inhalation of ambient 
 particulate matter ( pm ) causes respiratory 
 and cardiovascular health problems and mortality in humans .
pm may induce these effects by producing reactive oxygen species 
 ( ros ) , including hydrogen peroxide ( hooh ) and hydroxyl radical ( oh ) , in the body .
though less reactive 
 than oh , hooh is of interest because it is a signaling 
 molecule in vivo , has a relatively long lifetime , 
 can cross cell membranes , and is a precursor for oh . once deposited in the 
 lung lining fluid , redox - active species from inhaled pm can chemically 
 produce hooh at levels that far exceed those originally present in 
 the particles .
endogenous reductants 
 such as ascorbate , and other reducing species such as dithiothreitol 
 ( dtt ) , increase the production of hooh and oh from 
 pm or metal solutions ; thus the interactive chemistry between endogenous 
 reductants and deposited pm can increase the oxidant load in the body .
however , adverse effects 
 can occur when an overproduction of oxidants  for example , upon 
 pm exposure  overwhelms the body s anti - oxidative defenses .
it is unclear what concentration of hooh is necessary to elicit 
 adverse effects in the lung , as hooh likely affects each cell type 
 differently .
fibroblasts and human alveolar 
 cells exposed to hooh in the range of 10400 m exhibit 
 apoptosis , while higher concentrations induce necrosis .
human alveolar and bronchial epithelial cells released 40% of their 
 lactate dehydrogenase ( ldh ) in the presence of 100 and 1000 m 
 hooh , respectively , indicating alveolar cells may be more susceptible 
 to hooh than bronchial cells .
both 
 transition metals ( cu , zn , fe ) and quinones have been implicated 
 in hooh formation from pm .
zn and fe were identified via correlation between hooh production 
 and pm metal content ; however , many trace metals and quinones are 
 covariate , which confounds identifying the redox - active species responsible 
 for ros generation .
in addition , although 
 total ( acid - soluble ) metals are typically measured in these studies , 
 it is likely that the soluble metals drive the redox activity . for 
 example , while ros production from particles using the dichlorofluorescein 
 diacetate ( dcf - da ) assay showed a good correlation between ros production 
 and soluble cu ( r = 0.59 ) , there was 
 no correlation with total cu ( r = 0.02 ) .
there is also quantitative , mechanistic evidence 
 that specific particle components can generate hooh .
chung et al . measured quinone concentrations and hooh production 
 ( in the presence of 100 m dtt ) in ph 7.4 aqueous extracts of 
 ambient fine particles ( pm2.5 ) .
response 
 curves of the pure quinones , they concluded that the quinone content 
 of ambient pm2.5 could account for all hooh produced by 
 the particles .
in contrast , shen et al . found that soluble cu could explain essentially all hooh production 
 from ambient fine and coarse pm in ph 7.3 aqueous extracts containing 
 50 m ascorbate .
additionally , desferoxamine , a strong metal 
 chelator , halted hooh production , further indicating that hooh was 
 produced by metals .
given the uncertainties in our understanding 
 of hooh production 
 from ambient pm , our purpose is to quantify hooh formation from transition 
 metals and quinones ( both individually and in mixtures ) in a more 
 representative ( though still cell - free ) surrogate lung fluid ( slf ) . 

in this work we characterize an in vitro , cell - free assay to measure 
 the rate of hooh production from pm . given the important role of lung - lining 
 fluid antioxidants in ros formation , we include typical lung concentrations of four antioxidants : ascorbate ( asc ) , reduced 
 glutathione ( gsh ) , urate ( ua ) , and citrate ( cit ) .
asc , gsh , and ua 
 are naturally occurring in the lung fluid , while cit is a good proxy for proteins that mobilize iron in the 
 lung fluid . while we use ph and antioxidant 
 conditions similar to lung lining fluid
, it is impossible to reproduce 
 the complexity of particle  lung interactions using an in vitro assay .
this technique is intended as a useful screening 
 assay for the oxidative potential of ambient pm .
it also allows us 
 to identify the chemicals that can produce hooh in lungs and which 
 of these redox - active species are likely most important for hooh production 
 from inhaled ambient pm .
information 
 about chemicals and their purities , metal and quinone 
 stocks , detailed hooh measurement steps , and ambient pm samples is 
 given in the supporting information , section 
 s1 .
our slf consists of phosphate - buffered 
 saline ( pbs ) with four antioxidants .
the pbs contains 114 mm nacl , 
 7.8 mm sodium phosphate dibasic , and 2.2 mm potassium phosphate monobasic , 
 ph 7.27.4 , and is treated with chelex 100 resin ( sodium form , 
 bio - rad ) to remove trace metals .
each batch 
 of pbs is treated twice with chelex resin at a rate of one drop per 
 four seconds .
antioxidant stock solutions , made fresh each day , are 
 added to the pbs at the start of the reaction at final concentrations 
 of 200 m l - ascorbic acid sodium salt ( asc ) , 300 m 
 citric acid ( cit ) , 100 m reduced l - glutathione ( gsh ) , 
 and 100 m uric acid sodium salt ( ua ) .
analytical details 
 are given in the supporting information , section s1 . at time zero
, we mix the redox - active species into 
 5.0 ml of slf in a 7.0 ml fep bottle , seal it , and agitate it on a 
 shake table at setting 5 at room temperature .
we measure the hooh 
 concentrations in each reaction solution at 0 , 0.5 , 1 , and 1.5 h. 
 at each time point we remove a 0.50 ml aliquot , add 10.0 l 
 of 5.0 mm desferoxamine ( dsf ) to chelate metals and help stabilize 
 the hooh , and then immediately inject onto the hplc ( 50 l sample 
 loop ) .
fe(ii ) destroys hooh via the fenton reaction , and our method is especially sensitive to fe 
 contamination because of the presence of asc , which cycles inactive 
 fe(iii ) into active fe(ii ) . to mitigate this effect
, we adhere to 
 rigorous cleaning methods to maintain background fe concentrations 
 below 50 nm ; however , fe likely destroys some hooh even at these low 
 levels .
we use only fep bottles ( fisher scientific ) , and all are washed 
 in a freshly made 1 m nitric acid bath before use .
we calculate the rate 
 of hooh production from the concentrations of hooh measured at 0 , 
 0.5 , 1 , and 1.5 h. in many cases the rate of hooh production decreases 
 with time , causing a slight curvature for most data ( figure s1 ) and significant curvature for solutions with high 
 concentrations of fe ( figure s2 ) .
similar 
 behavior was observed by shen et al . for 
 both ambient pm and cu stocks , and by wang et al . for hooh production from coarse mode pm .
we calculate the 
 initial rate of hooh production using the b term 
 of a second - order polynomial : y = at + bt + c , where a , b , and c are fitted 
 constants .
the initial rate of production 
 between 0 and 1.5 h would not be affected by the choice of reaction 
 time for samples with slight curvature , but could be affected for 
 highly curved samples ( generally above 300 nm fe(ii ) , as shown in figure s2 ) if curvature occurs very early .
however , 
 significant curvature occurs only for laboratory samples where the 
 rate of hooh production is essentially zero .
ambient samples did not 
 show as much sensitivity to fe , as discussed 
 below , and ambient rates should be less sensitive to the choice of 
 time points .
we measure hooh production in a positive control 
 ( 250 nm cu(ii ) ) and blank ( slf containing four antioxidants ) on each 
 experiment day .
all sample rates are blank - corrected by subtracting 
 that day s blank rate .
if data have an error bar it indicates 
 that two or more replicates were measured and the data are reported 
 as the average  standard deviation of the blank - corrected initial 
 rates .
a small subset of data do not have replicates and are reported 
 without an error bar to identify that only one measurement was made . 

we could estimate the error from the standard error of the slope of 
 the rate regression , but this under - predicts the actual variability 
 of day - to - day replicates .
we estimate that the typical relative standard 
 deviation for our rates is 14% based on variability in the blank - corrected 
 positive control , which has an average ( 1 ) initial rate 
 of hooh formation of 1.99  0.28 m / h ( n = 18 ) , with a blank rate of 0.21  0.1 m / h . statistical 
 differences between means ( where n  2 ) are 
 calculated using the student s t - test , with p  0.05
as a first 
 step in identifying the components in pm that can produce hooh , we 
 start by screening hooh formation from individual chemicals at a concentration 
 of 500 nm in the slf .
as detailed in the supporting 
 information , section s2 , 500 nm is a reasonable concentration 
 for fe and cu but is over an order of magnitude higher than expected 
 for the quinones ( table s2 ) .
we start with 
 this relatively high concentration in order to identify any compound 
 that can produce hooh under our reaction conditions . of the 
 10 transition metals
tested , only cu(ii ) produces significant hooh 
 under our slf conditions , while fe(ii ) destroys background hooh , resulting 
 in a slightly negative rate of hooh production ( figure 1 ) .
pb produces hooh at a rate statistically different than 
 the blank , though extremely slowly .
the other seven metals  mn , 
 co , v , ni , zn , cd , cr  do not produce hooh , though there is 
 some evidence that v can destroy hooh . of the four quinones tested , 
 three produce hooh
 phenanthrenequinone ( pqn ) , 1,4-naphthoquinone 
 ( 1,4-nqn ) , and 1,2-naphthoquinone ( 1,2-nqn)but benzoquinone 
 ( bqn ) does not .
previous measurements of hooh production from 12 quinones 
 in a ph 7.4 extract solution containing 100 m dtt as a reductant 
 also found that the same three quinones produce hooh , while the nine 
 other quinones did not . at 500 nm ,
1,2-nqn 
 produces 1633 times more hooh than an equal concentration 
 of cu(ii ) , pqn , or 1,4-nqn ( figure 1 ) .
initial rates 
 of hooh production from 500 nm concentrations of 
 individual metals and quinones in a slf with four antioxidants .
asterisks mark rates that are statistically larger 
 than zero ( p < 0.05 ) .
the rate for 1,2-nqn ( 44 
  4 m / h ) is divided by 10 to fit on this scale . to quantify 
 hooh production from cu and quinones at concentrations relevant to 
 ambient pm ,
we next measured hooh rates as a function of concentration 
 for the four active compounds . as shown in figure 2 ,
the concentration responses of all three quinones are linear , 
 with slopes ( table 1 ) that indicate their relative 
 ability to produce hooh .
the relative reactivities of the quinones 
 in our slf are 23:2:1 , i.e. , 1,2-nqn  pqn > 1,4-nqn . a 
 previous 
 study by chung et al . of hooh production from quinones in ph 7.4 phosphate 
 buffer with 100 m dtt as a reductant showed a different relative 
 reactivity , pqn > 1,4-nqn = 1,2-nqn .
this 
 difference is likely due to the difference in antioxidant composition 
 and reductant .
our slf uses asc as the reductant and contains three 
 other antioxidants , while chung et al . used a pbs that contains only 
 dtt .
the reductive potential of dtt ( 0.33 v ) is much stronger than that of asc ( + 0.105 ) , and our antioxidant composition affects hooh 
 production ( see next section ) .
we have previously shown that pqn is 
 much more active in the dtt assay relative to cu than in our slf .
when both species are at a concentration of 
 500 nm , pqn produces the same rate of hooh production as cu ( figure 1 ) , while at the same concentration pqn causes 9 
 times more dtt loss than cu .
response 
 curves of the rates of hooh production 
 as a function of concentration of redox - active species .
regression 
 equations for the species are given in table 1 y is the initial 
 rate of hooh production ( m / h ) , and x is the 
 concentration of chemical species ( nm ) .
hooh production from cu goes to 
 zero at 3.4 nm ; therefore , hooh production should be assumed to be 
 zero at cu concentrations below 3.4 nm .
unlike the quinones , cu shows a 
 nonlinear concentration response 
 with a fast initial increase in hooh production that begins to level 
 off around 200 nm cu ( figure 2 ) .
we believe 
 this results from the loss of asc over time , which causes asc to become 
 the limiting reactant .
as we will describe below ( figure 3 ) asc acts as a reductant and is necessary for formation 
 of both hooh and oh in our slf .
once asc becomes 
 limiting , addition of more cu causes only small increases in the rate 
 of hooh production .
a result of the nonlinear behavior is that the 
 relative reactivity of cu compared to quinones changes depending on 
 the concentrations of each compound in solution .
we found a similarly 
 nonlinear concentration  response curve for cu in the dtt assay , 
 which measures the oxidative potential of pm by monitoring the oxidation 
 of dtt over time .
thus , this result is 
 not limited to the slf experimental conditions in the hooh assay . 

previous laboratory studies measured the concentration  response 
 curve for hooh production from cu(ii ) in a slf containing only 50 
 m asc was linear through 400 nm cu but began to plateau at 
 600 nm cu , the maximum concentration tested .
this indicates that the antioxidant mixture in the slf alters the 
 concentration  response behavior .
one implication of the nonlinear 
 cu curve in figure 2 is that after 200 nm , 
 large increases in cu concentration cause only small increases in 
 the rate of hooh production . at low concentrations ( < 50 nm ) , cu 
 and 1,2-nqn
have very similar reactivities , while at higher concentrations 
 1,2-nqn is much more reactive than cu .
however , this is tempered by 
 the differences in particle concentrations of these two species : for 
 typical ambient conditions ( table s2 ) , 
 the concentration of cu in a slf extract of pm will be approximately 
 1001000 times larger than that of 1,2-nqn .
while filter - based quinone 
 measurements may have both negative ( volatilization ) and positive 
 ( formation from ozone ) artifacts , the 
 magnitudes of these artifacts are unlikely to be large enough to make 
 1,2-nqn more significant than cu as a source of hooh for typical ambient 
 pm .
effect of antioxidant composition on the rate of hooh production 
 from 250 nm cu(ii ) in ph 7.3 pbs .
when present , the concentration 
 of each antioxidant is constant for all experiments : asc is 200 m , 
 cit is 300 m , and gsh and ua are each 100 m .
the final 
 solution composition , with all four antioxidants , is our slf condition 
 used in all other figures .
each sample rate is corrected by a blank 
 containing the same composition of antioxidants .
our slf includes 
 four antioxidants : asc , cit , gsh , and ua . in previous work using the 
 same slf we found that the antioxidant composition has a significant 
 effect on oh production from transition metals . because hooh is a precursor for oh
we test different mixtures of antioxidants to better characterize 
 our assay ; however , the base case is a slf with all four antioxidants . 

as expected , hooh production by 250 nm cu 
 is affected by the antioxidant composition ( figure 3 ) .
cu produces hooh in the presence of asc , and to a much 
 smaller extent in the presence of gsh only , but not in the presence 
 of cit only .
asc acts as the reductant in our system , cycling transition 
 metals from their oxidized to reduced forms and thereby allowing oxidant 
 production from cu(ii ) via pathways such as1234although cu with gsh produces hooh ( figure 3 ) , this mixture does not form oh . compared to asc only , the combination 
 of asc and cit doubles the rate of hooh production , to 43 m / h 
 for 250 nm cu(ii ) . under the conditions with asc and cit as the only 
 antioxidants , 100% of cu(ii )
thus , the cu(ii)-citrate complex is apparently more reactive than 
 free cu(ii ) , which is the dominant cu form in the asc - only condition . if we add gsh to the asc cit mixture , the production 
 of hooh plummets by a factor of 20 , to 2.2 m / h , which is similar 
 to the hooh production rate from our slf case with all four antioxidants . 

thus , ua does not affect hooh production from cu in the presence of 
 the other antioxidants .
overall , hooh production in our mixture with 
 all four antioxidants is substantially reduced because of gsh , likely 
 because gsh binds to cu and reduces its reactivity .
a similar suppression 
 by gsh was observed for oh production from cu(ii ) 
 in the same slf , and also in other studies 
 of oh from cu .
minteq speciation 
 modeling of a similar slf with the same four antioxidants shows that 
 gsh replaces cit as the primary ligand , and 100% of cu(ii ) is bound 
 to gsh under these conditions .
gsh is 
 well known as an important antioxidant in vivo , and 
 may be especially important in mitigating damage from hooh .
binding and deactivating cu may be one component 
 of this protective effect of gsh .
we also find that the antioxidant 
 mixture affects the ability of 
 quinones to generate hooh .
as shown in figure 
 s3 , compared to the case of asc only , hooh production in the 
 four antioxidant ( slf ) mixture is lower by factors of 2 and 6 for 
 500 nm pqn and 20 nm 1,2-nqn , respectively .
while hooh formation from 
 the quinones is less sensitive to antioxidant composition than is 
 cu , the impact on quinones is surprising and more work is necessary 
 to confirm this result and understand its mechanism .
ambient pm samples contain 
 a complex mixture of chemical species 
 that may produce hooh in a more complicated mechanism than in the 
 pure laboratory solutions measured here .
for example , quinones and 
 cu can act synergistically to produce hooh under some conditions : 
 semiquinone radicals can reduce cu(ii ) to cu(i ) , producing superoxide 
 that can react with cu(i ) to make hooh . to examine 
 this in our slf , we measured hooh production in mixtures of cu , quinones , 
 and/or fe ( figure 4 ) .
the gray bars in figure 4 represent the rate of hooh production measured 
 from the species mixed in the same bottle , while the colored stacked 
 bars are the sum of hooh production measured from the individual compounds .
initial 
 rates of hooh production in laboratory mixtures of quinones 
 and/or transition metals ( gray bars ) compared to the sum of the rates 
 from the individual redox - active species ( stacked colored bars ) .
error 
 bars of the colored stacked bars are the propagated errors of the 
 sum ( all have replicate samples ) .
the concentrations of metals and 
 quinones are constant : cu , fe , and pqn are at 500 nm , and 1,2-nqn 
 is at 20 nm . as shown by the first 
 two sets of bars ,
the rate of hooh production 
 in a mixture of a quinone and cu is the same as the sum of the rates 
 of the individual species ; i.e. , hooh production from cu and either 
 1,2-nqn or pqn is additive ( figure 4 ) .
thus , 
 reactions between cu and quinones appear to be negligible for hooh 
 formation in this system
. this may be due to the presence of asc , 
 which rapidly cycles cu(ii ) to cu(i ) and is present at a concentration 
 that is 40010,000 times higher than the quinones ; we expect 
 a similar asc dominance in lung lining fluid in vivo .
this likely 
 causes most cu(ii ) to react with asc instead of a semiquinone radical . 

in contrast to the additive behavior of cu with quinones , the addition 
 of 500 nm fe to slf containing cu and/or 1,2-nqn , greatly reduces 
 hooh formation , to 2030% of the rate measured without fe ( figure 4 ) .
this decrease in the rate of hooh formation might 
 be because fe is suppressing the formation of hooh from cu or quinones , 
 or because fe(ii ) is destroying hooh and converting it to oh via the fenton reaction .
we can not deduce which mechanism is at 
 work here , but plan to explore this in the future by looking at oh production from the same mixtures .
based on these 
 results , fe may have an important role in suppressing hooh generation 
 in ambient pm extracts since ambient concentrations of fe are high , 
 resulting in slf concentrations up to a few micromolar under the sampling 
 conditions described in the supporting information , section s2 .
we further investigated 
 the effect of fe by quantifying hooh production in 17 laboratory solutions 
 containing mixtures of fe with cu , 1,2-nqn,1,4-nqn and/or pqn ( summarized 
 in table s3 ) .
normalized 
 hooh production rate  , which is a value between 0 and 1 calculated 
 according tor1the numerator is the measured hooh rate in 
 the mixture ( including fe ) , while the denominator is the hooh rate 
 in the absence of fe , which was either measured directly or determined 
 based on the cu and/or quinone concentrations in conjunction with 
 the concentration  response curves in table 1 . as an example , a normalized hooh production value of 0.7 
 indicates the hooh rate in the presence of fe is 70% of the rate in 
 a similar solution without fe ( i.e , .
impact of iron on hooh production in laboratory solutions and suppression 
 of hooh production in extracts of ambient particles .
panel ( a ) shows 
 the normalized hooh production , i.e. , the measured rate of hooh formation 
 in laboratory mixtures containing copper and/or quinones and iron 
 divided by the expected ( calculated ) rate from just the copper and 
 quinones .
the rate of hooh production in the absence of fe is measured 
 directly for all cases except the three diamonds marked with a bold 
 outline , where hooh production was calculated based on the concentration 
 of cu .
black lines in panel ( a ) are the model fits to the data , with 
 equations noted on the lines .
panel ( b ) shows the normalized hooh 
 production for ambient particle extracts from fresno , ca , where the 
 expected rate was calculated using only the copper concentration . 

the last two panels compare the measured rates of hooh production 
 in ambient pm extracts with ( c ) the calculated hooh rates based on 
 the fit to the laboratory solutions in panel ( a ) , and ( d ) the calculated 
 rates based only on the concentrations of soluble cu in the ambient 
 pm extracts .
figure 5a shows that fe has a similar effect 
 on all of the laboratory mixtures : the normalized rate of hooh production 
 decreases with increasing fe concentration between 0 and 270 nm and 
 is relatively stable  at approximately 20% of the rate in the 
 absence of fe  at higher fe concentrations .
we applied the same 
 analysis to 39 ambient samples collected in fresno , ca , during 2008 
 and 2009 .
we measured both the concentration 
 of soluble metals and the initial rate of hooh production from these 
 samples , but did not measure the concentration of quinones ( supporting information , section s1 ) .
we calculate the normalized rate of hooh production 
 from the ambient extracts using the expected rate from soluble cu 
 since quinone concentrations are unknown ( figure 5b ) .
compared to the laboratory mixtures , fe appears to have 
 nearly the opposite effect on hooh production in the ambient pm extracts : 
 there is generally less suppression of hooh formation as the concentration 
 of fe increases ( figure 5b ) , but the relationship 
 is very scattered and there is no strong trend .
how could higher concentrations 
 of fe lead to a smaller suppression of hooh production by fe ?
one 
 possibility is that samples with higher fe concentrations also contain 
 higher amounts of organic ligands , which are binding to fe and reducing 
 its ability to reduce hooh formation . in our laboratory solutions 

cit is the dominant ligand for fe and the fe - cit complex 
 clearly suppresses hooh formation ( figure 5a ) .
it is also possible that cu produces hooh less efficiently in 
 ambient samples compared to in our laboratory solutions due to the 
 presence of organic ligands ( figure 2 ) .
if we apply the laboratory solutions model from figure 5a to ambient pm we can calculate the expected hooh 
 production based on the concentration of cu and fe in each sample . 
 when we plot the measured versus expected hooh production using this 
 method ( figure 5c ) , the modeled data are clustered 
 around the 1:1 line , but the r value 
 for this correlation is 0.01 , indicating the model has no predictive 
 ability .
we obtain a much better relationship ( r = 0.3 ) when we calculate the expected rate of hooh production 
 based on only the concentration of soluble cu using the cu concentration 
response 
 curve ( figure 2 ) , without considering suppression 
 by fe ( figure 5d ) . in this case
the expected 
 rate of hooh production is nearly always larger than the measured 
 rate ( i.e. , points are almost all above the 1:1 line ) .
this overestimate 
 of hooh rates suggests hooh production is suppressed in ambient pm 
 extracts , but that this suppression is not strongly tied to the soluble 
 fe concentration .
it is also possible that organic ligands from the 
 particles are binding to cu and suppressing its ability to form hooh . 

if we assume that cu controls hooh formation in figure 5d ( i.e. , if the contribution from quinones is negligible ) , 
 the average ( 1 ) suppression in hooh formation by cu in 
 ambient pm extracts is 44  22% ( median suppression = 40% ; range 
 = 3.793% .
more work is needed to determine whether this suppression 
 is due to fe or to a reduction in cu reactivity by organic ligands , 
 but the chemistry of hooh production in ambient particles is clearly 
 complicated .
however , the similarity between the measured rate of 
 hooh production and that predicted by cu ( figure 5d ) indicates that cu is likely a major contributor to hooh 
 production .
because 
 of the variable impact of fe on hooh production in ambient pm extracts , 
 and the potential for particulate ligands to reduce cu reactivity , 
 we can not predict the absolute rate of hooh production based on the 
 concentration of soluble metals in ambient pm .
however , mixtures of 
 cu and quinones ( the only species able to make hooh based in figure 1 ) show that the rate of hooh production from these 
 redox - active species is additive ( figure 4 ) 
 and that fe has a similar suppressive effect on hooh production from 
 both species ( figure 5a ) . thus
, in the case 
 that fe is responsible for suppressing hooh in ambient pm extracts 
 we can still estimate the  unsuppressed  rate of hooh 
 production based on the reported range of ambient pm2.5 quinone and soluble cu concentrations ( table 
 s2 ) . under these conditions cu produces the largest rate of 
 hooh production , 0.8 to 2.9 m hooh per hour , across the entire 
 range of redox - active concentrations reported in the literature .
quinones , 
 on the other hand , exhibit very low particle - phase concentrations 
 and generally do not contribute significantly to hooh production . 
 at the highest ambient concentrations , 1,2-nqn can produce on the 
 order of 0.5 m hooh per hour , while the rates for 1,4-nqn and 
 pqn are in the range 0.00.05 m / h , which is negligible 
 compared to production from cu . if we consider the lowest , median , 
 and highest concentrations of each redox - active species , cu accounts 
 for nearly all hooh production ( 100% , 96% , and 84% , respectively ) , 
 while 1,2-nqn accounts for 0% , 2% , and 14% of total hooh for these 
 three scenarios , while the other quinones make up the remainder . though 
 the total hooh rate in ambient pm extracts will be somewhat lower 
 than these calculated rates ,
we also consider the possibility 
 that reduced cu reactivity ( rather than reactions with fe ) accounts 
 for the hooh suppression we see in ambient pm extracts ( figure 5 ) . in this case
we repeat the rate calculations 
 above but with a 44% reduction in the rate of hooh formation by cu , 
 which is the average reduction needed to explain figure 5d . in this case
, cu is still the dominant source of hooh in 
 pm extracts , accounting for 100% , 93% , and 75% of hooh formation for 
 the lowest , median , and highest concentration scenarios .
thus , even 
 if cu reactivity is suppressed in ambient pm , we expect soluble cu 
 to dominate hooh production in most ambient pm samples .
this 
 result agrees with other studies that have identified cu as 
 important for ros generation using a variety of techniques , including 
 the dtt assay , a macrophage ros assay , and hooh and oh measurements .
these results 
 also agree with a recent epidemiological study that found the cu content 
 of pm was associated with mortality in california .
one important source of cu is likely traffic emissions , which have been consistently linked to adverse health effects .
for example , rats instilled with particles from multiple sites showed 
 a statistically significantly higher response for sites with higher 
 traffic emissions and a higher concentration of cu , but no association 
 with the pah content of pm .
taken together , 
 these diverse studies provide consistent evidence that cu is an important 
 component in the health effects from airborne particles .
 inhaled 
 ambient particulate matter ( pm ) causes adverse health effects , 
 possibly by generating reactive oxygen species ( ros ) , including hydrogen 
 peroxide ( hooh ) , in the lung lining fluid . 
 there are conflicting reports 
 in the literature as to which chemical components of pm can chemically 
 generate hooh in lung fluid mimics . 
 it is also unclear which redox - active 
 species are most important for hooh formation at concentrations relevant 
 to ambient pm . to address this 
 , we use a cell - free , surrogate lung 
 fluid ( slf ) to quantify the initial rate of hooh formation from 10 
 transition metals and 4 quinones commonly identified in pm . 
 copper , 
 1,2-naphthoquinone , 1,4-naphthoquinone , and phenanthrenequinone 
 all form hooh in a slf , but only copper and 
 1,2-naphthoquinone 
 are likely important at ambient concentrations . 
 iron suppresses hooh 
 formation in laboratory solutions , but has a smaller effect in ambient 
 pm extracts , possibly because organic ligands in the particles reduce 
 the reactivity of iron . 
 overall , copper produces the majority of hooh 
 chemically generated from typical ambient pm while 1,2-naphthoquinone 
 generally makes a small contribution . 
 however , measured rates of hooh 
 formation in ambient particle extracts are lower than rates calculated 
 from soluble copper by an average ( 1 ) of 44  22% ; 
 this underestimate is likely due to either hooh destruction by fe 
 or a reduction in cu reactivity due to organic ligands from the pm . 