechanism of formation and properties of thiodione. Biochemistry 2:537-543; 1963. 26. Hernandez, O.; Bhatia, A. V.; Walker, M. HPLC separation of the diastereomeric glutathione adducts of styrene oxide. J. Liquid Chromat. 6:1475-1489; 1983. 27. Boveris, A. Determination of the production of superoxide radical and hydrogen peroxide in mitochondria. Methods Enzymol. 105:429-435; 1984. 28. Sies, H.; Akerbom, T. P. M. Glutathione disulfide (GSSG). Efflux from cells and tissues. Methods Enzymol. 105:445-451 ; 1984.
Quinone epoxides
29. Ross, D.; Thor, H.; Orrenius, S.; Moldeus, P. Interaction of menadione (2-methyl-l,4-naphthoquinone) with glutathione. Chem. Biol. Interact. 55:177-184; 1985. 30. O'Brien, D. E The kinetics of the reaction of l-phenyl-lHtetrazole-5-thiol sodium salt with substituted 1,4-naphthoquinone 2,3-epoxides. J. Org. Chem. 33:262-265; 1968. 31. Brunmark, A.; Cadenas, E.; Lind, C.; Segura-Aguilar, J.; Ernster, L. DT-Diaphorase catalyzed two-electron reduction of quinone epoxides. Free Radic. Biol. Med. 3:181-188; 1987. 32. Silver, R. F.; Holmes, H. L. Synthesis of some 1,4-naphthoquinones and reactions relating to their use in the study of bacterial growth inhibition. Can. J. Chem. 46:1859-1864; 1968.
165
ABBREVIATIONS
Menadione--2-methyl- 1,4-naphthoquinone Phthiocol--2-methyl-3-hydroxy- 1,4-naphthoquinone Thiodione--2-methyl-3-glutathionyl-1,4-naphthoquinone H.p.l.c.--high performanceliquid chromatography GSH--glutathione GSSG--glutathione disulfide Lawsone--2-OH- 1,4-naphthoquinone
0891-5849/89 $3.00 + .00 © 1989 Pergamon Press plc
Original Contribution 1,4-REDUCTIVE ADDITION OF GLUTATHIONE TO QUINONE EPOXIDES Mechanistic studies with h.p.l.c, with electrochemical detection under anaerobic and aerobic conditions and Evaluation of chemical reactivity in terms of autoxidation reactions ANDERS BRUNMARK
and ENRIQUE C A D E N A S
Department of Pathology II, University of Linkfping, S-581 85 Linkfping, Sweden
(Received 4 February 1988; Accepted 6 April 1988)
Abstract--The nucleophilic addition of GSH to quinonoid compounds, characterized as a 1,4-reductive addition of the Michael type, was studied with p-benzoquinone- and 1,4-naphthoquinone epoxides with different degree of methyl substitution. Identification and evaluation of molecular products from the above reaction were assessed by h.p.l.c, with either reductive or oxidative electrochemical detection, based on the redox properties retained in the molecular products formed. It was found that the degree of methyl substitution of the quinone epoxide, from either the 1,4naphthoquinone- or p-benzoquinone epoxide series, determined their rate of reaction with GSH. The reductive addition implied the rearrangement of the quinone structure with opening of the epoxide ring yielding as the primary product a hydroxy-glutathionyl substituted adduct of either p-benzohydroquinone or 1,4naphthohydroquinone. The primary product undergoes elimination reactions and redox transitions which bring about a number of secondary molecular products. The distribution pattern of the latter depends on the degree of methyl substitution of the quinone epoxide studied and on the concentration of 02 in the solution. The occurrence of the hydroxy-substituent in position a, adjacent to the carbonyl group, enhances the autoxidation properties of the compound resulting in an augmented 02 consumption and H202 production. Therefore, it could be expected that the chemical reactivity of the products originating from the thiol-mediated nucleophilic addition to quinone epoxides would be of toxicological interest. Keywords--Quinone epoxides, Glutathione, Free radicals, H.p.l.c. with electrochemical detection, 1,4-Naphthoquinones, p-Benzoquinones, Quinone-glutathione conjugates, Autoxidation, H20~
of the redox chemistry of quinones are of particular interest, for they affect the likelihood and efficiency of [a] their reduction by different flavoproteins ~'6 as well as [b] their participation in autoxidation reactions. The redox properties of quinones were reported in some instances to sustain the toxicological effects observed in quinone-induced cell and tissue injury. 9-~ The latter feature, the addition chemistry of quinones, involves, among others, their reaction with 02 and sulphur nucleophiles. The nature of the new substituent in the quinone molecule will determine, in large measure, its subsequent chemical reactivity. 4 Addition reactions with 02 nucleophiles can be exemplified by the H202 (HOO-)-mediated oxidation of quinones to yield quinone epoxide products. This p r o cess is based on the known property that the - C = C -
INTRODUCTION
The cellular toxicity of quinones is partially based on two interrelated properties inherent to these compounds, their redox chemistry ~-3 and their addition chemistry.4 The former feature, in biological terms, implies, on the one hand, the cellular redox activation of quinones by either one- or two-electron transfer flavoproteins yielding semiquinone or hydroquinone derivatives, respectively t'3'~,6 and, on the other, subsequent autoxidation reactions with formation of O2 ~ , H202, and, in some cases, a triplet electronically-excited state of the quinone. 7'8 The substitution pattern of the quinone determines both the redox potential and the electron distribution in the quinonoid structure. These properties 149
150
A. BRUNMARKand E. CADENAS
double bonds of the quinonoid nuclei can be epoxidized. 4 At variance with the p-benzoquinone series, the adjacent benzene ring in the 1,4-naphthoquinone compounds precludes other possible sites for the H202mediated epoxidation. Moreover, the oxidation products of naphthoquinone series are less prone to fragmentation reactions than those of the p-benzoquinone series. 12 We have studied previously 8'~3 some aspects of the O2 addition reactions to quinonoid compounds, namely the oxidation by H202 ofp-benzoquinones and 1,4-naphthoquinones which yields the corresponding quinone epoxide products: 2,3-epoxy-p-benzoquinone and 2,3-epoxy-l,4-naphthoquinone derivatives, respectively. 13 The second order rate constants for the epoxidation reaction is similar for both unsubstituted quinone series, whereas it decreases with the increasing degree of methyl substitution of the quinones.~3 Addition reactions to quinones with sulphur nucleophiles could be characterized as 1,4-reductive additions of the Michael type 4 with formation of, for the case of GSH, the corresponding hydroquinone-glutathionyl conjugate. Recent mechanistic studies carried out with 1,4-naphthoquinone or menadione and GSH under anaerobic conditions suggested that the hydroquinone-thioether adduct underwent oxidation with formation of GSH-conjugated semiquinones intermediates. 14-16 Another recent finding indicates that the redox potential of the menadione-GSH conjugate (around - 0 . 1 9 2 volts) 17does not differ substantially from that of the parent quinone ( - 0.203 volts). ~8Therefore, the products resulting from reductive addition of GSH to quinones may hypothetically undergo autoxidation reactions, thus posing the question whether this feature of 1,4-reductive addition chemistry to quinones--involving thiol compounds--could be a potential source of 02 free radical species, with further deleterious effects on the cell. It was recently pointed that this conjugation reaction might promote cell injury in a fashion which is not linked to oxidative stress, 19,2°that is, without involving an autoxidation component. Thus, the 1,4-reductive addition of GSH to quinones provides new implications to quinone metabolism which might bear an antioxidant and/orprooxidant character. This research is concerned with the 1,4-reductive addition of GSH to quinone epoxides and mechanistic studies point to the reductive cleavage of the epoxide ring to yield as primary molecular product a 2-OHglutathionyl-hydroquinone conjugate. The hydroxy group in position e~, adjacent to the carbonyl group, introduces in the molecule a particular chemical reactivity, distinct from that originating from the GSHmediated reductive addition of the parent quinone compounds lacking the epoxide ring. This feature might broaden the biological relevance--in terms of the tox-
icological aspects--bound to the 1,4-reductive addition of GSH to quinone epoxides. These mechanistic studies were extended to 1,4-naphthoquinone epoxides with different degree of methyl substitution as well as p-benzoquinone epoxides. However, in the latter instances, identification of molecular products was complicated due to the lability of the intermediate compounds and their rapid decomposition to a variety of subproducts. The molecular products originating from the nucleophilic addition of GSH to quinone epoxides were assessed under anaerobic and aerobic conditions. The former approach does not involve autoxidation reactions and allows the evaluation of redox interactions within molecular products. The latter approach permits to determine the involvement of the different molecular products--identified under anaerobic conditions--in autoxidation reactions, that is, one- or two-electron transfer to 02 with formation of O2~ and H202, respectively. This research was carried out mainly by h.p.l.c. with electrochemical detection, taking advantage of the redox properties of all products formed under either anaerobic or aerobic situations. Further, this technique can measure minute amounts of quinones or hydroquinones present in reaction mixtures 8,21,22 and it provides--by means of the corresponding hydrodynamic voltamograms--the half-wave potential of the quinone/hydroquinone couples, a parameter which permits to predict the likelihood of redox interactions between different molecules. MATERIALS AND METHODS
Chemicals, biochemicals, and synthesis of quinone epoxides 1,4-naphthoquinone, 2,3-epoxy-, 1,4-naphthoquinone and 2-OH-l,4-naphthoquinone were obtained from Aldrich Chemical Co. (Steinheim, FRG). Scopoletin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals were from Merck (Darmstadt, FRG) and biochemicals from Boehringer (Mannheim, FRG). 2,3-Dimethyl-2,3-epoxy1,4-naphthoquinone was a gift from Dr. M. D. Threadgill (Pharmaceutical Sciences Institute, Aston University, Birmingham, UK). 2,3-epoxy-p-benzoquinone and 6-methyl-2,3-epoxyp-benzoquinone were prepared as described previously. 23 2-Methyl-2,3-epoxy-l,4-naphthoquinone (menadione epoxide) and 2-methyl-3-OH-1,4-naphthoquinone (phthiocol) were prepared according to the method described by Fieser 24and 2-methyl-3-glutathionyl- 1,4naphthoquinone (thiodione) by the procedure described by Nickerson et al. 25
Quinone epoxides
2-OH-3-glutathionyl-l,4-naphthoquinone was prepared by mixing 0.70-g 1,4-naphthoquinone epoxide dissolved in 14-ml ethanol with 1.23-g GSH dissolved in 70-ml distilled H20 and incubating at 37°C for 5 days. The reaction mixture was extracted with chloroform, lyophilized, and chromatographed on an 5/~m Spherisorb ODS-2 250 mm x 8 mm semi-preparative h.p.l.c, column (Jones Chromatography Ltd, Llanbradach, Mid Glamorgan, UK) with 25% methanol/75% H20 as the eluent. Fractions containing the desired product were lyophilized. The elemental analysis of the synthesised compound was performed by Mikro Kemi AB, Uppsala, Sweden; found: C, 48.8; H, 4.4; N, 8.7; S, 6.0, in agreement with that calculated for C20H22N309S: C, 50.0; H, 4.6; N. 8.7; S, 6.7. 3-Glutathionyl-l,4-naphthoquinone was prepared by the method designed by Nickerson et a1.25for thiodione and the product was purified from its corresponding diconjugate on the preparative chromatographic system described above, but with 40% methanol/60% H20 as the mobile phase. The elemental analysis data: found: C, 50.3; H, 4.8; N, 8.8; O, 27.3; S, 7.5, in agreement with that calculated for C20H22N308S: C, 51.7; H, 4.8; N, 9.0; O, 27.6; S, 6.9. Assay conditions
All reactions were carried out in 0.1 M potassium phosphate buffer, pH 7.65, containing 100--or 500 aM p-benzoquinone--or naphthoquinone epoxides, respectively, and equimolar amounts of GSH. Assay temperature was 37°C. Anaerobic conditions were obtained by purging the system with helium for 10 min and the reaction mixture was kept under helium atmosphere during the reaction period.
151
spectromonitor D (LDC, Riviera Beach, Florida, USA), operated at h = 220 nm, was connected in series with the electrochemical detector. Injection volume was 20 /tl. The mobile phases were selected in order to obtain optimal separation of the quinonoid derivatives studied and were as follows: (a) A mobile phase consisting of 35% 2-propanol/ 65% H20, buffered to pH 6.5 with 50 mM sodium phosphate, was used for the evaluation of 2,3dimethyl-2,3-epoxy- ! ,4-naphthoquinone, 2,3-epoxy-pbenzoquinone, and 6-methyl-2,3-epoxy-p-benzoquinone. Flow rate was 0.6 ml x min-~. (b) A modification of the mobile phase described by Hernandez et al. z6 was used. A buffer solution was made by diluting 5 ml of phosphoric acid to 1 litre with H20 and the pH was adjusted to 7.0 by addition of Tris-base. 10% of this buffer, 25% methanol, and 65% H20 containing 50 mM sodium sulphate was used as mobile phase for the characterization of 2,3-epoxy1,4-naphthoquinone,2-OH-3-glutathionyl- 1,4-naphthoquinone, 3-glutathionyl-1,4-naphthoquinone, and 2OH-1,4-naphthoquinone. Flow rate was 1.0 ml x min- ~. (c) A mobile phase similar to that described above but with a methanol content increased to 37% at expense of H20 was used for the evaluation of 2-methyl2,3-epoxy- 1,4-naphthoquinone, 2-methyl-3-OH- 1,4naphthoquinone, and 2-methyl-3-glutathionyl-l,4naphthoquinone. Flow rate was 1.0 ml x min -~. All the mobile phases were carefully degassed in vacuo, followed by at least 1 h of helium purging prior to analysis in order to eliminate oxidation by 02 of reduced products during h.p.l.c, runs.
H.p.l.c. with electrochemical detection
The concentration of quinones, quinone epoxides, and hydroxy- and glutathionyl quinone derivatives was measured by h.p.l.c, with either reductive or oxidative electrochemical detection. A 150 mm x 3.9 mm Novapak 4 / t m C18 reverse-phase column (Waters-Millipore AB, V. Fr61unda, Sweden) with an Uptight 20 mm × 2 mm C- 18 precolumn (Skandinaviska Genetec, Kungsbacka, Sweden) was connected to a Constametric III pump (LDC, Riviera Beach, Florida, USA). The eluent was monitored by an electrochemical detector unit consisting of a LC-4 potentiostat with a TL5A three electrode glassy carbon-flow cell and an Ag/ AgCI reference electrode (BAS, West Lafayette, IN, USA) operated at either - 0 . 9 volts (reductive mode) or +0.9 volts (oxidative mode). In experiments with 2,3-dimethyl-2,3-epoxy-l,4-naphthoquinone a LDC
02 uptake
02 consumption was measured in a thermostated cuvette filled with a Clark-type 02 electrode (Hansatech Ltd., Norfolk, UK).
H202 measurements H202 was measured with the scopoletin method27 as reduction of fluorescence intensity (hexcitation ---- 360 nm; hemission ---- 460 nm) on a Shimadzu 540 spectrofluorimeter (Shimadzu Corporation, Kyoto, Japan). Total GSH and GSSG measurements
Total GSH, expressed as glutathione equivalents, was measured as described in z8 with a Varian DMS-
152
A. BRUNMARKand E. CADENAS
100 spectrophotometer (Varian AB, Solna, Sweden). Samples were either diluted five-fold with distilled H20 and stored for a short time on ice or analyzed directly after withdrawal from the reaction mixture. GSSG was measured following NADPH oxidation ( E . 3 4 0 a m : 6.22 mM-Jcm-]). 28 RESULTS AND DISCUSSION
I. H.p.l.c. with electrochemical detection properties of 1,4-naphthoquinone- and menadione epoxide and hydroxy- and glutathionyl-substituted 1,4-naphthoquinones H.p.l.c. with electrochemical detection was used for the evaluation of products arising from the 1,4-nucleophilic addition of GSH to un- and methyl-substituted 1,4-naphthoquinone epoxides. This method is based on the redox properties retained in the molecular products originating from the above reaction and allows detection of minute amounts of products. 8'2~'22 Figure IA shows a chromatogram of the standards (see Materials and Methods section) used for the evaluation of the reaction between 2,3-epoxy-l,4naphthoquinone and GSH: 2-OH-3-glutathionyl-l,4naphthoquinone (peak a) was eluted first, followed by
the 2-OH- 1,4-naphthoquinone (lawsone) (peak b). The 3-glutathionyl-l,4-naphthoquinone adduct (peak c) was retained more strongly despite the hydrophilic character of the glutathionyl moiety; this may be due to the formation of ionic pairs between the compound and certain components in the mobile phase. Peak d corresponds to 2,3-epoxy- 1,4-naphthoquinone. Retention times (R,) and half-wave potentials (El/2) of the above quinonoid derivatives are listed in Table 1. Figure IB shows a chromatogram of the hydroxyand glutathionyl-adducts of the monomethyl-substituted 1,4-naphthoquinone (menadione) and of menadione epoxide. 2-Methyl-3-OH-1,4-naphthoquinone was eluted first followed by 2-methyl-3-glutathionyl-l,4naphthoquinone and finally--with the longest retention time--2-methyl-2,3-epoxy-1,4-naphthoquinone or menadione epoxide. Retention times (Rr) and half-wave potentials (Ej/2) are summarized in Table 1. It should be noted that a decreasing half-wave potential was observed in both series (unsubstituted and methyl-substituted naphthoquinones) with the following order: glutathionyl-substituted derivatives > hydroxy-substituted derivatives > quinone epoxides. This is illustrated in Figure 2 with the hydrodynamic voltamograms of 3-glutathionyl-l,4-naphthoquinone
O
(2@ °
SG
O [2 nn'l~s] O
O
O
0 o
I
2 1 ~ nA
o
SG 0 14 amok~]
B O
0 [4 nmalcsl
I~miami
;
O
o
O
Time (rain) Fig. 1. H.p.l.c. with reductive electrochemical detection chromatograms of 1,4-naphthoquinone- and menadione epoxides and hydroxy- and glutathionyl-substituted naphthoquinones. Assay conditions and synthesis of standard compounds as described in the Materials and Methods section. (A). Chromatogram of 1,4-naphthoquinone derivatives: (a) 2-OH-3-glutathionyl-1,4-naphthoquinone; (b) 2-OH-1,4-naphthoquinone; (c) 3-glutathionyl-1,4-naphthoquinone; (d) 2,3-epoxy-1,4-naphthoquinone. (B). Chromatogram of menadione derivatives: (a) 2-methyl-3-OH-1,4-naphthoquinone; (b) 2-methyl-3-glutathionyl-1,4-naphthoquinone; (c) 2-methyl-2,3-epoxy- 1,4-naphthoquinone. Applied potential - 0.9 volts.
Quinone epoxides
(1) Studies under aerobic conditions. Figure 3A shows the chromatogram corresponding to a reaction mixture containing 2,3-epoxy- 1,4-naphthoquinone obtained with h.p.l.c, with reductive electrochemical detection. The chromatogram obtained after 10-min incubation of the quinone epoxide with equimolar amounts of GSH (Fig. 3B) showed a large decrease in the intensity of the peak d corresponding to 2,3-epoxy-l,4-naphthoquinone and formation of three main products: peaks a, b, and c co-chromatographed with the standards shown in Figure 1A and possessed the same El/2 values listed in Table 1 and were identified as 2-OH-3-glutathionyl1,4-naphthoquinone, 2-OH-l,4-naphthoquinone, and 3-glutathionyl-l,4-naphthoquinone, respectively. The peak between peaks a and b in Figure 3B was not identified but it would correspond probably to a glutathione-diconjugate of 1,4-naphthoquinone. Analysis of the spent reaction mixture with oxidative applied potential (Fig. 3C) did not show significant amount of reduced products, except for a peak with a short retention time. This peak remained unaffected upon oxygenation and, therefore, we cannot ascribe it to a reduced form of any quinone. Figure 4 shows the time courses of GSH and 2,3epoxy-l,4-naphthoquinone consumption as well as product formation. Both reactants, GSH and 2,3-epoxy1,4-naphthoquinone were consumed at initial rates of about 240 I~M x min- ~, equivalent to a second-order rate constant of about 16 M-%-~. The total amount of GSH consumed was slightly lower than the amount of 2,3-epoxy-l,4-naphthoquinone consumed during the reaction ([GSH] . . . . . . . d / [quinone epoxide] ..... n~ = 0.93).
Table 1. H.p.l.c. with reductive electrochemical detection characteristics of un- and methyl-substituted 1,4-naphthoquinone derivatives Retention Time Rt(min)
Half-Wave Potential Etn(-volts)
1.8
0.68
4.6 10.1 17.8
0.55 0.22 0.72
2.3 4.1
0.50 0.30
13.3
0.79
Unsubstituted naphthoquinone derivatives 2-OH-3-glutathionyl-
1,4-naphthoquinone 2-OH-1,4-naphthoquinone 3-glutathionyl-1,4-naphthoquinone 2,3-epoxy-1,4-naphthoquinone Menadione derivatives 2-methyl-3-OH-1,4-naphthoquinone 2-methyl-3-glutathionyl1,4-naphthoquinone 2-methyl-2,3-epoxy1,4-naphthoquinone
153
Note. H.p.l.c. mobile phases used are specified in the Materials and Methods section.
(E,/2 0.22 volts), 2-OH-l,4-naphthoquinone (Ej/2 = - 0 . 5 5 volts), and 2,3-epoxy-l,4-naphthoquinone (E1/2 = - 0 . 7 2 volts).
H. GSH-mediated 1,4-reductive addition to 2,3-epoxy- 1,4-naphthoquinone The GSH-mediated 1,4-reductive addition to 2,3-epoxy-l,4-naphthoquinone was studied under aerobic- and anaerobic conditions in order to assess the importance of autoxidation reactions and potential interaction of secondary molecular products.
1.0
O
O
O °
$O o
0.5
0.2
0.4
0.6
01~
,i0
Appli~l volt~e (- ,,,olu)
Fig. 2. Hydrodynamic voltamograms of 3-glutathionyl-l,4-naphthoquinone, 2-OH-1,4-naphthoquinone, and 2,3-epoxy-l,4naphthoquinone. Assay conditions and synthesis of standards as described in the Materials and methods section.
154
A. BRUNMARK and E. CADENAS
ISOOnA
A
GSH, 85% (425/zM) quinone epoxide was consumed and the following quantitative distribution of molecular products was found: 51% (216 ktM) 2-OH-3-glutathionyl- 1,4-naphthoquinone, 20% (86/~M) 2-OH- 1,4naphthoquinone, and 22% (95/zM) 3-glutathionyl-1,4naphthoquinone (Table 2). The GSH-mediated reductive addition to 2,3-epoxy1,4-naphthoquinone was associated with 02 consumption, which was accounted for in terms of H202 formation (Fig. 5). The initial rates of 02 consumption and H202 formation were about 94- and 98 ltM × min -1, respectively (( - d[Oz]/dt)/(+ d[H202]/dt) 1). The distribution of molecular products found with h.p.l.c, with electrochemical detection (Fig. 3B) as well as the associated autoxidation reactions can be accounted for in terms of (a) a main reaction consistent with the reductive nucleophilic addition of GSH to the naphthoquinone epoxide and (b) secondary pathways involving elimination reactions and redox transitions.
-'V" 0
10
20
i
B
c SOOnA
I
SOOmA
b
1
0
10
20
0
5
10
(a) Main reaction: 1,4-reductive nucleophilic addition of GSH to quinone epoxide. The 1,4-reductive addition of GSH to 2,3-epoxy-l,4-naphthoquinone results in epoxide ring opening and formation of the primary molecular product 2-OH-3-glutathionyl-l,4-naphthohydroquinone (reaction 1).
Time (rain)
Fig. 3. H.p.l.c. with electrochemical detection analysis of products from the reaction of 2,3-epoxy-1,4-naphthoquinone with GSH under aerobic conditions. Assay conditions as described in the Materials and methods section. (A) H.p.l.c. with reductive electrochemical detection (applied potential - 0 . 9 volts) chromatogram of 500/tM 2,3-epoxy-l,4-naphthoquinone. (B) H.p.l.c. with reductive electrochemical detection chromatogram of 500 pM 2,3-epoxy- 1,4-naphthoquinone after 10-min incubation with equimolar amount of GSH. (C) H.p.l.c. with oxidative electrochemical detection (applied potential + 0.9 volts) chromatogram of the spent reaction mixture described in (B). Labelled peaks were identified as: (a) 2-OH-3-glutathionyl-1,4naphthoquinone; (b) 2-OH-1,4-naphthoquinone; (c) 3-glutathionyl1,4-naphthoquinone; (d) 2,3-epoxy-1,4-naphthoquinone. Peak (a) in Figure 3C could not be ascribed to a reduced form of any quinone; see explanations in the text.
The spent reaction mixture contained about 30 /tM GSSG, probably arising from the oxidation of GSH by H202, a reaction that has been reported29 in order to account for GSSG production during the interaction of GSH with menadione. The time courses of molecular product formation are illustrated in Figure 4B, C, and D for 2-OH-3glutathionyl-1,4-naphthoquinone, 2-OH- 1,4-naphthoquinone, and 3-glutathionyl-l,4-naphthoquinone, respectively. After 10-min incubation of 500 ~M 2,3epoxy- 1,4-naphthoquinone with equimolar amounts of
c+° O
+ GS-
--
O O2H +
(1)
O (b) Secondary reactions: elimination reactions and redox transitions. The secondary reactions give rise to the molecular products identified by h.p.l.c, with reductive electrochemical detection shown in Figure 3B. These involve elimination reactions and redox transitions. Elimination reactions. Peak c in Figure 3B co-chromatographed with the 3-glutathionyl-1,4-naphthoquinone standard (Fig. 1A). Its formation can be rationalized in terms of an elimination reaction as illustrated in reaction 2 and as previously proposed for the inter-
155
Quinone epoxides Table 2. Relative contribution of molecular products during the aerobic- and anaerobic GSH-mediated reductive addition to 2,3epoxy- 1,4-naphthoquinone
0
O
0
o Aerobiosis Anaerobiosis
C so
0
0
O
51 43
20 29
22 --
-29
O°-
+ 2H+ SG O 0
+ OHSG
(2)
0
Redox transitions. The primary molecular product, 2-OH-3-glutathionyl- 1,4-naphthohydroquinone (as shown in reaction 1) can transfer electrons to either unreacted 2,3-epoxy- 1,4-naphthoquinone (reaction 3) or 02 (reaction 4). The former reaction is accompanied by the opening of the epoxide ring and is similar to the two-electron enzymatic reduction catalyzed by DT-diaphorase. 3~ The molecular products are 2-OH-3-glutathionyl-l,4naphthoquinone (identified as peak a in the chromatogram in Fig. 3B) and 2-OH-naphthohydroquinone (reduced lawsone).
the bulk of quinone epoxide is consumed via reaction 1, whereas a part of it is consumed in redox transitions within the frame of reaction 3. This is in agreement with the observed slight excess of quinone epoxide consumed over GSH consumed (Fig. 4A, B). The latter reaction, 02 consumption and consequent H 2 0 2 formation (Fig. 5), is accounted for by further redox transitions represented by electron transfer to 02 from either the 2-OH-3-glutathionyl-l,4-naphthoquinone (reaction 4) or 2-OH-l,4-naphthohydroquinone (reaction 5) (the latter compound being a product of reaction 3). 2-OH-1,4-naphthoquinone, a product of reaction 5, is identified as peak b in the chromatogram of Figure 3B. O+
02
+2H +
O-
~
O O- + H202 SG
(4)
+ H202
(5)
O O"
O
°+ O2+2H +
O SG O-
SG
0
action of l-phenyl-lH-tetrazole-5-thiol with substituted 2,3-epoxy- 1,4-naphthoquinones. 3°
O
o
O-
O
0 0
0-
+ SG O
+ H+ (3)
O
O-
Reactions 1 and 3 represent two different pathways for quinone epoxide consumption. It is assumed that
It is suggested that reactions 4 and 5 represent the main autoxidation reactions associated with the GSHmediated nucleophilic addition to 2,3-epoxy-l,4-
156
A. BRUNMARKand E. CADENAS
A
B 50~
!N)"
500
Y
|
==
125
2so ;/3
!
[ |
o
,.4
Time(rain)
Time(min)
0 150
150
o
100 ,3
¢
..=
f~
0
Time(mini
Time(rain)
Fig. 4. Time courses for reactant consumption and product formation during the reaction of 2,3-epoxy-1,4-naphthoquinone with GSH under aerobic and anaerobic conditions. Assay conditions as described in the Materials and methods section. Open and closed symbols correspond to assays carried out under anaerobic and aerobic conditions, respectively. (A) Consumption of total glutathione equivalents; (B) Consumption of 2,3-epoxy-1,4-naphthoquinone and formation of 2-OH-3-glutathionyl-1,4-naphthoquinone product. (C) Formation of 2-OH-1,4-naphthoquinone product. (D) Formation of 3-glutathionyl-1,4-naphthoquinone and its reduced counterpart.
naphthoquinone. Most likely, electrons are transfered directly to O2 from 2-OH-3-glutathionyl-1,4-naphthohydroquinone and 2-OH- 1,4-naphthohydroquinone. This is based on the observation that the addition of 100 /iM 2-OH- 1,4-naphthoquinone or 3-glutathionyl- 1,4naphthoquinone to a reaction mixture consisting of GSH plus 2,3-epoxy-l,4-naphthoquinone did not increase significantly the rate of 02 uptake by the system. This could be the expected if the above compounds were required intermediates for electron transfer to 02. Reactions 4 and 5 are written as a two-electron transfer to O2 with direct formation of H202. It is likely though, that production of HzO2 (Fig. 5) proceeds in two one-electron transfer steps with formation of glutathione-semiquinone intermediate ~4-16and 02 ~. However, we cannot provide ESR evidence for the occurrence of a glutathione-semiquinone intermediate and
reactions 4 and 5 reflect solely the results summarized in Figure 5. The relative contribution of the autoxidation reactions (reactions 4 and 5) to H202 formation could be analyzed on the basis of the quantitative data presented in Figs. 4 and 5 and summarized in Table 2. About 190/IM H202 is formed during the reductive addition of GSH to 2,3-epoxy-l,4-naphthoquinone; approximately 86 ~M 2-OH-1,4-naphthoquinone is detected in the spent reaction mixture and this would correspond (according to reaction 5) to an equimolar amount of H202. The remaining H202 should be formed out of the autoxidation of 2-OH-3-glutathionyl-1,4-naphthohydroquinone (reaction 4), that is, about 104 ,uM. Because about 216 /tM 2-OH-3-glutathionyl-l,4naphthoquinone is detected in the reaction mixture, the remaining product should originate from the redox
Quinone epoxides
.~:~:ZZ ............................................................................. 200
v
loo
J
0
10
T
20
Time (min) Fig. 5. Time courses of 02 consumption and H,_O2 formation upon supplementation of 2,3-epoxy-l,4-naphthoquinone epoxides with GSH. Assay conditions as described in the Materials and methods section. --., ---, and - - indicate O: consumption for 2,3-dimethyl2,3-epoxy-l,4-naphthoquinone, 2-methyl-2,3-epoxy-l,4-naphthoquinone, and 2,3-epoxy-l,4-naphthoquinone, respectively....e..., --e--, and - e - indicate H20: production for 2,3-dimethyl-2,3-epoxy1,4-naphthoquinone, 2-methyl-2,3-epoxy-l,4-naphthoquinone, and 2,3-epoxy- 1,4-naphthoquinone, respectively.
157
to 3-glutathionyl-l,4-naphthoquinone was also observed. Analysis of the spent reaction mixture with oxidative electrochemical detection (Fig. 6B) showed a main peak (peak c) corresponding to 3-glutathionyl-1,4-naphthohydroquinone, the compound with the highest redox potential in the system (Table 1). The intensity of this peak decreased instantly and was abolished completely following oxygenation of the reaction mixture. Similarly to what commented for peak a in Figure 3C, we cannot ascribe the analogous peak in Figure 6B to a reduced form of any quinone and, accordingly, this peak remained unaffected upon oxygenation. The time courses of GSH and 2,3-epoxy-l,4naphthoquinone consumption as well as product formation under anaerobic conditions are summarized in Figure 4. The total amount of GSH consumed during the reductive addition to 2,3-epoxy-l,4-naphthoquinone did not change appreciably (Fig. 4A) in comparison with experiments carried out under aerobiosis, whereas the total amount of quinone epoxide consumed exceeded that observed in the presence of 0 2 (A[quinone epoxide] ...... bio~iJA[quinone epoxide]acrobio~ = 1.15) (Fig. 4B). Moreover, under anaerobic conditions, 2,3epoxy- 1,4-naphthoquinone was consumed in large ex-
I
5OO n A
transition shown in reaction 3. In summary, the 216 ~uM2-OH-3-glutathionyl- 1,4-naphthoquinone detected in the reaction mixture should be formed with similar efficiency from [a] redox transition with unreacted epoxide (providing about 112 pM; reaction 3) and [b] autoxidation reactions (providing about 104 pM; reaction 4). (2) Studies under anaerobic conditions. The study of the GSH-mediated nucleophilic addition to 2,3-epoxy1,4-naphthoquinone under anaerobic conditions brings about the following advantages: it precludes electron transfer to 02 and, thereby, it facilitates the evaluation of possible interactions within the molecular products. H. p. l.c. with reductive electrochemical detection of the spent reaction mixture carried out under anaerobic conditions showed two peaks (Fig. 6A): peak a and b co-chromatographed with the standards shown in Figure 1A and corresponded to 2-OH-3-glutathionyl-1,4naphthoquinone and 2-OH-1,4-naphthoquinone, respectively. A very small peak (peak c) corresponding
_._3,_ 6
~
5OOnA
A
~ 1'o
2~
o
5
10
Time (min) Fig. 6. H.p.l.c. with electrochemical detection chromatograms of the reaction between 2,3-epoxy-1,4-naphthoquinone and GSH under anaerobic conditions. Assay conditions as described in the Materials and methods section. Figure shows chromatograms obtained after 10-min incubation of 500 pM 2,3-epoxy-l,4-naphthoquinone and equimolar amounts of GSH under anaerobic conditions analyzed with reductive (A) and oxidative (B) applied potential ( - 0 . 9 volts and + 0.9 volts, respectively). Labelled peaks [a-d] are the same as in Figure 3 and co-chromatograph with the standards in Figure IA.
158
A. ]3RUNMARKand E. CADENAS
cess over GSH: (A[quinone epoxide] - A[GSH] = 108/~M). Under anaerobic conditions 97% (486 ItM) of 2,3epoxy-1,4-naphthoquinone was consumed, whereas 76% (378/zM) GSH was consumed. This uneven molar ratio for consumption of reactants suggested the interaction of derived molecular products with 2,3-epoxy-l,4naphthoquinone which had not reacted with the thiol. The quantitative distribution pattern of products under anaerobiosis was as follows: 43% (211 /~M) 2-OH-3glutathionyl-l,4-naphthoquinone, 29% (145 /tM) 2OH-1,4-naphthoquinone, and 29% (142 ltM) 3-glutathionyl- 1,4-naphthohydroquinone. The differences between the distribution of molecular products under anaerobiosis and aerobiosis during the thiol-mediated reductive addition of 2,3-epoxy-l,4naphthoquinone are listed in Table 2. The excess of 2,3-epoxy-l,4-naphthoquinone consumed over that of GSH under anaerobic conditions could be interpreted as reaction 3 prevailing over reaction 4, for autoxidation reactions are negligible under these conditions. The molecular mechanism for the formation of the products identified during the GSH-mediated reductive addition to 2,3-epoxy-l,4-naphthoquinone under anaerobiosis, that is, 2-OH-1,4-naphthoquinone and 2OH-3-glutathionyl-l,4-naphthoquinone, is implied in reaction 1 followed by redox transitions as that of reaction 3.3-Glutathionyt- 1,4-naphthoquinone would be formed through an elimination reaction similar to reaction 2. This compound would display a main oxidant activity, substituting for 02 (as in reactions 4 and 5)
in the oxidation of reduced lawsone (reaction 6) or 2OH-3-glutathionyl-l,4-naphthohydroquinone (Fig. 6B). O-
O
O-
O
SG
O
O(6)
O
O-
111. GSH-mediated nuclephilic addition to methyl-substituted naphthoquinone epoxides The h.p.l.c, with reductive electrochemical detection chromatogram of 500 /~M 2-methyl-2,3-epoxy1,4-naphthoquinone is shown in Figure 7A (peak a) and that of the spent reaction mixture (after 20-min incubation of menadione epoxide with equimolar amounts of GSH) in Figure 7B. The peak corresponding to 2-methyl-2,3-epoxy-l,4-naphthoquinone decreased substantially upon incubation of the methylsubstituted quinone epoxide with GSH resulting in the formation of a compound with a shorter retention time and more hydrophilic character (peak b; Fig. 7B). Peak b was identified as 2-methyl-3-glutathionyl-1,4naphthoquinone (thiodione), which was the sole molecular product in this reaction. H.p.l.c. with oxidative
A
B
1000 nA
5
10
15
0
5
1'0
1'5
Time (min) Fig. 7. H.p.l.c. with electrochemical detection analysis of products originating from the 1,4-nucleophilic addition of GSH to 2-methyl-2,3-epoxy-l,4-naphthoquinone or menadione epoxide under anaerobic conditions. Assay conditions as described in the Materials and methods section. (A) Chromatogram obtained with reductive applied potential (-0.9 volts) of a solution containing 500/tM 2,3-epoxy-2-methyl-l,4-naphthoquinone(peak a). (B) Chromatogramobtained with reductive applied potential of a solution of 500/tM 2-methyl-2,3-epoxy-l,4-naphthoquinoneafter 20-min incubation with equimolar amounts of GSH: peak a decreases at expense of product formation (= peak b, identified as 2-methyl-3-glutathionyl-l,4-naphthoquinone (thiodione) after comparison with standards in Fig. 1B).
Quinone epoxides
electrochemical detection did not reveal any reduced quinonoid compound even under anaerobic conditions (not shown). Figure 8 illustrates the time course for menadione epoxide and GSH consumption and concomitant formation of the molecular product 2-methyl-3-glutathionyl-l,4-naphthoquinone (thiodione), which was produced in equimolar amounts. 2-Methyl-2,3-epoxy1,4-naphthoquinone or menadione epoxide and GSH react with an initial rate of about 123 ,uM x min -l, that is, at half the rate observed with the unsubstituted 1,4-naphthoquinone epoxide (Fig. 4A,B). Menadione epoxide was consumed at identical rates regardless whether the assay was carried out under aerobic- or anaerobic conditions. Reaction 7 intends to rationalize the findings observed during the reductive addition of GSH to menadione epoxide over an intermediate which undergoes a rapid elimination reaction to yield 2-methyl-3-glutathionyl-l,4-naphthoquinone. The latter compound corresponds to peak b in Figure 7B and represents the main or sole molecular product. The addition of nbutanethiol to menadione epoxide gave a similar product, that is, 2,n-butylthio-3-methyl-l,4-naphthoquinone or menadione-thioether. 32 O
OH
0
0
~
O CH3 +
OH-
(7)
SG O
In accordance with the mechanism illustrated in reaction 7, autoxidation reactions with formation of H202 (reactions 4 and 5 above) would be of little significance. Consequently, the amount of 02 consumed and H202 produced during the GSH-mediated nucleophilic addition to 2-methyl-2,3-epoxy-1,4-naphthoquinone was negligible in comparison with that observed with the unsubstituted naphthoquinone epoxide (Fig. 5). At the moment we cannot provide a satisfactory explanation for the formation of H202; it is clear that mechanisms other than that comtemplated in reaction 7 may operate as a source of H202 during the above reaction. A small amount of GSSG (about 10/~M) was found in the reaction mixture. It is likely that redox transitions involving glutathionyl radicals, as those proposed by Ross et a1.,29 would be operative in this system. Of
159
| E,h e~
0
110
210
Time(min) Fig. 8. Time courses for reactant consumption and product formation during the l-4-reductive addition of GSH to 2-methyl-2,3epoxy- 1,4-naphthoquinone or menadione epoxide. Assay conditions as described in Fig. 7 and in the Materials and methods sections. Points correspond to samples withdrawn at different intervals from the ongoing reaction mixture.
note, however, is that these reactions should be considered secondary to the primary thiol-mediated reductive addition to menadione epoxide described in Figures 7 and 8 and rationalized in reaction 7. 2,3-Dimethyl-2,3-epoxy-l,4-naphthoquinone reacted with GSH at a rate that was far slower (18 ItM x min- l) than that of other epoxides in the naphthoquinone series. The rate of consumption of GSH was higher than that of quinone epoxide (not shown). The system did not interact with 02 as could be seen from the total absence of O2 consumption and H202 production (Fig. 5). A UV detector, connected in series with the electrochemical detector, showed a signal which increased in intensity with time during the reaction (not shown). This signal appeared close to the void volume, which suggests the presence of a hydrophilic molecule, probably containing the glutathionyl rest. No products, however, were detected by h.p.l.c, with electrochemical analysis, indicating the formation of a compound which did not possess the redox properties required for its evaluation with this method, most likely due to loss of the quinonoid structure of the product. The loss of the quinonoid structure can be understood as a consequence of a change in the electron configuration in the molecule produced upon GSH conjugation. To regain a quinonoid structure two substituents on the quinone ring would be required to leave, that is, hydroxyl
160
A. BRUNMARK and E. CADENAS
anion (HO-) and glutathionyl anion ( G S ) , which would be equivalent to a four-electron oxidation. The hypothetical molecule possesses no reducing equivalents and, thus, it will not undergo rearrangement into a quinonoid structure. A speculative mechanism is described in reaction 8, where the product should be considered as an intermediate compound which may undergo further rearrangements involving scission between C2 and C3. 0
0 +
v
GS-
(8)
"1]" CH3 O
~k~cHSG O
O" H 3 C ~ O" ~+ G S "
IV. Reaction of un- and methyl-substituted p-benzoquinone epoxides with GSH The reductive addition of GSH to p-benzoquinone epoxides was studied with 2,3-epoxy-p-benzoquinone and 6-methyl-2,3-epoxy-p-benzoquinone. Figure 9A, B shows the time courses for GSH and p-benzoquinone epoxide consumption. 2,3-Epoxy-pbenzoquinone was consumed at a rate 10-fold higher than 6-methyl-2,3-epoxy-p-benzoquinone (117- and 1 ! /~M × min- ~, respectively). This large difference may be due to the electron-donating properties of the methyl substituent in 6-methyl-2,3-epoxy-p-benzoquinone, which would decrease considerably its rate of reaction with GSH. Reaction 9 exemplifies the mechanism for the 1,4-reductive addition of GSH to p-benzoquinone epoxides. O H3C~o +
GS-
O O-
H3C ~
two additions each followed by oxidation. 4 The excess of GSH consumed cannot be accounted for in terms of GSSG formation. The latter observation may indicate secondary reactions (secondary to the initial 1,4-reductive addition of GSH to p-benzoquinone epoxide; reaction 9), probably comprising a redox transition between the hydroxy-p-benzohydroquinone-thioether and p-benzoquinone epoxide (reaction 10). This reaction (similar to reaction 3) would lead to epoxide ring opening yielding an unconjugated 2-OH-p-benzohydroquinone, a compound shown to possess strong autoxidation properties? ~ O H 3 C ~
O"
O
O O
O-
GS - "-~ O
O-
Consequently, the overall addition reaction of GSH to p-benzoquinone epoxides was associated with a substantial consumption of 02, which was largely accounted for as H202 production (Fig. 9C). A lag phase was observed to precede the maximal rate of 02 uptake and H202 production, thus suggesting that electrons were mainly transferred to 02 by an intermediate accumulating in the reactioo mixture. The hydroxy-hydroquinone derivatives resulting from secondary interactions, as that illustrated in reaction 10, seem to support the autoxidation features of this system, unlike the stable p-benzohydroquinone conjugate 8,~9 formed during the reductive addition of GSH to p-benzoquinone lacking an epoxide ring.
O'+ 2H+
(9)
GS" " ( / O-
The time courses in Figure 9A, B also indicate that (a) the amount of GSH consumed is higher than that of either p-benzoquinone epoxide tested and (b) the consumption of the unsubstituted p-benzoquinone epoxide still proceeds after GSH has been totally consumed. The former observation may indicate a second site of conjugation, thus involving the usual sequence of
CONCLUSIONS AND COMMENTS The 1,4-reductive addition of GSH to the unsubstituted quinone epoxides (from either the p-benzo- or 1,4-naphthoquinone epoxide series) studied here implies predominantly epoxide ring opening and leads primarily to the formation of a hydroxy-glutathionylhydroquinone derivative. Further reactions following the primary formation of a 2-OH-3-glutathionyl-hydroquinone derivative are determined by (a) the degree of methyl substitution of the quinone epoxide and (b) the 02 concentration in the reaction mixture, parameters which are by no means
Quinone epoxides A
161 C
B
100,
100
]
[,,0
,.v
l
0
150L
10
20
0
10
20
0
0
10
20
Tlrlae (rain)
Fig. 9. Time courses of reactant consumption, 02 consumption, and HzO2 formation during reaction between GSH and un- or methyl-substituted p-benzoquinone epoxide. Assay conditions as described in the Materials and methods section. (A) Consumption of 2,3-epoxy-p-benzoquinone ( 0 ) or 6-methyl-2,3-epoxy-p-benzoquinone ( • ) during its reaction with GSH~ (B) Consumption of GSH during its reaction with 2,3-epoxy-p-benzoquinone ( 0 ) or 6-methyl-2,3-epoxy-p-benzoquinone ( 0 ) . (C) O2 uptake (traces) and H20: (symbols) production during the nucleophilic addition of GSH to 2,3-epoxy-p-benzoquinone ( - - and 0 ) and 6-methyl-2,3-epoxy-p-benzoquinone (--- and • ) .
independent of each other. They will both influence to different extent the interaction of secondary products leading to an ultimate pattern of molecular product distribution, which is characteristic for every quinone epoxide studied.
(a) Influence of the degree of methyl substitution on the 1,4-reductive addition of GSH to quinone epoxides The rate of the 1,4-reductive addition of GSH to pbenzoquinone- and 1,4-naphthoquinone epoxides decreases with increasing degree of methyl substitution of the quinone epoxide (Table 3). Parent quinones (lacking epoxide groups) react more rapidly with GSH than those containing an epoxide ring. Similar considerations apply to the differences found between the 1,4-naphthoquinone- and p-benzoquinone epoxide seTable 3. Initial rates of quinone epoxide consumption upon nucleophilic attack by GSH. Dependence on degree of methyl substitution in the quinone epoxide molecule -d[Quinone epoxide]/dt ,uM x rain -t 1,4-Naphthoquinone series 2,3-epoxy- 1,4-naphthoquinone 2-methyl-2,3-epoxy! ,4-naphthoquinone 2,3-dimethyl-2,3-epoxy1,4-naphthoquinone p-Benzoquinone series 2,3-epoxy-p-benzoquinone 6-methyl-2,3-epoxy-p-benzoquinone
240 123 18 117 11
Note. Assay conditions as described in the Materials and Methods section.
ries, for the p-benzoquinone offer a - C ~ C - double bond opposite to the site possessing the epoxy group. The former (with a lower electron density) will be a more favorable site for the nucleophilic addition than the latter (with a higher electron density). The decreased rate of 1,4-reductive addition of GSH to quinone epoxides with increasing degree of methyl substitution can be interpreted on the basis of the substituent in C2: the electron-donating properties of the - C H 3 groups will decrease accordingly the electrophilicity in C3 and, hence, its ability to react with GSH. A hydroxy substituent in C:, with a high electron density, such as that in lawsone or 2-OH-1,4-naphthoquinone, will prevent definitely the nucleophilic addition of GSH. 25 We have previously shown that the increasing degree of methyl-substitution of quinone epoxides was related to a decrease in the half-wave potential value. ~3 It could be expected that the redox potential of a quinone epoxide would influence the rate of reaction of the latter with GSH, assuming the requirement of an intermediate electron transfer complex within the reaction. However, it is difficult at the moment to draw a conclusive relationship between, on the one hand, the rate of GSH-mediated 1,4-reductive addition to naphthoquinone epoxides with different degree of methyl substitution and, on the other, their corresponding redox potential (as reflected by the half-wave potential13). Preliminary approaches, though, indicate that the rate of 1,4-reductive addition decreases exponentially with the decrease of the redox potential of the quinone epoxide. In summary, it is proposed that the rate of addition
162
A. BRUNMARKand E. CADENAS
of a quinone or quinone derivative with a sulphur nucleophile (GS-) will decrease in following order: quinone > methyl-substituted quinone > quinone epoxide > methyl-substituted quinone epoxide.
and 5). These relationships are summarized in the scheme in Figure 10. The absence of 02 in the system will prevent autoxidation of the primary molecular product (2-OH-3glutathionyl-l,4-naphthohydroquinone). This will be reflected in the prevailance of elimination reactions (reaction 2) and redox transitions with unreacted naphthoquinone epoxide (reaction 3) with respective formation of 3-glutathionyl-l,4-naphthoquinone and 2-OH-1,4-naphthohydroquinone (reduced lawsone). Under these circumstances, 3-glutathionyl- 1,4-naphthoquinone will substitute for 02 in reactions 4 and 5 as the ultimate oxidizing species, because it possesses the highest redox potential among the quinonoid compounds present in the system. Its reaction with both 2OH-3-glutathionyl- 1,4-naphthohydroquinone and 2-OH1,4-naphthohydroquinone will bring about the distribution of molecular products observed during the 1,4reductive addition of GSH to 2,3-epoxy-1,4-naphthoquinone under anaerobic conditions. These mechanistic relationships are summarized in the scheme in Figure 11. Qualitative alterations in the pathways drawn in the scheme in Figure 10 (under aerobic conditions) are expected to be determined by the increasing degree of
(b) Interaction with 02 subsequent to the 1,4-reductive addition of GSH to quinone epoxides The primary product resulting from the GSH nucleophilic addition to 2,3-epoxy-l,4-naphthoquinone, namely, 2-OH-3-glutathionyl-l,4-naphthohydroquinone, is endowed with a high reducing power and it can be oxidized by either (a) unreacted quinone epoxide (reaction 3) or (b) 02 with formation of H202 (reaction 4). Another possibility is an elimination reaction (reaction 2). The presence of 02 in the reaction mixture favors the autoxidation of the primary product, thereby decreasing the contribution to the formation of final molecular products through both elimination reactions and quinone/hydroquinone derivative redox transitions. Autoxidation reactions are relevant under aerobic conditions and the main sources of H20: (Fig. 5) would involve electron transfer to 02 from 2-OH-3glutathionyl- 1,4-naphthohydroquinone and 2-OH- 1,4naphthohydroquinone (reduced lawsone) (reactions 4
I'IO"
0-
GS-
0
SG
o-
~
O
o
o
°
o-
o
o
o O2
H202
SG
02
~H202 o
o
Fig. 10. Scheme of reactions for the GSH-mediated 1,4-reductive addition to 2,3-epoxy-l,4-naphthoquinoneunder aerobic conditions. Boxes below the chemical structures indicate the peak identified in the spent reaction mixture shown in Figurc 3B and identified after comparison with retention times and half-wavepotentials in Figure 1A and Table 1.
Quinone epoxides o
O"
GS"
O
O
o-
0
163 I-K)-
SG
O
O
SG
o-
/ ½so O
O
O
o
Fig. 11. Scheme of reactions for the GSH-mediated 1,4-reductive addition to 2,3-epoxy-l,4-naphthoquinone under anaerobic conditions. Boxes below the chemical structures indicate the peak identified in the spent reaction mixture shown in Figure 6 and identified after comparison with retention times and half-wave potentials in Figure IA and Table 1.
methyl-substitution of 2,3-epoxy-l,4-naphthoquinone. The GSH nucleophilic addition to the unsubstituted naphthoquinone epoxide yielded substantial amounts of H202, whereas the addition to menadione epoxide (2-methyl-2,3-epoxy-l,4-naphthoqui. none) showed significantly lower H202 production, and that to the dimethyl-substituted 1,4-naphthoquinone epoxide showed neither 02 consumption nor H202 production. In terms of their relative contribution to autoxidation associated with the nucleophilic addition to 1,4-naphthoquinone epoxides, following order could be observed: 2,3-epoxy-l,4-naphthoquinone > 2-methyl-2,3-epoxy-1,4-naphthoquinone > 2,3-dimethyl-2,3-epoxy- 1,4-naphthoquinone. The primary product formed upon GSH addition to menadione epoxide is disubstituted in C3 and the compound is not to be considered a hydroquinone. To regain a quinonoid structure a H O - has to be eliminated from the intermediate compound within an oxidation reaction (reaction 7). Consequently, the formation
of 2-methyl-3-glutathionyl-l,4-naphthoquinone occurs mainly through loss of a H O - rather than a redox transition with O2 with formation of H202. A mechanism as that proposed by Nickerson et al. 25 for H202 production is not applicable within the context of these experiments, for their experimental model consisted of [menadione]/[GSH] molar ratio of 2, and the ultimate autoxidizing species was reported to be the reduced form of menadione. 25 The mechanistic implications related to the 1,4-reductive addition of GSH to 2,3-dimethyl-2,3-epoxy1,4-naphthoquinone are difficult to interpret. The structure drawn in reaction 8 is merely hypothetical and should be considered as an intermediate compound prone to undergo scission reactions. The decreased participation in redox reactions of the products originating from the GSH nucleophilic addition to monomethyl- (reaction 7) and dimethyl(reaction 8) substituted 1,4-naphthoquinone can be understood in terms of: (a) the occurrence of inter-
164
A. BRUNMARKand E. CADENAS
m e d i a t e p r o d u c t s w i t h d i s u b s t i t u t e d c a r b o n a t o m s , (b) the c o n s e q u e n t p r e v a i l a n c e o f e l i m i n a t i o n or f r a g m e n tation r e a c t i o n s , and, t h e r e b y (c) the i m p o s s i b i l i t y o f r e a r r a n g e m e n t into a r e d u c e d q u i n o n o i d structure. A l i k e the u n s u b s t i t u t e d 1 , 4 - n a p h t h o q u i n o n e e p o x ide, the G S H - m e d i a t e d r e d u c t i v e a d d i t i o n to p - b e n z o q u i n o n e e p o x i d e s s t u d i e d h e r e w o u l d l e a d indisting u i s h a b l y to the f o r m a t i o n o f a H O - s u b s t i t u t e d pb e n z o h y d r o q u i n o n e . S u c h a c o m p o u n d , as s h o w n in F i g u r e 9, is p r o n e to u n d e r g o r e d o x r e a c t i o n s , in s o m e instances implying autoxidation. F i n a l l y , it can be c o n c l u d e d that the r e a c t i o n o f q u i n o n e e p o x i d e s with G S H u n d e r a e r o b i c c o n d i t i o n s p r o d u c e s h y d r o x y - and g l u t a t h i o n y l - q u i n o n e d e r i v a tives, c o m p o u n d s m o r e prone to autoxidation than those r e s u l t i n g f r o m the n u c l e o p h i l i c a d d i t i o n to q u i n o n e s l a c k i n g an e p o x i d e g r o u p . T h e m a i n feature o f this r e a c t i o n is the o c c u r r e n c e o f a h y d r o x y substituent in p o s i t i o n e~, a d j a c e n t to the c a r b o n y l g r o u p , w h i c h appears to w i t h s t a n d the e n h a n c e d a u t o x i d a t i o n o b s e r v e d , T h i s h i g h e r c h e m i c a l r e a c t i v i t y m a y be o f t o x i c o l o g i c a l interest. H o w e v e r , it s h o u l d be b e a r e d in m i n d , as d e s c r i b e d a b o v e , that the m e t h y l s u b s t i t u t i o n o f quin o n e e p o x i d e s l o w e r e d the rate o f t h e i r c h e m i c a l addition w i t h s u l p h u r n u c l e o p h i l e s , t h e r e b y p r o d u c i n g less r e a c t i v e m o l e c u l a r p r o d u c t s , at least in t e r m s o f autoxidation reactions. This lower chemical reactivity m a y not translate d i r e c t l y into a low t o x i c i t y , for a less r e a c t i v e c o m p o u n d is, b e c a u s e o f its l o n g e r l i f e t i m e , m o r e l i k e l y to be t r a n s p o r t e d a w a y f r o m its g e n e r a t i o n loci to an a d e q u a t e m o l e c u l a r target. Acknowledgements--Supported by grants 7679 and 4481 from the
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ABBREVIATIONS
Menadione--2-methyl- 1,4-naphthoquinone Phthiocol--2-methyl-3-hydroxy- 1,4-naphthoquinone Thiodione--2-methyl-3-glutathionyl-1,4-naphthoquinone H.p.l.c.--high performanceliquid chromatography GSH--glutathione GSSG--glutathione disulfide Lawsone--2-OH- 1,4-naphthoquinone