dna alkylating agents 
 have been used in the clinic for more than 
 60 years for the treatment of cancer and are still considered first - line 
 medication .
their mode of action involves covalent modification 
 to dna by forming genotoxic dna adducts that can interfere with replication 
 of cells , especially in rapidly dividing cancer cells .
however , a 
 drawback is the typically poor selectivity of these drugs to target 
 cancer cells , which usually results in a wide range of side effects 
 in normal cells of treated patients and arises from processes that 
 are often poorly understood .
the use of drug combinations is a common 
 strategy to limit the dose required for each single agent .
in addition , 
 reduction of unwanted toxicity can be achieved by developing drugs 
 that are selectively activated by conditions prevalent in cancer , 
 but not in normal tissues such as hypoxia , aberrant expression 
 of metabolic enzymes , or oxidative stress .
pr104a is an experimental dna - alkylating 
 hypoxia - activated prodrug 
 ( hap ) .
this drug is administered as the 
 corresponding phosphate ester pr104 , which is systemically hydrolyzed 
 to the alcohol prodrug pr104a .
pr104a undergoes metabolic nitroreduction 
 to generate cytotoxic hydroxylamine ( pr104h ) and amine ( pr104 m ) metabolites 
 ( scheme 1 ) .
one - electron 
 reductases such as cypor , mtrr , ndor1 , and nos2a mediate this activation 
 in a process that is inhibited by the presence of oxygen .
in fact , the initial radical that results from this reduction ( 1 , scheme 1 ) can be reoxidized by oxygen , whereas under hypoxic conditions , 
 further reduction generates the two major metabolites pr104h and pr104 m . 
 these metabolites induce cross - links in dna .
it has been found recently 
 that pr104a also can be activated in 
 an oxygen - independent manner by the two - electron aldo - keto reductase 
 1c3 ( akr1c3 , scheme 1 ) .
evidence for pr104a activation by 
 akr1c3 includes correlation of protein expression and oxic metabolism 
 of pr104a and enhancement of pr104 antitumor activity 
 in akr1c3-negative tumor xenograft models engineered to overexpress 
 akr1c3 . in a recent study , preconditioning of ht-29 
 colon cancer cells with a low nontoxic dose of sulforaphane ( sf ) , 
 an isothiocyanate from cruciferous vegetables with the ability to 
 induce xenobiotic - metabolizing and antioxidant enzymes , up - regulated the level of 18 proteins . among 
 them , akr1c3 was seven - fold more abundant in sf - treated cells . sf - preconditioning prior to pr104a treatment 
 sensitized ht-29 cells to pr104a and decreased the ec50 for cell death by a factor of 3.6 but did not sensitize the immortalized , 
 nontransformed normal human colonic epithelial cells ( hcec ) .
the study focused on the relationship between 
 akr1c3 levels and cytotoxicity , but the impact of sf on dna damage 
 induced by pr104a was not investigated .
there is clear evidence that dna interstrand cross - links contribute 
 to pr104a cytotoxicity , as demonstrated by increased sensitivity of 
 cell lines with genetic defects in cross - link repair and correlations between cytotoxicity and cross - link formation as 
 determined by the alkaline comet assay in cell lines in vitro and 
 in xenografts .
however , these studies 
 also demonstrated that in cell lines with low rates of metabolic activation 
 of pr104a , cytotoxicity is not accounted for by cross - link formation . 

thus , more direct methods for quantitation of dna damage by pr104a 
 are needed to better understand its mode of toxicity . as an initial 
 step
, we recently developed a high - resolution / accurate - mass ( hram ) 
 lc  ms dna adductomic method for screening for pr104a - derived dna adducts .
both mono and cross - linked dna adducts induced 
 by the two metabolites , pr104h and pr104 m , and by direct alkylation 
 of dna by pr104a were identified in dna extracted from cancer cells 
 treated with pr104a . however , to our 
 knowledge , an approach for quantifying pr104a - derived dna adducts 
 in biological samples does not exist .
studies involving the 
 application of a strategy for quantifying 
 pr104a adducts are expected to lead to improved understanding of the 
 selectivity factors driving the efficacy of metabolism - activated dna 
 alkylating agents .
therefore , a goal of this study was to quantify 
 pr104a - derived dna adducts in cells and evaluate how factors that 
 modulate the responsiveness of cells are reflected in dna adduct profiles . 

the strategy was based on the creation of a stable isotope - labeled 
 adduct mixture ( silam ) as a reference standard by enzymatic reduction 
 of pr104a in the presence of dna .
it was used as an internal standard 
 for relative quantitation of adducts by stable isotope dilution mass 
 spectrometry .
the silam was generated and characterized , and its use 
 as reference standard was validated by measuring adduct levels in 
 purified dna and in dna extracted from cells treated with pr104a . 

lc  ms analysis using the silam was carried out via a selected 
 reaction monitoring ( srm ) approach targeting 19 dna adducts induced 
 by pr104a and its metabolites pr104h and pr104 m previously detected 
 in cells via dna adductomic screening .
we used the silam - srm approach 
 to test the hypothesis that enhanced cytotoxicity observed by sf - preconditioning 
 of ht-29 cells is induced by increased level of dna adducts by measuring 
 their levels in colon cancer cells preconditioned with sf .
calf thymus dna ( ctdna ) was purchased 
 from worthington biochemical corporation ( lakewood , nj ) .
akr1c3 was 
 purchased from united states biological life sciences ( salem , ma ) . 

the reference standard [ pyridine - d4]o-[4-(3-pyridyl)-4-oxobutyl-1-yl]-2-deoxyguanosine 
 ( d4-o - pob - dg ) was prepared 
 as previously described ( isotopic purity , 98 at .
cell culture medium and supplements were purchased from invitrogen 
 ( life technologies , switzerland ) .
all solvents used for high performance 
 liquid chromatography ( hplc ) and ms analysis were of the purest grade 
 commercially available ( 99.9% ) .
ht-29 
 cells were obtained from the leibniz - institute 
 dsmz gmbh in january 2012 ( braunschweig , germany ) .
ht-29 cells were 
 grown in dmem medium with glutamax containing 10% fetal bovine serum 
 and 1% penicillin / streptomycin . immortalized normal diploid human 
 colonic epithelial cells ( clone hcec1ct , abbreviated hcec ) were obtained 
 in august 2011 from prof .
jerry shay and grown in a humidified incubator 
 containing 5% co2 at 37 c .
the two cell lines have 
 regularly been confirmed to be mycoplasma free by a luminescence detection 
 assay ( mycoalert mycoplasma detection kit , lonza , switzerland ) .
full details regarding cytotoxicity analysis , reactions of ctdna 
 with pr104a or d4-pr104a , validation experiment , 
 and quantitation of dguo by hplc for this study can be found in the supporting information .
cells were seeded in six - well 
 plates at a density of 5.0  
 10 cells / well and were allowed to attach overnight .
afterward , 
 the medium was replaced with fresh medium containing a final concentration 
 of 2.5 m sf or 0.1% dmso ( solvent control ) .
after 48 h , the 
 medium was removed , the wells were washed with pbs , and cells were 
 treated with medium containing a final concentration of 150 m 
 pr104a or 0.1% dmso ( solvent control ) and incubated at 37 c 
 for 4 h. thereafter , the dna from intact cells was isolated by extraction 
 with a dna isolation kit for cells and tissues ( roche , switzerland ) . 
 in
short , cells were washed with pbs and scraped to one side of the 
 well using a scraper .
lysis buffer ( 750 l ) was added to each 
 well , cell lysates were collected and sonicated with a vibra - cell 
 sonicator ( sonics , ct ) using the following conditions : 40% amplitude , 
 15 cycles at 10 s on and 15 s off .
one microliter of proteinase k 
 solution was added to the cell lysates , and samples were incubated 
 for 1 h at 65 c .
samples were allowed to cool to room temperature , 
 50 l of rnase solution was added , and samples were incubated 
 at 37 c for 1 h. care should be taken in removing rna from the 
 sample since adducts from the bases common to dna and rna are indistinguishable . 

a sample of 330 l of protein precipitation solution was added 
 and samples mixed vigorously , cooled on ice for 5 min , and centrifuged 
 at 16  900 g for 4 min to form a protein pellet . 

supernatants were transferred in clean 2 ml eppendorf tubes , and 1 
 ml cold isopropanol was added .
samples were gently mixed until the 
 dna pellet was visible and centrifuged at 4 c , 16  900 g for 10 min .
supernatants were removed without disturbing 
 dna pellets , which were washed with 1 ml cold etoh by centrifugation . 

the resulting dna pellets were allowed to dry at room temperature 
 for 30 min .
dna concentrations of the resulting solutions were determined 
 with a nanodrop 1000 spectrometer ( thermo scientific , waltham , ma ) . 
 the 10 mm tris - hcl/5 mm mgcl2 buffer ( ph 7 ) was added to 
 a final volume of 900 l .
samples were spiked with the silam 
 ( 1.5 l ) and thermally hydrolyzed at 80 c for 1 h. the 
 sample containing the highest amount of dna was used as reference 
 to calculate the units ( u , corresponding to the amount of the enzyme 
 that catalyzes the conversion of 1 mol of substrate per min ) 
 of enzymes for enzyme hydrolysis .
then 600 u / mg dna of dnase i was 
 added , and samples were incubated overnight at 37 c .
afterward , 
 additional dnase i ( 600 u / mg dna ) was added together with phosphodiesterase 
 i ( 20 mu / mg dna ) and alkaline phosphatase ( 240 u / mg dna ) , followed 
 by incubation at 37 c overnight .
the enzymes were removed by 
 centrifugation using a centrifree ultrafiltration device ( mw cutoff 
 30  000 atomic mass unit ( amu ) ; merck millipore , cork , ireland ) . 

an aliquot ( 60 l ) was taken from each sample for the analysis 
 of deoxyguanosine ( dguo ) by hplc .
a solution of the same buffer and 
 enzymes was used as a negative control .
hydrolysates were purified 
 by solid - phase extraction ( spe ) on a strata - x cartridge ( 30 m , 
 phenomenex , torrance , ca ) .
the samples were loaded 
 on the cartridge , washed with 1 ml of 40% ch3oh , and eluted 
 with 600 l of 80% ch3oh in h2o .
prior to lc  ms analysis , samples were dissolved in 20% ch3oh in h2o to a final volume of 10 l .
corresponding to 
 dna amounts ranging from 18 g ( cell samples ) or 1550 
 g ( ctdna samples ) , 0.5 , 2 , or 5 l of hydrolyzed , spe - purified , 
 and reconstituted sample was injected onto a nanoacquity uplc ( waters 
 co. , milford , ma ) system equipped with a 5 l injection loop . 

separation was performed with a capillary column ( 75 m i d , 
 10 cm length , 15 m orifice ) created by hand packing a commercially 
 available fused - silica emitter ( new objective , woburn ma ) with 5 m 
 luna c18 bonded separation media ( phenomenex , torrance , ca ) .
the flow 
 rate was 1000 nl / min for 5.5 min , then decreased to 300 nl / min with 
 a 50 min linear gradient from 250% ch3cn in 5 mm 
 nh4oac aqueous buffer ( ph 5.5 ) , an increase to 98% ch3cn in 3 min , with a 2 min hold and a 5 min re - equilibration 
 at 1000 nl / min at 2% ch3cn .
the injection valve was switched 
 at 5.5 min to remove the sample loop from the flow path during the 
 gradient .
data - dependent cnl - ms scanning with nanoelectrospray was carried out using 
 an ltq orbitrap velos instrument ( thermo scientific , waltham , ma ) . 

the nanoelectrospray source voltage was 2.0 kv , and the capillary 
 temperature was 350 c .
the ion focusing and transfer elements 
 of the instrument were adjusted for maximum signal intensity by using 
 the automated instrument tuning feature while the background ion signal 
 of m / z 371.1 ( decamethylcyclopentasiloxane ) 
 was monitored to create the tune file used for data analysis .
data - dependent ms analysis 
 was performed with repeated full scan detection followed by ms acquisition and constant neutral loss triggering of ms fragmentation .
full scan ( 2002000 da ) detection was 
 performed using the orbitrap detector at a resolution of 60  000 
 ( at m / z 400 ) with one microscan 
 ( one mass analysis followed by ion detection ) , automatic gain control 
 ( agc ) target settings of 1  10 , and a maximum ion 
 injection time setting of 100 ms .
ms fragmentation was 
 performed in the ion trap on the three most intense full scan ions 
 listed in a parent mass list with orbitrap detection at a resolution 
 of 7500 , agc of 2  10 , one microscan , maximum ion 
 injection time of 100 ms , and full scan injection waveforms enabled . 

the parent mass list was composed of 298 masses corresponding to [ m 
 + h ] ions of anticipated mono and cross - linked dna adducts 
 from reaction of the four dna bases with pr104a , pr104h , pr104 m , or 
 the corresponding d4-analogues .
ms fragmentation parameters 
 were as follows : 3 amu isolation width , normalized collision energy 
 of 35 , activation q of 0.25 , and activation time 
 of 10 ms .
data - dependent parameters were as follows : triggering threshold 
 of 10 000 , repeat count of one , exclusion list size of 500 , exclusion 
 duration of 60 s , and exclusion mass width of 5 ppm .
ms hcd fragmentation ( 2 amu isolation width , normalized collision 
 energy of 35 , activation time of 0.1 ms ) with orbitrap detection at 
 a resolution of 7500 was triggered upon observation of neutral losses 
 ( 5 ppm ) of 116.0474 , 151.0494 , 135.0545 , 126.0429 , and 111.0433 
 amu between the parent ion and one of the 50 most intense product 
 ions from the ms spectrum , provided a minimum signal of 
 1000 was observed .
the following ms parameters were used : 
 one microscan , agc target setting 2  10 , maximum 
 ion injection time of 100 ms . all spectra were acquired using the 
 background ion signal of m / z 371.10124 
 amu ( decamethylcyclopentasiloxane ) as a lock mass .
instrument 
 sensitivity was checked before each analysis by injection of 10 fmol 
 of labeled standard ( d4-o - pob - dg ) 
 and by integration of the observed peak in the extracted ion chromatogram 
 of the exact mass of its parent ion . for srm scanning ,
samples 
 were analyzed by nanoelectrospray in srm mode on a tsq vantage instrument 
 ( thermo scientific , waltham , ma ) .
the nanoelectrospray source voltage 
 was 1.6 kv , and the capillary temperature was 300 c .
q2 cid 
 gas pressure , 1.0 mtorr ; collision gas , argon ; scan width , m / z 0.100 ; scan time , 0.050 s ; collision 
 energy , 20 v ( base adducts ) or 15 v ( nucleoside adducts ) ; q1 peak 
 width , 0.70 amu ; and q3 peak width , 0.70 amu .
mass transitions monitored 
 were m / z 446.2311.1 , m / z 462.2311.1 , m / z 476.2341.1 , m / z 492.2341.1 , m / z 494.1359.1 , m / z 510.1359.1 , m / z 554.1403.0 , m / z 562.2446.2 , m / z 563.2428.1 , m / z 579.2428.1 , and 579.2444.1 , m / z 592.2476.1 , m / z 595.2444.1 , m / z 608.2492.2 , m / z 611.3460.1 , m / z 625.2474.2 , m / z 695.3579.2 , m / z 711.3595.2 , and m / z 741.3625.2 , 
 and the 19 isotope labeled corresponding standards ( + 4 amu ) .
the 
 tune file used for the analysis was created by maximizing signal at 
 conditions similar to those present during dna adduct elution ( 300 
 nl / min , 50% ch3cn , mass range 400800 amu ) . instrument 
 sensitivity was checked before each analysis by injection of the same 
 sample containing seven pr104a - induced dna adducts and by comparison 
 of the peak areas .
a t test was 
 used to verify whether sf - preconditioned and control samples were 
 statistically significantly different from each other ( graphpad prism 
 6 ) .
the silam to be used for relative 
 quantitation was generated by reacting purified dna from calf thymus 
 ( ctdna ) with d4-labeled pr104a in the 
 presence of akr1c3 and nadph . following a 24 h reaction , the dna was 
 precipitated , the residual d4-pr104a removed 
 by chloroform / isoamyl alcohol extraction , the dna hydrolyzed , and 
 the hydrolysate enriched by solid phase extraction .
the resulting 
 mixture then was characterized by a previously reported hram lc  ms dna adductomic approach .
briefly , repeated full scan detection followed by ms acquisition and constant neutral loss triggering of ms fragmentation ( data - dependent scanning ) to confirm the presence 
 of a dna adduct was performed by nanoelectrospray on an ltq orbitrap 
 velos .
data - dependent ms fragmentation was performed on 
 the three most intense full scan ions listed in a parent mass list 
 composed of d4-labeled masses of the [ m 
 + h ] ions of anticipated mono and cross - linked dna adducts 
 induced in the four dna bases by pr104a , pr104h , and pr104 m . ms fragmentation was performed upon observation of the accurate 
 mass neutral loss of the four dna bases ( g , 151.0494 amu ; a , 135.0545 
 amu ; t , 126.0429 amu ; and c , 111.0433 amu ) or of the 2-deoxyribose 
 ( 116.0474 amu ) .
a total of 33 isotope - labeled 
 mono and cross - linked dna adducts induced by pr104a , pr104h , and pr104 m 
 were detected in the silam by the presence of an ms fragmentation 
 event ( figure 1 , base 
 peak accurate mass extracted ion chromatograms for the 33 detected 
 adducts are shown in figure s1 in the supporting information ) .
their abundances in the silam , estimated by integration 
 of the peak areas , varied by three orders of magnitude between the 
 most to the least abundant adducts ( figure 1 ) .
for example , the deaminated nucleoside 
 monoadduct that resulted from cytosine alkylation by pr104a ( m / z 635.1245 , analyte 29 in figure 1 ) was among the less abundant 
 adducts , whereas the hydrolyzed monoadduct that resulted from n7-guanine 
 alkylation by pr104a ( m / z 496.1837 , 
 analyte 7 in figure 1 ) was among the most abundant adducts present . d4-labeled 
 pr104a dna adducts detected by ms fragmentation present 
 in the stable isotope - labeled adduct mixture 
 ( silam ) .
the silam - based method 
 for relative quantitation of pr104a - derived dna adducts was validated 
 by comparing calculated and measured dna adduct concentrations in 
 samples obtained by diluting a single ctdna sample that had been reacted 
 with pr104a into four samples containing increasing amounts of dna . 

all samples were spiked with the same amount of the silam , processed 
 as described in the materials and methods section , 
 and analyzed by lc 
results of this quantitation validation 
 experiment involving purified dna are shown in figure 2 for a representative dna cross - link adduct 
 ( m / z 625 ) in samples analyzed with 
 two different dna adductomic approaches : the hram lc  ms approach and a new developed srm approach targeting 19 dna 
 adducts induced by pr104a , pr104h , or pr104 m .
for the pr104a - induced 
 cross - linked dna adduct , the slope and r estimates were extrapolated and a very good correlation between 
 calculated dna concentration and measured adduct concentration was 
 found for both lc  ms approaches used ( figure 2 ) .
in addition to the cross - link adduct of m / z 625 , 8 versus 12 additional adducts 
 were present in the ctdna samples and quantified by the hram and srm 
 approaches , respectively ( structures , correlation plots , slopes , and r values for these adducts can be found in figures 
 s2s4 in the supporting information ) .
these adducts comprised mono and cross - linked adducts induced 
 by the two metabolites or by direct alkylation by pr104a .
for all 
 these adducts and for both the analytical approaches ( hram vs srm ) , 
 as expected , a very good correlation was found between calculated 
 dna and measured adduct concentrations ( slope and r estimates ranged from 0.921.12 and 0.971.00 
 for the hram validation , and from 0.981.16 and 0.891.00 
 for the srm targeted validation , figures s2s4 ) .
validation of the silam for relative quantitation of the pr104a - derived 
 dna adducts ( shown here for the dna cross - link with m / z 625 as a representative example ) in purified 
 dna treated with pr104a ( 100 m final concentration ) for 24 
 h. analysis was performed with both a high - resolution analysis ( hram ) 
 and with a nominal mass targeted analysis ( srm ) .
because of the higher number of adducts detected and the 
 ease of 
 use and sensitivity of quantitation of triple quadrupole instrumentation 
 operating in srm mode , we decided to use this system with the silam 
 for relative quantitation .
thus , the accuracy of quantitation of this 
 approach was evaluated for dna extracted from pr104a - treated cells . 
 in an analogous experiment to
that performed with purified dna , ht-29 
 cells were treated with pr104a ( 500 m final concentration ) , 
 and the dna was extracted and divided into two samples with one containing 
 50% more dna than the other .
these samples were spiked with the same 
 amount of the silam , the dna adducts enriched by solid phase extraction , 
 and samples were analyzed by nanolc 
a total of eight dna adducts induced by pr104a and pr104 m were 
 quantified .
deviations of the measured dna adduct concentrations from 
 the calculated dna amounts were between 0.6 and 5.5% ( table s1 ) .
the newly developed silam approach 
 was used to compare levels of pr104a - derived dna adducts in colon 
 cancer cells preconditioned with sf .
first , the experiments demonstrating 
 increased sensitivity of ht-29 cells for pr104a treatment upon sf - preconditioning 
 and the lack of alteration in sensitivity of hcec cells , which was 
 observed previously , were repeated , and the results confirmed ( figure s5 ) .
there 
 was an increase in sensitivity of ht-29 cells toward pr104a ( ec50 value 20.5 m vs 7.9 m for the nontreated vs 
 sf - treated cells ) , which represented a 2.6-fold decrease , while there 
 was no change in the case of hcec cells ( figure s5 ) .
however , the relationship between adduct formation and 
 the positive interaction of sf and pr104a was unknown .
pr104a - derived 
 dna adducts were therefore quantified in ht-29 and hcec cells preconditioned 
 with a sf concentration corresponding to the ec10 value 
 derived for sf in ht-29 cells when individually tested .
the analysis 
 was performed by spiking the samples containing dna extracted from 
 ht-29 or hcec cells preconditioned with sf and treated with pr104a 
 with the silam prior to hydrolysis and dna adduct enrichment by solid 
 phase extraction .
the analysis was performed in srm mode targeting 
 19 pr104a- , pr104h- , and pr104m - induced dna adduct masses .
the relative 
 amount of each dna adduct was calculated by dividing the ratio of 
 the peak areas corresponding to the unlabeled and labeled adducts 
 by the total dna concentration of each sample .
the total dna was determined 
 by quantitation of 2-deoxyguanosine ( dguo ) by hplc .
negative 
 control samples were samples containing buffer and enzymes used for 
 the dna hydrolysis and a sample containing dna extracted from cells 
 treated with sf only . none of the 19 targeted adducts was observed 
 in these samples .
a total of seven mono and cross - linked dna 
 adducts resulting from 
 pr104a , pr104h , or pr104 m were observed and quantified in both cell 
 lines ( figure 3 ) .
some 
 adducts were detected in both cell lines ( m / z 462 , 711 , 492 , 510 , 625 , and 741 ) , whereas two adducts 
 could be detected in one cell line only ( m / z 695 in ht-29 , and m / z 476 in hcec , figure 3 ) .
dna adducts detected included both mono and cross - linked adducts 
 from pr104a ( m / z 476 , 492 , 510 , 
 625 , and 741 ) and its reactive pr104h and pr104 m metabolites ( m / z 462 , 695 , and 711 , figure 3c ) .
sf - preconditioning of ht-29 
 cells increased the amount of dna adducts induced by the two metabolites 
 pr104h and pr104 m , but not of the dna adducts resulting from direct 
 alkylation by pr104a ( figure 3a ) .
this increase was statistically significant for two of 
 the metabolite - induced dna adducts quantified in ht-29 cells ( figure 3a ) .
on the other 
 hand , no alteration of metabolite - induced dna adduct levels upon sf - preconditioning 
 was observed in hcec cells ( figure 3b ) .
these data support a direct role of dna adducts 
 in pr104a toxicity and , moreover , support a mechanistic model for 
 the basis of how sf - preconditioning sensitizes ht-29 cells .
pr104a - derived 
 dna adduct levels and fold change relative to sf - untreated 
 controls in ( a ) ht-29 and ( b ) hcec cells upon sf - preconditioning versus 
 controls .
asterisks above the bars represent statistical significance 
 ( t test , p < 0.05 ) , whereas nd 
 represents nondetects .
( c ) proposed structures of pr104a - derived dna 
 adducts quantified in ht-29 and hcec cells .
a potential strategy to further our understanding 
 of the mechanism 
 of action of the prodrug pr104 and to support biomarker - based personalized 
 strategies for its clinical use involves using dna adducts as drug - specific 
 biomarkers of efficacy .
dna adducts have been used as mechanism - based 
 biomarkers of exposure to carcinogens for hazard identification and 
 risk assessment and for the estimation of human exposure to occupational , 
 environmental , and dietary chemicals .
examples of anticancer drug classes for which the relationship between 
 dna adducts and response were studied in vitro and in vivo include 
 platinum - based drugs , nitrogen mustards , and minor groove binding 
 agents . the advantage of monitoring drug 
dna 
 adducts as biomarkers of efficacy relies on their drug - specificity 
 and stability in dna .
the challenge relies on the ability to identifying 
 and quantifying them in biological samples since dna adducts are usually 
 present in animal and human tissues in extremely low abundance compared 
 to unmodified nucleobases ( on the order of 0.0110 adducts 
 per 10 unmodified nucleobases if induced by carcinogens , and on the order of 11000 adducts per 10 unmodified 
 nucleobases if induced by anticancer drugs ) . in this study , a dna 
 adductomic approach for the quantitation of previously detected pr104a - derived 
 dna adducts in cells was developed and 
 validated for allowing relative quantitation of pr104a - dna adducts 
 in purified dna and in dna extracted from cells treated with pr104a . 
 with the developed approach ,
dna adduct levels were measured in cells 
 preconditioned with the bioactive food compound sulforaphane ( sf ) , 
 and the relationship between pr104a - derived dna adducts and cell viability 
 response in vitro was investigated .
we found a 2.4-fold increase 
 in the level of dna adducts induced by the two metabolites pr104h 
 and pr104 m in sf - preconditioned ht-29 cells .
this increase was in 
 good agreement with the 2.6-fold increase in cytotoxicity in ht-29 . to circumvent limitations to rapid development and validation of 
 dna adduct monitoring strategies for clinical applications , we used 
 an unconventional strategy for adduct quantitation that does not require 
 synthesis of labeled standard for each adduct , but rather involves 
 the creation of a stable isotope - labeled adduct mixture ( silam ) .
quantitation 
 of dna adducts usually involves spiking the samples at an early stage 
 of the sample preparation with a chemically synthesized stable isotope - labeled 
 internal standard of a dna adduct of interest . the isotope - labeled internal standard accounts for losses 
 occurring during sample preparation , ion suppression from the sample 
 matrix , and instrument variability during lc  ms analysis .
addition of a known concentration of the stable isotope labeled 
 standard allows one to calculate the accurate concentration of the 
 adduct of interest in the sample , which results in absolute quantitation .
absolute quantitation approaches depend on the 
 availability of stable isotope labeled internal standards for adducts 
 of interest , the synthesis of which can be time - consuming and challenging 
 depending , for example , on adduct structure and stability and on the 
 cost of the stable isotope - labeled starting material .
the results 
 described herein demonstrate that using a silam reference standard 
 can allow for the simultaneous relative quantitation of several pr104a - derived 
 dna adducts in the same sample and account for adduct losses during 
 sample preparation and ion suppression from sample matrix during lc 
ms 
 analysis . on the basis of triggered ms fragmentation 
 events for 
 neutral loss of a dna adduct feature , and absence of this fragmentation 
 event in the control samples ,
the silam contained 33 detectable isotopically 
 labeled mono and cross - linked dna adducts induced by pr104a including 
 adducts ascribed to alkylation by the metabolites pr104h and pr104 m 
 ( figure 1 ) . the assignment 
 of adducts as pr104a versus pr104h or pr104 m adducts was on the basis 
 of their accurate masses and fragmentation patterns .
the composition 
 of the silam suggests the potential for deriving relative amounts 
 of up to 33 pr104a - derived dna adducts simultaneously depending on 
 whether they are present in biological samples .
the use of silam for 
 relative quantitation of pr104a - derived dna adducts was demonstrated 
 by the strong correlation between calculated and measured dna adduct 
 concentrations in a validation experiment involving pr104a - treated 
 purified dna and dna extracted from pr104a - treated cells ( figures 2 and s2s4 , and table s1 ) .
the hram orbitrap 
 analysis used for the initial pr104a - adduct discovery 
 work also provided good relative quantitation 
 in purified dna samples spiked with silam , in which nine adducts from 
 the list of target adducts were quantified .
this result is comparable 
 to the 13 adducts measured by triple quadrupole srm - targeted analysis 
 and is encouraging for future combined adduct discovery / relative quantitation 
 investigations of this type .
however , the silam triple quadrupole 
 srm - targeted approach provided for relative quantitation of more of 
 the targeted adducts and is a simpler , more widely available technology 
 and therefore was chosen for the evaluation of pr104a - derived adducts 
 in treated cells .
its accuracy and precision was evaluated for dna 
 extracted from pr104a - treated cells , and the results further supported 
 the suitability of the silam for relative quantitation of pr104a - derived 
 dna adducts ( table s1 ) .
the silam - srm 
 approach has the potential to be used for monitoring 
 dna adducts induced by dna alkylating drugs in patient samples . for 
 this purpose ,
biological samples such as tumor biopsy , circulating 
 cancer cells , and surrogate tissues could be collected from patients 
 undergoing chemotherapy and dna adducts analyzed by silam - srm .
alternatively , 
 the dna adducts can be quantified by the silam - srm approach in peripheral 
 blood mononuclear cells or tumor biopsy isolated from patients treated 
 with a microdose of the dna alkylating drug , whereas another potential 
 scenario for sensitivity testing might involve ex vivo exposure of 
 cancerous or normal surrogate cells to the dna alkylating drug with 
 evaluation of adduct formation .
we further evaluated the method in the context of characterizing 
 dna adduct profiles in colon cancer cells preconditioned with sf prior 
 to pr104a treatment .
we previously reported that the increase in pr104a 
 activity in ht-29 cells upon preconditioning with sf results from 
 an increase in abundance and activity of the enzyme akr1c3 .
increase in gene expression of akr1c3 , as for 
 the other xenobiotic - metabolizing and antioxidant enzymes modulated 
 by sf , is mediated by the transcription factor nuclear factor 
 erythroid 2-related factor 2 ( nrf2 ) .
thus , 
 sf reacts with cysteine residues of the nrf2 repressor keap1 , which 
 results in nuclear translocation of nrf2 and binding of the transcription 
 factor to dna .
higher amount of akr1c3 
 is thought to increase the level of drug activation and the production 
 of reactive hydroxylamine ( pr104h ) and amine ( pr104 m ) metabolites , 
 as demonstrated previously by analysis of the metabolites by lc 
the expectation was that sf - induced akr1c3 would lead to increased 
 metabolite - induced dna adducts ; on the other hand , dna adducts resulting 
 from direct alkylation by pr104a should not depend on the level of 
 akr1c3 .
therefore , we expected the levels of direct adducts to be 
 invariant between sf - preconditioned and directly treated cells .
this model was indeed supported by the results of this study , in 
 which preconditioning ht-29 cells with sf led to an increase in the 
 levels of dna adducts induced by the two metabolites pr104h and pr104 m 
 but did not affect the level of dna adducts resulting from direct 
 alkylation by pr104a ( figure 3a ) .
the average increase in metabolite - induced dna adducts 
 relative to sf - untreated control in ht-29 cells ( 2.4 fold ) 
 matched closely the 2.6-fold decrease observed in the ec50 value derived from the cell viability dose  response curve 
 in ht-29 cells ( figure s5 ) . among the three 
 sf - modulated metabolite - induced dna adducts quantified in ht-29 cells , 
 the one with m / z
462 was attributed 
 to a hydrolyzed monoadduct induced by the reaction of pr104 m with 
 a guanine , whereas the other two adducts with m / z 695 and 711 were attributed to cross - linked adducts induced 
 by pr104h with two adenines , and by pr104 m with two guanines , respectively 
 ( figure 3c ) .
an evaluation 
 of the individual contribution of these adducts to cytotoxicity and 
 therefore a conclusion regarding which of these adducts are more biologically 
 relevant for the observed cytotoxicity is challenging , due to the 
 heterogeneity of dna alkylation , that is , it is not possible to induce 
 and observe the cytotoxic effects of one adduct at a time , rather 
 all are formed in concert . despite the ability of interstrand cross - links 
 to prevent dna strand separation and block dna replication and transcription , their formation is much lower than that of monoadducts . 
 therefore , the overall cytotoxicity may result from a combination 
 of effects from mono and cross - linked adducts , and further research 
 outside the scope of the current study , such as using cells with the 
 selective capacity to remove particular adducts , would be of interest 
 to resolve this open question .
finally , it was anticipated that 
 there would be no change in adduct 
 levels when hcec cells were preconditioned with sf given the slight 
 akr1c3 induction and no significant increase in akr1c3 activity in 
 these nontumorigenic colonic mucosal cells .
again , this assertion could be confirmed ( figure 3b ) . to investigate reasons for different 
 cytotoxicity responses in ht-29 versus hcec cells , erzinger et al . 

measured the cellular uptake of sf but found no significant differences 
 in sf uptake between the two cell lines .
therefore , one may speculate 
 that differences in induction of akr1c3 in ht-29 and hcec cells result 
 from differences in the level of nrf2 , or in the ability to translocate 
 nrf2 into the nucleus to reach dna .
nevertheless , the absence of change 
 in adduct levels in hcec cells upon sf - preconditioning measured in 
 this study further supports the causative relationship between induction 
 of pr104a - derived adducts and cytotoxicity .
in 
 conclusion , we established in this study evidence for a direct 
 relationship between sf - preconditioning of cells , the abundance of 
 dna adducts derived from the experimental nitrogen mustard prodrug 
 pr104a , and pr104a cytotoxicity in a cancer and in a noncancerous 
 cell line .
this insight was enabled by the development of a stable 
 isotope - labeled dna adductomic approach involving srm data acquisition 
 for relative quantitation of dna adducts induced by pr104a in cells . 

the analytical approach based on nanolc  ms - srm targeting and 
 use of a silam reference mixture was useful to detect changes in the 
 abundance of pr104a - derived dna adducts at pharmacologically relevant 
 levels .
the results of the quantitation of pr104a - derived dna adducts 
 in cell lines suggest that these adducts contribute to pr104a potency , 
 underlying the possibility of using them as biomarkers of efficacy 
 to improve its chemotherapeutic selectivity and to stratify patients 
 on the basis of their susceptibility to the drug .
moreover , the general 
 dna adductomic - silam approach could be adapted for relative quantitation 
 of dna adducts induced by any dna alkylating agent using a stable 
 isotope - labeled analog of the agent , but without preparation of individual 
 labeled adducts , as a tool to support drug discovery and improve the 
 efficacy of existing anticancer drugs .
 pr104a 
 is an experimental dna - alkylating hypoxia - activated prodrug 
 that can also be activated in an oxygen - independent manner by the 
 two - electron aldo - keto reductase 1c3 . 
 nitroreduction leads to the 
 formation of cytotoxic hydroxylamine ( pr104h ) and amine ( pr104 m ) metabolites , 
 which induce dna mono and cross - linked adducts in cells . 
 pr104a - derived 
 dna adducts can be utilized as drug - specific biomarkers of efficacy 
 and as a mechanistic tool to elucidate the cellular and molecular 
 effects of pr104a . toward this goal , a mass spectrometric bioanalysis 
 approach based on a stable isotope - labeled adduct mixture ( silam ) 
 and selected 
 reaction monitoring ( srm ) data acquisition for relative 
 quantitation of pr104a - derived dna adducts in cells was developed . 
 
 use of this silam - based approach supported simultaneous relative quantitation 
 of 33 pr104a - derived dna adducts in the same sample , which allowed 
 testing of the hypothesis that the enhanced cytotoxicity , observed 
 by preconditioning cells with the transcription - activating isothiocyanate 
 sulforaphane , is induced by an increased level of dna adducts induced 
 by pr104h and pr104 m , but not pr104a . by applying the new silam - srm 
 approach 
 , we found a 2.4-fold increase in the level of dna adducts 
 induced by pr104h and pr104 m in ht-29 cells preconditioned with sulforaphane 
 and a corresponding 2.6-fold increase in cytotoxicity . 
 these results 
 suggest that dna adduct levels correlate with drug potency and underly 
 the possibility of monitoring pr104a - derived dna adducts as biomarkers 
 of efficacy . 