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Mutation Research 646 (2008) 17–24

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /molmutCommuni ty address : www.e lsev ier .com/ locate /mutres

uantification of aristolochic acid-derived DNA adducts inat kidney and liver by using liquid chromatography–electrospray ionizationass spectrometry

an Chana, Hao Yuea, Wing Tat Poonb, Yan-Wo Chanb, Oliver J. Schmitzc,aniel W.J. Kwonga, Ricky N.S. Wongd, Zongwei Caia,∗

Department of Chemistry, Hong Kong Baptist University, Hong Kong, ChinaHospital Authority Toxicology Reference Laboratory, Princess Margaret Hospital, Hong Kong, ChinaDepartment of Analytical Chemistry, University of Wuppertal, GermanyDepartment of Biology, Hong Kong Baptist University, Hong Kong, China

r t i c l e i n f o

rticle history:eceived 8 April 2008eceived in revised form 6 August 2008ccepted 26 August 2008vailable online 4 September 2008

a b s t r a c t

Aristolochic acid (AA), derived from the herbal genus Aristolochia and Asarum, has recently been shown tobe associated with the development of nephropathy. Upon enzyme activation, AA is metabolized to thearistolactam-nitrenium ion intermediate, which reacts with the exocyclic amino group of the DNA basesvia an electrophilic attack at its C7 position, leading to the formation of the corresponding DNA adducts.The AA-DNA adducts are believed to be associated with the nephrotoxic and carcinogenic effects of AA.

eywords:ristolochic acidristolochic acid nephropathyNA adductsC–MS

In this study, liquid chromatography coupled with electrospray ionization mass spectrometry (LC–MS)was used to identify and quantify the AA–DNA adducts isolated from the kidney and liver tissues of theAA-dosed rats. The deoxycytidine adduct of AA (dC–AA) and the deoxyadenosine–AA adduct (dA–AA)were detected and quantified in the tissues of rats with one single oral dose (5 mg or 30 mg AA/kg bodyweight). The deoxyguanosine adduct (dG–AA), however, was detected only in the kidney of rats that weredosed at 30 mg AA/kg body weight for three consecutive days. The amount of AA–DNA adducts found in

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. Introduction

Aristolochic acid (AA), a mixture of structurally relateditrophenanthrene carboxylic acids, derived from the herbalenus Aristolochia and Asarum [1–3], has recently been showno be associated with the development of certain renal dis-rder, namely, aristolochic acid nephropathy (AAN) and thealkan endemic nephropathy (BEN). Major components of AA

nclude aristolochic acid I (AAI, 8-methoxy-6-nitrophenanthro(3,4-)-1,3-dioxolo-5-carboxylic acid) and aristolochic acid II (AAII,-nitrophenanthro(3,4-d)-1,3-dioxolo-5-carboxylic acid) that dif-

ers from AAI by lacking a methoxy group (Fig. 1). AA is not only anown nephrotoxin [4,5] but also one of the most potent carcino-ens in the Carcinogenic Potency Database [6]. But, AA-containingerbs had been widely used to treat tumors, snake bites, obstetric

∗ Corresponding author at: Department of Chemistry, Hong Kong Baptist Univer-ity, Kowloon Tong, Kowloon, Hong Kong, China. Tel.: +852 34117070;ax: +852 34117348.

E-mail address: zwcai@hkbu.edu.hk (Z. Cai).

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027-5107/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2008.08.012

e dosage.© 2008 Elsevier B.V. All rights reserved.

ilments, rheumatism, small pox and pneumonia [7,8] until AA wasound to be a potent carcinogen in rats [9].

During a slimming regimen in Belgium in the early 90s, about00 cases of renal disease were reported due to the misuse of AA-ontaining herbs in the preparation of the slimming drugs [5]. Thebserved renal disorder was termed aristolochic acid nephropathy10], a unique rapidly progressive renal fibrosis associated with therolong intake of AA-containing herbs. AAN cases have also beeneported in France, Germany, Spain, United Kingdom, the Unitedtates, Japan and China [10]. AA–DNA adducts were detected in theidney and ureter of patients who suffered from AAN [11], evenears after the discontinued use of AA-containing herbs [12]. Itas reported that AA–DNA adducts might be associated with theevelopment of renal interstitial fibrosis and cancer in rats and inumans [12,13].

In 2001, the Food and Drug Administration (FDA) advised

onsumers to stop using any herbal products containing or are sus-ected of containing AA. The use of Aristolochia genus in herbaledicine is currently no longer permitted in the US, Canada, Aus-

ralia, most European and Asian countries. Though being bannedn many countries, Aristolochia drugs are still widely used in folk

18 W. Chan et al. / Mutation Research 646 (2008) 17–24

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edicine all over the world [10] and AA-containing herbs con-inue to be available on the internet [14]. The most recent case ofAN associated with the use of Chinese herbal preparations waseported in UK in July 2006 [15].

Balkan endemic nephropathy, a peculiar renal disease first seenn farmers along the Danube River over 50 years ago, was foundo have similar clinical and histopathological features with AAN.hough being extensively studied ever since its discovery, therothelial cancer associated with BEN was only recently shown toe the result of a chronic dietary poisoning of AA, derived fromristolochia clematitis whose seeds got mixed with the wheat grainuring harvest [16,17]. AA–DNA adducts were identified in the renalNA samples of the BEN patients [16,18], which highlighted thearcinogenic property of AA in human beings.

In the past, 32P-postlabeling assay has been extensively usedor the analysis of AA–DNA adducts [19–27]. Although the assay

as very sensitive, it gives no information regarding the chemi-

al identity of the detected DNA adducts. Furthermore, due to thetrong �-emitting property of the needed �-32P labeled ATP, 32P-ostlabeling detection of DNA adducts was limited to laboratoriesith facilities that can handle radioactive materials.

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NA adduct formation of AA.

Liquid chromatography coupled with electrospray ionizationass spectrometry (LC–MS), which combines the high separation

fficiency of HPLC and the sensitive and specific detection capa-ility of MS, has emerged as an alternative analytical tool for DNAdduct analysis [28–31]. Recently, an LC–ESI-MS method was devel-ped by Grollman et al. for the analysis of AA–DNA adducts in theenal tissue of the patients suffered from BEN [16]. Multiple stageass spectrometric analyses (MS/MS and MS3) on a 2D-QIT MSere performed for the peak identification. The AA–DNA adducts

howed characteristic fragmentation loss of a deoxyribose moi-ty with 116 Da. Characteristic fragment ions at m/z 262 and m/z92 were detected for the DNA adducts induced by AAII and AAI,espectively. Singh and Farmer reviewed the use of LC–MS for DNAdduct detection and pointed out that it was possible to determineNA adducts by LC–MS with high sensitivity [32]. Thus, LC–MSas applied to the characterization and quantification the AA–DNA

dducts isolated from rat kidney and liver in this study.Because of its persistence, AA–DNA adducts have been used as

iomarkers for AA exposure and as model compounds for investi-ating the mutagenic and carcinogenic properties of AA [16–27].ecently, we have characterized a variety of AA–DNA adducts,

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amely, dA–AAI, dA–AAII, dG–AAI, dG–AAII, dC–AAI and dC–AAIIroduced by various in vitro activation systems by using LC–ESI-S/MS technique [29]. The study is now extended to the in vivo

haracterization and quantification of AA–DNA adducts in kidneynd liver tissues from the AA-dosed rats. Dose-dependent yields ofA–AAI and dA–AAII were observed in the kidney of AA-dosed rats,ut dC–AAII was only quantified in the kidney tissue from the ratsosed with AA at high levels.

. Materials and methods

.1. Caution

Aristolochic acid is mutagenic and carcinogenic and should be handled withare.

.2. Chemicals

Aristolochic acid (96% purity), a 1:1 mixture of AAI and AAII, was purchasedrom Acros (Morris Plains, NJ, USA). Calf thymus DNA, 2-deoxyadenosine (dA),-deoxyguanosine (dG), 2-deoxycytidine (dC), DNase I, phosphodiesterase I andlkaline phosphatase were obtained from Sigma (St. Louis, MO, USA). HPLC-gradeethanol and acetonitrile were purchased from Tedia (Fairfield, OH, USA). Wateras produced from a Milli-Q Ultrapure water system with the water outlet operat-

ng at 18.2 M� (Millipore, Billerica, MA, USA). Authentic standards, namely, dA–AAI,A–AAII, dG–AAI, dG–AAII, dC–AAI and dC–AAII, were prepared and characterizedy high-resolution MS (HRMS) and MS/MS analyses as described previously [29].

.3. Preparation of dA–AA adducts

The authentic dA–AA adducts were prepared following the method described bychmeiser et al. [23] but with some modifications: To 100 mg of AA (mixture of AAInd AAII) in 20 mL of methanol, 300 mg of dA was added. This mixture was allowedo stir at room temperature for 10 min before 50 mL of water (with 1% acetic acid)as added. The solution was stirred for another 10 min and then 100 mg of zincust was added with continuous stirring. After stirring for 1 h at room temperature,0 mL of acidic water (containing 1% acetic acid) and 200 mg of zinc dust were added

n sequence. The resulting mixture, after stirring for another 1 h, was incubated at7 ◦C for 6 h in dark. The mixture was extracted three times with equal volume ofthyl acetate. The combined organic fractions, after drying with anhydrous sodiumulphate, were evaporated under reduced pressure to dryness at 30 ◦C. The residueas then dissolved in 5 mL of methanol, centrifuged and taken for preparative HPLC

eparation.The separation, carried out on a Waters Alliance 2695 HPLC system equipped

ith a 2996 PDA detector (Milford, MA, USA), was monitored at 254 nm. Aliquots100 �L) of the methanolic solution were applied to a preparative HPLC column150 mm × 6.0 mm, 5 �m, RP-18, YMC) and eluted at a flow rate of 1.5 mL/min withhe solvent mixture of 0.3% aqueous acetic acid (A) and methanol (B) using theollowing solvent gradient: initially at 30% B, raised to 80%. B in 7 min and then heldor 3 min before re-conditioning. Fractions of dA–AAII were rechromatographed onn analytical column (Hypersil BDS C18, 250 mm × 4.6 mm, 5 �m) with the sameolvent mixture but using the following solvent gradient: initially 30% B, raised to0% B in 30 min before re-conditioning. The collected dA–AAII was characterized byV absorption, fluorescence and HRMS analyses.

.4. Animal experiments

Male Sprague–Dawley rats (n = 9), weighing 180–200 g were used in this study.hey were kept in a temperature- and humidity-controlled room with artificialark/light cycles. The rats were divided into three groups and acclimatized for 5 daysrior to dosing. Groups of three rats received a single oral dose of 0, 5 or 30 mg/kgody weight of AA in 1% NaHCO3 solution, respectively. Rats were sacrificed 24 hfter the AA dosing by decapitation. Kidney and liver samples were collected andtored at −80 ◦C until DNA extraction using Trizol reagent according to the procedurerescribed by the manufacturer (Invitrogen, CA, USA).

.5. DNA digestion and adduct enrichment

The DNA isolated from tissues of the AA-dosed rats were subjected to enzymaticydrolysis as described previously [21,24]: 3.5 mL of 0.01 M Tris buffer, 5 mM of

odium chloride (pH 7.5), 150 �L DNase I (1 mg/mL) and 350 �L of 0.01 M magnesiumhloride and 0.01 M Tris buffer (pH 7.0) were added to 1 mg of modified DNA andncubated at 37 ◦C for 1 h. After addition of 4 mL of 0.2 M Tris buffer (pH 9.0) and.15 units of phosphodiesterase, the incubation was continued for another 48 h.fter that, 110 �L of alkaline phosphatase (3.5 unit/mL) was added and incubated

or another 24 h.

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arch 646 (2008) 17–24 19

The digested DNA sample was subjected to solid phase extraction (SPE) using a18 Sep-Pak cartridge (Plus, Waters) connected to a vacuum manifold. The columnas initially conditioned with 8 mL of methanol followed by 8 mL of water. Theigested DNA sample (8 mL) was then loaded onto the column and washed sequen-ially with 8 mL of water, 8 mL of methanol/water (5:95, v/v) and 5 mL of methanol.he methanol fraction was collected and evaporated to dryness under a stream ofitrogen at 37 ◦C and the residue obtained was dissolved in 50 �L of methanol prioro LC–MS analysis.

.6. LC–ESI-MS analysis

HPLC experiments were conducted on a HP 1100 capillary system equippedith an auto-sampler and a micro-pump (Agilent Technologies, Palo Alto, CA, USA).everse phase column (Phenomenex, Lunar C18, 150 mm × 2.0 mm, 5 �m) was usedo separate the AA–DNA adducts from the unmodified nucleosides. Injection vol-me was 8 �L. The mobile phase consisted of two components, with component IA) being 0.2% acetic acid, component II (B) being acetonitrile. The flow rate was sett 200 �L/min. In the analysis of AA–DNA adducts, the solvent gradient began at 20%and held for 5 min, then increased to 80% B in 5 min, and held for another 15 min.

n the first 10 min, effluent from the LC was diverted to waste in order to minimizeontamination of the ESI source. For the analysis of the unmodified nucleosides, theollowing solvent gradient program was used: initially, 20% B was held for 4 min,hen raised to 100% B in 1 min and held for another 4 min before re-conditioning.

HRMS and MS/MS analyses were conducted on a hybrid quadrupole time-of-ight (Qq-TOF) tandem mass spectrometer (API Q-STAR Pulsar i, MDS Sciex, Toronto,anada). Positive ion mode ESI-MS was used for the analysis, with the TurboIonsprayarameters for AA–DNA adducts optimized as follows: ionspray voltage (IS), 4800 V;eclustering potential I (DPI), 20 V; declustering potential II (DPII), 15 V and focus-

ng potential (FP), 50 V. The mass range chosen was m/z 400–650. The ion sourceas I (GSI), gas II (GSII), curtain gas (CUR), collision gas (CAD) and the tempera-ure of GSII were set at 30, 15, 30, 3 and 300 ◦C, respectively. The HRMS analysis,hich employs a TOF mass spectrometer for the accurate mass determination of

hemical compounds, provided the mass difference of less than 10 ppm betweenhe measured and corresponding theoretical values for all targeted molecular ions(measured m/z − theoretical m/z)/theoretical m/z × 106].

.7. Quantitative analysis of AA–DNA adducts in rat kidney and liver tissues

The DNA adduct dA–AAII was used as the standard to quantify the AA–DNAdducts in the rat tissues (kidney and liver). Different amounts of the dA–AAIItandard (ranged from 0.7 to 84.4 pmol on column) were spiked to blank CT–DNAigestion extract and analyzed by LC–MS. The calibration line, obtained by plottinghe peak areas of the extracted ion chromatograms (XIC) vs. the amount of the stan-ard on column, was used for quantification of AA–DNA content in the rat tissues.he concentration of the unmodified nucleosides was determined by LC–MS analysisf the diluted DNA digest, which was obtained by diluting 50 �L of the digested DNAamples with 450 �L of methanol/water (50:50, v/v). The AA–DNA adduct concen-rations ([AA–DNA]) were expressed in terms of adducts per normal 109 nucleotides.

AA–DNA] = No. of AA–DNA adductsNo. of normal nucleotides

× 109

. Results

.1. Characterization of 7-(deoxyadenosin-N6-yl)-aristolactam IIdA–AAII)

Reduction of AA in the presence of DNA leads to the for-ation of AA–DNA adducts [13] (Fig. 1). The preparation of

eoxynucleoside-AA adducts from 2-deoxynucleosides and AAIr AAII using xanthine oxidase (XO) and hypoxanthine has beeneported previously with the product yields for dA–AAI and dG–AAIs low as 0.1–0.4% [33,34]. Under these (enzymatic) conditions, AAas found to be either not-reacted or reduced to aristolactams.owever, by using zinc-acetic acid (1%) as the activation system,

he reduction of AAII could reach completion in 6 h, giving a yieldf 1.8% for dA–AAII with the remaining AA reduced to aristolactams.

The dA–AAII adduct was isolated from the reaction mixture by

reparative HPLC and purified by analytical HPLC. The purifieddduct, after freeze-drying, was dissolved in methanol and ana-yzed by UV absorption, fluorescence spectroscopy and HRMS. TheV spectrum of dA–AAII showing absorption maxima at 267, 282,93 and 396 nm was identical to that reported in the literature [34].

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he fluorescence spectrum (in methanol) showed a broad emissionaximum at 464 nm (�ex 291 nm) and three excitation maxima at

91, 340 and 394 nm (�em 464 nm). The HRMS analysis showedon peaks at m/z 513.1533 and m/z 397.1057, corresponding to theM+H]+ and [M−deoxyribose+H]+ ions.

.2. Quantitative analysis of AA–DNA adducts

The DNA samples that were extracted from rat kidney and liverissues after a single oral administration of AA were digested,PE enriched and analyzed by LC–MS. The recovery of AA–DNAdducts in the SPE enrichment process (Section 2.5) as deter-ined by spiking 50 pmol of dA–AAII into 8 mL of blank CT–DNA

igest was determined to be 96.9 ± 6.6% (n = 5). The reproducibil-ty of the LC–MS method developed, assessed by seven replicatednalysis of dA–AAII (0.7 pmol dA–AAII/injection) in blank CT–DNAigest, showed a relative standard deviation (R.S.D., standard devi-tion/mean × 100%) of 8.2%. The limit of detection, defined as themount of dA–AAII that generated a response that was three timeshe standard deviation of the baseline noise (i.e., S/N = 3), was.04 pmol/injection.

Three AA–DNA adducts, namely, dA–AAII (Fig. 2B and C), dA–AAIFig. 2F and G) and dC–AAII (Fig. 3), were detected in kidney tissuef rats dosed with 5 and 30 mg/kg of AA by LC–MS. The identifica-ion of the AA–DNA adducts in the sample extracts was based onhe chromatographic retention time and HRMS data. No AA–DNAdducts were detected in the samples taken from the kidney andiver tissues of the control rats. By using the purified dA–AAII astandard, the concentrations of dA–AAII, dA–AAI and dC–AAII inhe tissues of the AA-dosed rats were determined. The determina-ion was conducted by assuming that dA–AAI and dC–AAII had theame ESI-MS response as dA–AAII.

The levels of total AA–DNA adducts in the kidney tissue of ratsxposed to 5 and 30 mg/kg of AA were found to be 2.5 and 11.4/109

ormal nucleotides, respectively (Table 1). Although the dC–AAIIdduct was detected in the tissue samples from the kidney of ratsosed with 5 mg/kg of AA with the S/N > 3, its concentration waselow the limit of quantification defined at S/N > 10. The level ofA–AAII adduct found in the kidney samples was higher than thatf dA–AAI and much higher than that of dC–AAII. For the rats dosedith 30 mg/kg of AA, the analysis of kidney DNA showed that the

oncentration of dA–AAII was 1.6 and 5.2 times of dA–AAI andC–AAII, respectively.

The dA–AAII and dA–AAI adducts were also detected and quan-ified in the liver tissue of rats dosed with 30 mg/kg of AA. Whilehe dA–AAII level was significantly higher than that of dA–AAI inidney tissue, the levels of these two DNA adducts were similar iniver tissue. The total AA–DNA adduct concentration was 3.6/109

able 1A–DNA adduct concentrations in the kidney and liver tissues of rats treated withsingle oral dose of AA with different dosing levels

issue Adduct Aristolochic acidsa (mg/kg)

5 30

idney dA–AAII 1.6 ± 0.2b 6.2 ± 1.0dA–AAI 0.9 ± 0.1 4.0 ± 0.5dC–AAII NQc 1.2 ± 0.4

iver dA–AAII NDd 1.6 ± 0.2dA–AAI ND 2.0 ± 0.1

Total adduct level in kidney and liver 2.5 15.0

a Aristolochic acids containing AAI and AAII (1:1).b Mean ± standard deviation for adducts/109 normal nucleotide (n = 3).c Not quantified.d Not detected.

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arch 646 (2008) 17–24

ormal nucleotides in the liver of rats exposed to 30 mg/kg of AA,hich was three to four times lower than that in the kidney tissue.o AA–DNA adducts were detected in the liver of rats dosed withA at 5 mg/kg.

The dG–AA adducts were not detected in the DNA samples ofhe rats after a single oral dose of AA. dG–AAI and dG–AAII wereetected in rats kidney samples after 30 mg/kg dosage of AA forhree consecutive days (Fig. 4). It was however, they were not quan-ified because the peaks were below the limit of quantificationefined at S/N > 10.

. Discussion

As nitrophenanthrene carboxylic acid alkaloids, AAI and AAIIndergo nitro-reduction to form reactive aristolactam-nitrenium

ons. Electrophilic attack of aristolactam-nitrenium ion via its7 position to the exocyclic amino group in the DNA bases ledo the formation of major adducts (Fig. 1) [13]. The AA–DNAdducts were identified spectroscopically as 7-(deoxyadenosin-N6-l)-aristolactam I (dA–AAI), 7-(deoxyguanosin-N6-yl)-aristolactam(dG–AAI), 7-(deoxyadenosin-N6-yl)-aristolactam II (dA–AAII)

nd 7-(deoxyguanosin-N6-yl)-aristolactam II (dG–AAII). While theeoxyadenosine adducts dA–AAI and dA–AAII exhibited iminoharacter, the deoxyguanosine adducts dG–AAI and dG–AAII dis-layed a secondary amine type of bonding [33,34]. Characterizationnd quantification of the adducts are important for elucidatinghe pathway of activation as well as the carcinogenic and nephro-oxic effects of AA [13]. In our previous in vitro study of the DNAdducts induced by AA, the HRMS and MS/MS capability of a Qq-OF MS were demonstrated to be efficient for the characterizationf AA–DNA adducts [29].

The AA–DNA adducts were detected in forestomach, glandulartomach, liver, kidney and urinary bladder of rodents [13]. Evenhough the same adduct pattern was found, AA only targeted kidneynd forestomach for tumor induction [26]. Thus, AA exhibited aigher carcinogenicity in the kidney and forestomach [27]. In thistudy, the DNA adduct formation was investigated by LC–MS in bothiver (non-target tissue) and kidney (target tissue) of the rats dosed

ith AA to better understand the genotoxicity of AA. Three AA–DNAdducts, namely, dA–AAII (Fig. 2B and C), dA–AAI (Fig. 2F and G) andC–AAII (Fig. 3), were detected in the kidney tissue of rats dosedith 5 and 30 mg/kg of AA. Though dC–AA adducts were detected

n previous in vitro studies by 32P-postlabeling [35,36] and LC–MS29] analyses, the detection of dC–AA in vivo has not been reported.o our best knowledge, this is the first report of dC–AA adductsetected in vivo. The detection of dC–AAII demonstrated that LC–MSight be superior to the 32P-postlabeling assay for DNA adduct

nalysis because of its capability for the structure characterizationf unknown DNA adducts.

dA–AAII was synthesized via an in vitro reaction and used ashe standard for the quantitative analysis of the tissue samples.n this study, standards of dA–AAI and dC–AAII were not avail-ble because of their low reaction yield even under the optimizedynthetic protocol (Section 2.3). Because dA and dC showed simi-ar proton affinity [37], it was assumed that dA–AAII, dA–AAI andC–AAII had the same ESI-MS response for the quantification ofA–AAI and dC–AAII. Higher concentrations of the DNA adductsere found in the kidney tissue of rats dosed with higher level ofA (Table 1). The concentrations of dA–AAII and dA–AAI in the kid-ey samples of rats dosed with 30 mg/kg of AA were 3.9 and 4.4

imes higher than those of the rats dosed with 5 mg/kg AA, respec-ively. The relative abundance of the AA–DNA adducts observed inidney was dA–AAII > dA–AAI > dC–AAII. This pattern agrees withhe reported observation in recent studies [26,27] that AAII gen-rated a higher level of DNA adducts than AAI in the target tissue

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kidney). Given that the content ratio of AAI and AAII was 1:1 in theA dosing standard, the reason of the detected different abundancef the DNA adducts might be attributed to the different repair effi-iencies towards DNA adducts derived from AAI and AAII and/orhe different rates of conversion of AAI and AAII to their reactiveristolactam-nitrenium ion by the nitroreductase enzymes [27].

The DNA adduct dA–AAI detected in this study was found toe at concentration of 0.9 adducts/109 normal nucleotides in kid-ey DNA samples of the rats that were dosed with AA (mixturef AAI and AAII) at 5 mg/kg. The result was different from thatas reported Bieler et al. in which dA–AAI was detected approx-

mately as 70 adducts/109 normal nucleotides when Wister ratsere given AAI at 5 mg/kg of body weight [11]. A higher concen-

ration of dA–AAI was also detected in the study by Fernando et al.hen male Wister rats were given a single oral dose of 13.8 mmol

f AAI [38]. Apart from the lowered dosage that was used in ourtudy (2.5 mg AAI/kg), the discrepancy may have been arose by theifferent type of rats that were used (S.D. vs. Wister). The possibil-

ty of mutual competition of AAI and AAII in DNA binding sites or

n active sites of the enzymes may be the other causal factors forhe observed discrepancy.

In the liver tissue samples from the rats dosed with AA 5 mg/kg,o AA–DNA adducts were detected (Table 1). However, dA–AAII

asDb

ig. 2. Extracted ion chromatograms of dA–AAII (m/z 513.0–513.2) obtained from LC–MSell as the kidney tissue samples of rats after a single dosage of AA at 30 mg/kg (B), 5 mg

m/z 543.0–543.2) obtained from LC–MS analyses of the sample extract from in vitro inespectively. High-resolution ESI-MS spectra inserted.

arch 646 (2008) 17–24 21

nd dA–AAI were identified in the liver samples with the dosagef 30 mg/kg. The concentration of dA–AAI in the liver tissue was.0/109 normal nucleotide, which was slightly higher than that ofA–AAII (1.6/109 normal nucleotide). The observation of slightlyigher levels of dA–AAI compared to dA–AAII in rat liver tissue haseen reported by Mei et al. [26] and Dong et al. [27].

The levels of dA–AAI and dA–AAII in kidney were found toe 2.0 and 3.9 times higher than those in the liver, respectively,hich was similar to the results reported in the literature [27].

he observed difference might have been attributed by the dif-erent repair efficiencies and/or different activation rates of thenzymes, e.g., nitroreductase(s), cytochrome P450 1A1 and P450A2, prostaglandin H synthase in these organs [13].

dG–AAI and dG–AAII adducts were not detected in both kid-ey and liver tissue samples from the rats receiving one singleral dose of AA. It was reported that the concentration ratios ofG–AA:dA–AA adducts were 1:2.6–1:33 in the internal organs ofA-dosed rats and the kidney and ureteric tissue of CHN patients

11,25–27]. Lower level of dG–AA adducts compared to dA–AA

dducts was also observed in the in vitro experiment [22,23,39,40],uggesting a lower dG–AA yield in the reaction between AA andNA. The different yield might be the result of the different accessi-ility of the AA-reactive amino group between dG and dA in the DNA

analyses of dA–AAII standard (0.7 pmol) in blank CT–DNA digestion extract (A) as/kg (C) and 0 mg/kg (D). (E), (F), (G) and (H) showed the chromatograms of dA–AAIcubation as well as the kidney tissue samples of rats dosed at 30, 5 and 0 mg/kg,

22 W. Chan et al. / Mutation Research 646 (2008) 17–24

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ig. 3. Extracted ion chromatograms of the dC–AAII (m/z 489.0–489.2) obtained fromf rats dosed with AA at 30 mg/kg (B), 5 mg/kg (C) and 0 mg/kg (D). High-resolution

elical structure. The exocyclic amino group of dG was found in theather narrow minor groove of DNA, whereas that of dA was in theore openly accessible major groove [41]. The more easily acces-

ible amino group on dA might account for the higher dA–AA yieldbserved. The same explanation might also be applied to the detec-ion of dC–AA adduct, where the reactive amino group is located inhe major groove.

To accumulate sufficient amount of dG–AA adduct for the LC–MSnalysis, additional experiment using rats multiply dosed withA at 30 mg/kg for three consecutive days was performed. Under

he identical experimental conditions and instrument settings, theG–AAI and dG–AAII adducts were detected at S/N > 3. However, thedducts were not quantified in the kidney samples (Fig. 4) becausehe peak was below the limit of quantification defined at S/N > 10.

o dG–AA adducts were detected in the liver tissue samples. Theon-detection of dG–AA adducts in the liver DNA samples couldave been attributed by the relative lowered adduct concentrationhen compared with the kidney DNA samples. This is supported by

he observation that the concentration of dA–AA adducts was three

i

lae

S analysis of the in vitro incubation sample (A) as well as the kidney tissue samplesS spectra were inserted.

o four times lower than that in the kidney tissue (see Section 3).he use of more sensitive/selective mass spectrometry e.g., tripleuadrupole mass spectrometer may allow better identification anduantification of the dG–AA adducts.

Chronic [4,42] and acute [43] nephrotoxicity have been observedn laboratory rodents upon the AA dosing. Dose-dependent renalesion was observed in AA-dosed rats [4]. It was suggested that theA–DNA adducts somehow trigger the fibrotic process that pro-ressively destroys the kidney of the AAN and BEN patients [13].he current study demonstrated the significant higher concentra-ion of AA–DNA adducts detected in kidney than in liver tissuend the dose-dependence of AA–DNA concentration in the kidneyNA (Table 1). The obtained results supports the postulation thatA–DNA adducts might have been associated with the nephrotox-

city of AA.The amount of DNA samples required for this study is relative

arge when compared with that required for previous LC–ESI-MSnalysis, in which a triple quadrupole MS was used [44]. It is how-ver, the high resolution and MS/MS capability of the Qq-TOF MS

W. Chan et al. / Mutation Research 646 (2008) 17–24 23

F from Ls g (C)( ampleH

iw

s[atrwa

C

A

ChasaF(

R

[

[

[

ig. 4. Extracted ion chromatograms of the dG–AAII (m/z 529.0–529.3) obtainedamples of rats that were multiply dosed with AA at with 30 mg/kg (B) and 0 mg/km/z 559.0–559.3) for the in vitro incubation sample as well as the kidney tissue sigh-resolution ESI-MS spectra were inserted.

s excellent for structural elucidation of the DNA adduct, especiallyhen it comes to the study of unknown/new DNA adducts.

Strong fluorescence was observed for the AA–DNA adducts,imilar to the nitroreduction derivatives of AA or aristolactams3]. Therefore, HPLC with fluorescence detection might provide anlternative method for the analysis of AA–DNA adducts other thanhe LC–MS and radioactive labeling assays. Further investigation ofat urine and other internal organs of rats dosed with AA by HPLCith fluorescence detection might provide additional information

bout the carcinogenicity of AA.

onflict of interest

The authors declare that there are no conflicts of interest.

cknowledgements

We are grateful to Dr. Jian Zhen Yu of the Department ofhemistry, Hong Kong University of Science and Technology forer suggestions in doing the quantitative analysis of the AA–DNAdducts. The supports of the Research Grant Council, Univer-ity Grants Committee of Hong Kong (HKBU2459/06M), the Foodnd Health Bureau and Health and Health Services Researchund (05060141) of Hong Kong and the Sino-German CorporationGZ364) on this study are acknowledged.

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