quinones form the second 
 largest class of antitumor agents approved 
 for clinical use in the u.s . , and several antitumor quinones are in 
 different stages of clinical and preclinical development
a common feature of quinone - containing drugs 
 is their ability to undergo reversible redox reactions to form semiquinone 
 and oxygen radicals .
one - electron reduction of a quinone 
 ( q ) gives the semiquinone radical ( q or 
 qh ) , whereas two - electron reduction gives the hydroquinone 
 ( qh2 ) .
the semiquinone can 
 also be formed by a comproportionation reaction between a quinone 
 and a hydroquinone ( reaction 1 ; the opposite 
 of reaction 1 is the semiquinone disproportionation 
 reaction).1the catalytic enhancement of ascorbate 
 ( asch ) oxidation by quinones has been previously 
 observed .
in addition , we have previously 
 observed that 
 quinones enhance the rates of ascorbate reduction of nitric oxide and s - nitrosothiols . in each of those works ,
the enhancement activity of quinones increases 
 with its one - electron reduction potential within a certain range of 
 one - electron reduction potential values .
thus , the semiquinone , the 
 one - electron - reduced species , is postulated to be the actual intermediate 
 responsible for reducing those targets . furthermore , antitumor activity 
 enhancement by quinones in the presence of ascorbate has been reported .
hydrogen peroxide has important roles as 
 a signaling molecule in 
 the regulation of a variety of biological processes . hydrogen peroxide also plays an important role in aging and cancer . although quinone - enhanced 
 ascorbate reduction reactions of the species mentioned above have 
 been reported , very few works report the possibility of a semiquinone - mediated 
 reduction of h2o2 .
in fact , koppenol and butler suggested that quinones with reduction 
 potentials between 330 and 460 mv could be involved 
 in metal - independent fenton reaction 2.2however , there has not been 
 any study comparing 
 the reactivities among quinones with regard to reaction 2 with their dependence on the quinone one - electron reduction 
 potential .
reaction 2 acquires more biomedical 
 importance in view of the facts that parenteral administration of 
 ascorbate generates h2o2 in tissues and that quinones enhance the antitumor activity 
 of ascorbate .
these observations suggest 
 that quinones may be acting as h2o2 production 
 promoters and/or as enhancers of hydroxyl radical production .
the metal - independent production of hydroxyl radicals by the reaction 
 of h2o2 with halogenated quinones in the absence 
 of reducing agents has been reported .
this type of reaction 
 was not detected for non - halogenated quinones , indicating the need 
 for electron - withdrawing substituents at the quinone ring for this 
 reaction to occur . in this work ,
we have determined the relative extent 
 of hydroxyl radical production in the presence and absence of non - halogenated 
 quinones , using ascorbate as the reducing agent .
hydroxyl radicals 
 can be determined by spin trapping using nitrones , and the resulting 
 nitroxide concentration can be measured by electron paramagnetic resonance 
 ( epr ) spectrometry .
however , nitroxides 
 are prone to reduction to the epr - silent hydroxylamine in the presence 
 of reducing agents .
therefore , in this work , we have determined the 
 relative ability of quinones to enhance the rate of ascorbate reduction 
 of h2o2 by measuring the amount of methane produced 
 from the reaction of hydroxyl radicals with dmso .
a similar procedure 
 for the selective detection of oh radical production , where dmso is 
 the hydroxyl radical scavenger and the produced methane gas is detected 
 by gas chromatography ( gc ) , has been previously used in several works .
the mechanism of this reaction has been previously determined using 
 epr and radiolysis techniques .
these quinones were selected 
 because they have a wide range of one - electron reduction potentials , 
 are commercially available , and were found to be active in the enhanced 
 reduction of oxygen by ascorbate .
methane ( 99.0% pure ) was purchased in 
 a gas lecture bottle from sigma - aldrich corp .
stock solutions of quinones 
 were prepared in water and used on the same day of their preparation . 

deionized and chelex - treated water was used in the preparation of 
 all stock and sample solutions .
chelex treatment of water was monitored 
 using the ascorbate test , as described by buettner .
stock solutions of ascorbic acid , phosphate buffer 
 ( ph 7.4 ) , detapac , 
 h2o2 , dmso , and quinone were deareated by purging 
 with high - purity n2 and equilibrated to 37 c in a 
 temperature - controlled water bath .
the reaction mixture most frequently 
 used contained 10 m quinone , 100 m detapac , 100 m 
 neocuproine , 10 mm ascorbate , and 2 mm h2o2 in 
 a 1:3 dmso / phosphate buffer ( 20 mm , ph 7.4 ) ( v / v ) mixture .
the reason for adding detapac and neocuproine 
 was to further inhibit transition metal catalysis in addition to that 
 mediated by chelex treatment of solutions .
nitrogen - saturated 
 amber 3.00 ml septum - capped bottles were used with continuous spinning 
 bar stirring .
the reaction bottles were placed in a water - jacketed 
 beaker with a constant temperature of 37 c before addition of 
 reagents . after the reaction ,
a single aliquot of 50 l was 
 withdrawn from the gas phase on top of the reaction mixture and injected , 
 using a gastight syringe , into a gas chromatograph for analysis .
the 
 chromatographic peak retention time of ch4 was established 
 by using a ch4 standard .
the chromatograph used was 
 an agilent 6890 with thermal conductivity detection and an alltech 
 16226 porapak q column .
chromatographic conditions were as follows : 
 flow rate of 5.0 ml of he / min , column temperature of 35 c , and 
 injector and detector temperatures of 120 and 150 c , respectively .
alternatively , the detection of hydroxyl radicals was also done 
 by measuring the hydroxylation products of salicylic acid . a reaction mixture containing 10 m phq 
 or dq or no quinone , 100 m detapac , 100 m neocuproine , 
 10 mm ascorbate , 1 mm salicylic acid , and 2 mm h2o2 in 20 mm phosphate buffer , ph 7.4 , was incubated for 40 min 
 followed by hplc determination of 2,3- and 2,5-dihydroxybenzoic acid 
 ( 2,3-dhb and 2,5-dhb , respectively ) in the sample .
the hydroquinone of phq was 
 prepared , as described elsewhere , by reducing the quinone with nabh4 . for this purpose ,
a n2-saturated 
 hydroquinone aqueous stock solution was prepared by mixing a n2-saturated phq solution in methanol with solid nabh4 .
the solution was then dried with a n2 stream followed 
 by addition of a n2-saturated water / dmso ( 1:1 v / v ) mixture . 

the solution ph was decreased to 3 by hcl addition to destroy excess 
 nabh4 and to stabilize the hydroquinone solution .
half - wave reduction potentials were determined 
 in nitrogen - purged acetonitrile solutions containing 1 mm quinone 
 and 0.1 m tetra - n - butylammonium perchlorate ( tbap ) 
 using differential pulse voltammetry ( dpv ) .
a bas cv 50w voltammetric 
 analyzer using a glassy carbon working electrode was used for these 
 determinations .
an ag / agcl(sat ) electrode was used as the reference 
 electrode ( e = + 0.22 v vs nhe ) , and a platinum 
 wire was used as the counter electrode .
differential pulse voltammograms 
 were obtained in the potential range from 2.00 to 0.00 v , 
 using a 50 mv pulse amplitude and 20 mv / s of scan rate . the reduction 
 potential values were obtained from the dpv peak potential maxima . 

these are similar to the half - wave one - electron reduction potentials , e1/2 , in normal polarographic measurements .
hplc analyses were 
 done using a hp zorbax 
 c-18 ( 4.6  250 mm ) column and eluted using a gradient from 100% 
 ammonium phosphate ( ph 3.5 ) to 100% methanol .
an agilent 1100 analytical hplc system with absorption 
 detection at 300 nm was used .
the retention times of the corresponding 
 phq , salicylic acid , and 2,3- and 2,5-dhb peaks were determined using 
 commercial standards .
all determinations were repeated at least three 
 times , and the average of these determinations  sd is reported .
upon adding 
 a n2-saturated millimolar ascorbate solution to a n2-saturated solution containing detapac , neocuproine , and h2o2 ( millimolar ) in a 1:3 dmso / phosphate buffer 
 ( 20 mm , ph 7.4 ) ( v / v ) mixture and incubating that solution for 40 
 min , a gc peak with a retention time of 5.4 min was detected .
the 
 identity of that peak was established by injecting pure methane to 
 the gc .
if an identical sample was prepared that instead contained 
 10 m phq , then a methane gc peak was also detected that was 
 24 times more intense than that observed in the absence of quinone 
 ( figure 2 ) .
thus , phq strongly enhances the 
 rate of hydroxyl radical formation , even at micromolar concentrations .
gas chromatograms 
 of the gaseous phase of samples after 40 min 
 of incubation .
samples contained ( a ) 100 m detapac , 100 m 
 neocuproine , 10 mm ascorbate , and 2 mm h2o2 in 
 1:3 dmso / phosphate buffer ( 20 mm , ph 7.4 ) ( v / v ) ; ( b ) the same composition 
 as in panel ( a ) but with 10 m phq ; and ( c ) 10 m fecl2 , 10 mm ascorbate , and 2 mm h2o2 in 
 1:3 dmso / phosphate buffer ( 20 mm , ph 7.4 ) ( v / v ) .
as indicated earlier , the indirect selective detection of 
 oh radical 
 production , where dmso is the hydroxyl radical scavenger and the produced 
 methane gas is detected by gas chromatography ( gc ) , has been previously 
 used in several works .
the mechanism of this reaction has been previously determined using 
 radiolysis and epr techniques .
although ethane could be another 
 observed product , its production is very limited at the high dmso 
 concentration used in this work ( ca .
3.5 m ) and thus the major gaseous 
 product is methane . if phq was replaced by other 
 quinones , then production of hydroxyl 
 radicals was also detected in a manner that depends on the quinone 
 one - electron reduction potential ( figure 3 ) . 

incubation times of 4 to 20 h were previously used in determining 
 henry s constants for methane in water .
the incubation time of 40 min used in the present work 
 was selected in order to detect a relatively strong gc signal as well 
 as to discriminate reactivity differences among the quinones used . 

a time course for ch4 generation by a sample , using phq 
 as the quinone , is shown in figure 4 .
although the gc ch4 peak areas do not correspond to the 
 maximum areas that can be measured upon reaching methane solubility 
 equilibrium , they reveal the catalytic effect of quinones on the production 
 of hydroxyl radicals from h2o2 + ascorbate as 
 well as their relative abilities to do so .
relative ch4 concentrations determined after a 40 min 
 incubation of n2-saturated samples containing 10 m 
 quinone , 100 m detapac , 100 m neocuproine , 10 mm ascorbate , 
 and 2 mm h2o2 in 1:3 dmso / phosphate buffer ( 20 
 mm , ph 7.4 ) ( v / v ) . the symbols used to represent a blank sample without 
 quinone , phqh2 , and fe are for solutions without 
 quinone , with 10 m phqh2 , or with 10 m fecl2 , respectively , and correspond to the methane concentration , 
 but not to the one - electron reduction potential , indicated by the 
 position of the symbol .
the symbols ascribed to phq correspond 
 to the same sample composition as that listed (  ) , with 10 
 m dfo added (  ) , or with 100 m bathophenanthroline 
 disulfonate and 100 m ferrozine added (  ) .
time dependence of ch4 concentrations determined 
 from 
 the headspace of a n2-saturated sample containing 10 m 
 phq , 10 mm ascorbate , 2 mm h2o2,100 m 
 detapac , and 100 m neocuproine in 1:3 dmso / phosphate buffer 
 ( 20 mm , ph 7.4 ) ( v / v ) . in order to further evidence hydroxyl radical formation , 
 another 
 probe for hydroxyl radicals was used , i.e. , the hydroxylation of salicylic 
 acid to form 2,3- and 2,5-dhb . because 
 the formation of 2,3-dhb is more prominent under our conditions , its 
 chromatographic area was used as a measure of hydroxyl radical production . 
 the ratio of the 2,3-dhb chromatographic peak area corresponding to 
 a phq - containing sample to that of a dq - containing sample ( 4.0  
 0.2 ) was essentially the same as the ratio of the ch4 chromatographic 
 peak area corresponding to a phq - containing sample to that of a dq - containing 
 sample ( 3.8  0.3 ) .
thus , the observed behavior between these 
 2 quinones is independent of the assay used to detect hydroxyl radical 
 production . in the absence of ascorbate , the ch4 gc 
 peak areas obtained 
 for samples containing phq , ubq , mnq , and dmobq are essentially the 
 same as that of a blank sample in the absence of quinone .
this indicates 
 that the reaction reported for halogenated quinones involving the 
 nucleophilic addition of hoo to the quinone ring 
 followed by hydroxyl radical production is not occurring .
this is 
 consistent with a previous report of the absence of hydroxyl radical 
 production upon mixing h2o2 with dmbq or dq .
varying chelating agent concentrations 
 from 0 to 400 m in 
 samples containing the same amounts of ascorbate , h2o2 , and phq , respectively , produced only a variation of up to 
 ca .
furthermore , 
 addition of 10 m of the iron chelator deferroxamine ( dfo ) or 
 100 m of each of the nonhydroxamate iron chelators bathophenanthroline 
 disulfonate and ferrozine to the sample in the presence of 100 m 
 detapac and 100 m neocuproine did not decrease the methane 
 gc peak area compared to that in the absence of these chelators ( figure 3 ) .
the concentration of dfo used should be able 
 to chelate any trace iron present after chelex treatment of the water 
 or solution .
thus , the dependence of the 
 extent of methane production on the quinone one - electron reduction 
 potential and the absence of variations in methane production as a 
 function of transition metal chelator concentration indicate that 
 the observed catalytic activity of the quinones is not metal - mediated . 

substitution of phq with the same concentration of fecl2 as that of the quinone , but excluding metal chelators in the incubation 
 sample , exceeds the reactivity of 10 m phq in producing hydroxyl 
 radicals by 14% ( figure 3 ) , as detected from 
 the methane gc peak area . the fenton reaction rate constant ( fe + h2o2 ) is reported to be 2.0  
 10 m s in phosphate 
 buffer .
assuming instantaneous semiquinone 
 formation ( see below ) and similar rate constants for reactions after 
 the hydroxyl radical production step , we can estimate the rate constant 
 for the phq - assisted fenton reaction ( reaction 2 ) from the gc peak area obtained from the phq - containing 
 sample , compared to that of the fe - containing sample , 
 as k2 = ( 0.86 ) ( 2.0  10 ) = 1.7  10 m s. thus , phq reactivity is comparable to that of fe . 
 because phq is a common toxin present in diesel combustion smoke , 
 the possibility that phq - mediated catalysis of hydroxyl radical formation 
 is similar to that of fe adds another important pathway 
 to the modes in which phq can execute its toxicity . in an identical 
 manner , values of k2 can be estimated 
 for dmbq , mnq , and dq as 1.3  10 , 7.4  10 , and 4.6  10 m s , respectively .
rate constants , k2 , for the electron transfer from the semiquinones of 
 dmbq , mnq , and dq to oxygen to form superoxide are 8.8  10 , 2.4  10 , and 2.2  10 m s , respectively .
therefore , at equal concentrations of oxygen and h2o2 , semiquinone reduction of oxygen is expected for these quinones 
 instead of h2o2 reduction .
thus , h2o2 reduction by semiquinones should acquire more importance 
 in hypoxic tissues whenever h2o2 concentrations 
 exceed that of oxygen by several orders of magnitude .
relative ch4 concentrations determined after a 40 min 
 incubation of n2-saturated samples containing ( a ) 10 m 
 phq , 10 mm ascorbate , 2 mm h2o2 , and various 
 concentrations of detapac and neocuproine ; ( b ) 10 m phq , 100 
 m detapac , 100 m neocuproine , 2 mm h2o2 , and various concentrations of ascorbate ; ( c ) 10 m 
 phq , 100 m detapac , 100 m neocuproine , 10 mm ascorbate , 
 and various concentrations of h2o2 ; and ( d ) 
 100 m detapac , 100 m neocuproine , 10 mm ascorbate , 2 
 mm h2o2 , and various concentrations of phq in 
 1:3 dmso / phosphate buffer ( 20 mm , ph 7.4 ) ( v / v ) . replacing 
 10 m phq with 10 m of its hydroquinone in a sample containing 
 100 m detapac , 100 m neocuproine , 10 mm ascorbate , and 
 2 mm h2o2 in a 1:3 dmso / phosphate buffer ( 20 
 mm , ph 7.4 ) ( v / v ) mixture and incubating the mixture for 40 min produced 
 a ch4 peak area that is only 14  2% of that obtained 
 in the phq - containing sample ( figure 3 ) . although 
 this value is larger than that obtained in the absence of quinone , 
 2  1% , it is consistent with the semiquinone , not the hydroquinone , 
 being largely responsible for the enhanced production of hydroxyl 
 radicals . in order 
 to obtain further evidence that the semiquinone is the actual h2o2-reducing species , a phq solution containing 
 dmso , detapac , h2o2 , neocuproine , and phosphate 
 buffer ( ph 7.4 ) ( v / v ) was mixed with an equal volume of a phqh2 solution in the absence of oxygen and ascorbate .
final 
 concentrations after mixing were 5 mm phq , 5 mm phqh2 , 
 100 m detapac , 100 m neocuproine , and 2 mm h2o2 in 1:3 dmso / phosphate buffer ( 20 mm , ph 7.4 ) ( v / v ) . 

the phqh2 concentration used contains the same amount of reducing equivalents 
 as those involved in the one - electron oxidation of 10 mm ascorbate . 

the methane gc peak area obtained ( 93  8 arbitrary units ) after 
 incubation for 40 min was essentially the same as that obtained when 
 h2o2 reduction occurs by reacting ascorbate 
 with phq ( 97  4 arbitrary units , figure 3 ) . because most para - semiquinones have pka values for their protonated form , qh
, between 3 and 5 and those corresponding to ortho - semiquinones , 
 between 4 and 5 , the anion radical species 
 is the most abundant species at physiological ph and thus is the carrier 
 of electrons from ascorbate to h2o2 .
thus , it is very unlikely 
 that michael addition of hoo to the quinones occurs 
 at the ph used for this work because very low concentrations of this 
 anion will be present .
furthermore , the phq relative hplc peak area 
 of a h2o2-containing sample and that of a sample 
 with no h2o2 and no ascorbate added , both containing 
 phosphate buffer , dmso , detapac , and neocuproine , after 40 min of 
 incubation , was 103.4  0.5 and 100  1 relative units , 
 respectively .
the latter shows that phq is not being transformed to 
 another type of molecule during the incubation time in the presence 
 of h2o2 .
such a transformation would have made 
 the dependence of the ch4 area on the e1/2 values of the quinones very erratic , as their identities 
 in the reaction samples would have been different from those present 
 during the e1/2 measurements .
the rate of ch4 formation increases 
 linearly with an increase in concentration of all reagents , i.e. , 
 phq , asch , and h2o2 ( keeping 
 the other reagents at constant concentrations ) ( figure 5 ) , indicating first - order kinetics with respect to each of 
 these species . the fact that this rate depends on h2o2
concentration is in contrast to the zero - order behavior of 
 the ascorbate autoxidation rate with respect to o2 .
if the semiquinone reduction of h2o2 is postulated to be the slow step in the mechanism , 
 a rate equation can be obtained which is first - order with respect 
 to each species , phq , asch , and h2o2 ( scheme 2 ) .
a constant steady - state 
 concentration of the ascorbyl radical , asc , has been observed in previous works during the quinone - enhanced as well as in the iron- and methylene blue - catalyzed ascorbate oxidation reactions .
, quinones reduce h2o2 in the presence 
 of ascorbate in a quinone redox - potential - dependent manner and independent 
 of transition metal traces .
the most active quinone in the studied 
 series , phq , is only 14% less active than the classic fenton reagent 
 cation , fe , at the same concentration .
these observations 
 are relevant to the antitumor - enhancing activity of quinones in the 
 presence of ascorbate and to the toxic activity of environmental quinone 
 contaminants .
 the quinones 1,4-naphthoquinone ( nq ) , 
 tetramethyl-1,4-benzoquinone 
 ( dq ) , 2-methyl-1,4-naphthoquinone ( mnq ) , 2,3-dimethoxy-5-methyl-1,4-benzoquinone 
 ( ubq-0 ) , 2,6-dimethylbenzoquinone ( dmbq ) , 2,6-dimethoxybenzoquinone 
 ( dmobq ) , and 9,10-phenanthraquinone ( phq ) enhance the rate of h2o2 reduction by ascorbate , under anaerobic conditions , 
 as detected from the amount of methane produced after hydroxyl radical 
 reaction with dimethyl sulfoxide . 
 the amount of methane produced increases 
 with an increase in the quinone one - electron reduction potential . 
 
 the most active quinone in this series , phq , is only 14% less active 
 than the classic fenton reagent cation , fe2 + , at the same 
 concentration . 
 since phq is a common toxin present in diesel combustion 
 smoke , the possibility that phq - mediated catalysis of hydroxyl radical 
 formation is similar to that of fe2 + adds another important 
 pathway to the modes in which phq can execute its toxicity . 
 because 
 quinones are known to enhance the antitumor activity of ascorbate 
 and because ascorbate enhances the formation of h2o2 in tissues , the quinone - mediated reduction of h2o2 should be relevant to this type of antitumor activity , 
 especially under hypoxic conditions . 
Introduction
Materials
and Methods
Results and Discussion