Quantification of aristolochic acid-derived DNA adducts in rat kidney and liver by using liquid chromatography–electrospray ionization mass spectrometry

Mutation Research 646 (2008) 17–24

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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres

Quantification of aristolochic acid-derived DNA adducts in rat kidney and liver by using liquid chromatography–electrospray ionization mass spectrometry Wan Chan a , Hao Yue a , Wing Tat Poon b , Yan-Wo Chan b , Oliver J. Schmitz c , Daniel W.J. Kwong a , Ricky N.S. Wong d , Zongwei Cai a,∗ a

Department of Chemistry, Hong Kong Baptist University, Hong Kong, China Hospital Authority Toxicology Reference Laboratory, Princess Margaret Hospital, Hong Kong, China c Department of Analytical Chemistry, University of Wuppertal, Germany d Department of Biology, Hong Kong Baptist University, Hong Kong, China b

a r t i c l e

i n f o

Article history: Received 8 April 2008 Received in revised form 6 August 2008 Accepted 26 August 2008 Available online 4 September 2008 Keywords: Aristolochic acid Aristolochic acid nephropathy DNA adducts LC–MS

a b s t r a c t Aristolochic acid (AA), derived from the herbal genus Aristolochia and Asarum, has recently been shown to be associated with the development of nephropathy. Upon enzyme activation, AA is metabolized to the aristolactam-nitrenium ion intermediate, which reacts with the exocyclic amino group of the DNA bases via 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. 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 the AA-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 body weight). The deoxyguanosine adduct (dG–AA), however, was detected only in the kidney of rats that were dosed at 30 mg AA/kg body weight for three consecutive days. The amount of AA–DNA adducts found in the rats correlated well with the dosage. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Aristolochic acid (AA), a mixture of structurally related nitrophenanthrene carboxylic acids, derived from the herbal genus Aristolochia and Asarum [1–3], has recently been shown to be associated with the development of certain renal disorder, namely, aristolochic acid nephropathy (AAN) and the Balkan endemic nephropathy (BEN). Major components of AA include aristolochic acid I (AAI, 8-methoxy-6-nitrophenanthro(3,4d)-1,3-dioxolo-5-carboxylic acid) and aristolochic acid II (AAII, 6-nitrophenanthro(3,4-d)-1,3-dioxolo-5-carboxylic acid) that differs from AAI by lacking a methoxy group (Fig. 1). AA is not only a known nephrotoxin [4,5] but also one of the most potent carcinogens in the Carcinogenic Potency Database [6]. But, AA-containing herbs had been widely used to treat tumors, snake bites, obstetric

∗ Corresponding author at: Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong, China. Tel.: +852 34117070; fax: +852 34117348. E-mail address: [email protected] (Z. Cai). 0027-5107/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2008.08.012

ailments, rheumatism, small pox and pneumonia [7,8] until AA was found to be a potent carcinogen in rats [9]. During a slimming regimen in Belgium in the early 90s, about 100 cases of renal disease were reported due to the misuse of AAcontaining herbs in the preparation of the slimming drugs [5]. The observed renal disorder was termed aristolochic acid nephropathy [10], a unique rapidly progressive renal fibrosis associated with the prolong intake of AA-containing herbs. AAN cases have also been reported in France, Germany, Spain, United Kingdom, the United States, Japan and China [10]. AA–DNA adducts were detected in the kidney and ureter of patients who suffered from AAN [11], even years after the discontinued use of AA-containing herbs [12]. It was reported that AA–DNA adducts might be associated with the development of renal interstitial fibrosis and cancer in rats and in humans [12,13]. In 2001, the Food and Drug Administration (FDA) advised consumers to stop using any herbal products containing or are suspected of containing AA. The use of Aristolochia genus in herbal medicine is currently no longer permitted in the US, Canada, Australia, most European and Asian countries. Though being banned in many countries, Aristolochia drugs are still widely used in folk

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Fig. 1. Metabolic activation and DNA adduct formation of AA.

medicine all over the world [10] and AA-containing herbs continue to be available on the internet [14]. The most recent case of AAN associated with the use of Chinese herbal preparations was reported in UK in July 2006 [15]. Balkan endemic nephropathy, a peculiar renal disease first seen in farmers along the Danube River over 50 years ago, was found to have similar clinical and histopathological features with AAN. Though being extensively studied ever since its discovery, the urothelial cancer associated with BEN was only recently shown to be the result of a chronic dietary poisoning of AA, derived from Aristolochia clematitis whose seeds got mixed with the wheat grain during harvest [16,17]. AA–DNA adducts were identified in the renal DNA samples of the BEN patients [16,18], which highlighted the carcinogenic property of AA in human beings. In the past, 32 P-postlabeling assay has been extensively used for the analysis of AA–DNA adducts [19–27]. Although the assay was very sensitive, it gives no information regarding the chemical identity of the detected DNA adducts. Furthermore, due to the strong ␤-emitting property of the needed ␥-32 P labeled ATP, 32 Ppostlabeling detection of DNA adducts was limited to laboratories with facilities that can handle radioactive materials.

Liquid chromatography coupled with electrospray ionization mass spectrometry (LC–MS), which combines the high separation efficiency of HPLC and the sensitive and specific detection capability of MS, has emerged as an alternative analytical tool for DNA adduct analysis [28–31]. Recently, an LC–ESI-MS method was developed by Grollman et al. for the analysis of AA–DNA adducts in the renal tissue of the patients suffered from BEN [16]. Multiple stage mass spectrometric analyses (MS/MS and MS3 ) on a 2D-QIT MS were performed for the peak identification. The AA–DNA adducts showed characteristic fragmentation loss of a deoxyribose moiety with 116 Da. Characteristic fragment ions at m/z 262 and m/z 292 were detected for the DNA adducts induced by AAII and AAI, respectively. Singh and Farmer reviewed the use of LC–MS for DNA adduct detection and pointed out that it was possible to determine DNA adducts by LC–MS with high sensitivity [32]. Thus, LC–MS was applied to the characterization and quantification the AA–DNA adducts isolated from rat kidney and liver in this study. Because of its persistence, AA–DNA adducts have been used as biomarkers for AA exposure and as model compounds for investigating the mutagenic and carcinogenic properties of AA [16–27]. Recently, we have characterized a variety of AA–DNA adducts,

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namely, dA–AAI, dA–AAII, dG–AAI, dG–AAII, dC–AAI and dC–AAII produced by various in vitro activation systems by using LC–ESIMS/MS technique [29]. The study is now extended to the in vivo characterization and quantification of AA–DNA adducts in kidney and liver tissues from the AA-dosed rats. Dose-dependent yields of dA–AAI and dA–AAII were observed in the kidney of AA-dosed rats, but dC–AAII was only quantified in the kidney tissue from the rats dosed with AA at high levels.

The digested DNA sample was subjected to solid phase extraction (SPE) using a C18 Sep-Pak cartridge (Plus, Waters) connected to a vacuum manifold. The column was initially conditioned with 8 mL of methanol followed by 8 mL of water. The digested DNA sample (8 mL) was then loaded onto the column and washed sequentially with 8 mL of water, 8 mL of methanol/water (5:95, v/v) and 5 mL of methanol. The methanol fraction was collected and evaporated to dryness under a stream of nitrogen at 37 ◦ C and the residue obtained was dissolved in 50 ␮L of methanol prior to LC–MS analysis.

2. Materials and methods

HPLC experiments were conducted on a HP 1100 capillary system equipped with an auto-sampler and a micro-pump (Agilent Technologies, Palo Alto, CA, USA). Reverse phase column (Phenomenex, Lunar C18 , 150 mm × 2.0 mm, 5 ␮m) was used to separate the AA–DNA adducts from the unmodified nucleosides. Injection volume was 8 ␮L. The mobile phase consisted of two components, with component I (A) being 0.2% acetic acid, component II (B) being acetonitrile. The flow rate was set at 200 ␮L/min. In the analysis of AA–DNA adducts, the solvent gradient began at 20% B and held for 5 min, then increased to 80% B in 5 min, and held for another 15 min. In the first 10 min, effluent from the LC was diverted to waste in order to minimize contamination of the ESI source. For the analysis of the unmodified nucleosides, the following solvent gradient program was used: initially, 20% B was held for 4 min, then 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-offlight (Qq-TOF) tandem mass spectrometer (API Q-STAR Pulsar i, MDS Sciex, Toronto, Canada). Positive ion mode ESI-MS was used for the analysis, with the TurboIonspray parameters for AA–DNA adducts optimized as follows: ionspray voltage (IS), 4800 V; declustering potential I (DPI), 20 V; declustering potential II (DPII), 15 V and focusing potential (FP), 50 V. The mass range chosen was m/z 400–650. The ion source gas I (GSI), gas II (GSII), curtain gas (CUR), collision gas (CAD) and the temperature of GSII were set at 30, 15, 30, 3 and 300 ◦ C, respectively. The HRMS analysis, which employs a TOF mass spectrometer for the accurate mass determination of chemical compounds, provided the mass difference of less than 10 ppm between the measured and corresponding theoretical values for all targeted molecular ions [(measured m/z − theoretical m/z)/theoretical m/z × 106 ].

2.1. Caution Aristolochic acid is mutagenic and carcinogenic and should be handled with care. 2.2. Chemicals Aristolochic acid (96% purity), a 1:1 mixture of AAI and AAII, was purchased from Acros (Morris Plains, NJ, USA). Calf thymus DNA, 2-deoxyadenosine (dA), 2-deoxyguanosine (dG), 2-deoxycytidine (dC), DNase I, phosphodiesterase I and alkaline phosphatase were obtained from Sigma (St. Louis, MO, USA). HPLC-grade methanol and acetonitrile were purchased from Tedia (Fairfield, OH, USA). Water was produced from a Milli-Q Ultrapure water system with the water outlet operating at 18.2 M (Millipore, Billerica, MA, USA). Authentic standards, namely, dA–AAI, dA–AAII, dG–AAI, dG–AAII, dC–AAI and dC–AAII, were prepared and characterized by high-resolution MS (HRMS) and MS/MS analyses as described previously [29]. 2.3. Preparation of dA–AA adducts The authentic dA–AA adducts were prepared following the method described by Schmeiser et al. [23] but with some modifications: To 100 mg of AA (mixture of AAI and AAII) in 20 mL of methanol, 300 mg of dA was added. This mixture was allowed to stir at room temperature for 10 min before 50 mL of water (with 1% acetic acid) was added. The solution was stirred for another 10 min and then 100 mg of zinc dust was added with continuous stirring. After stirring for 1 h at room temperature, 30 mL of acidic water (containing 1% acetic acid) and 200 mg of zinc dust were added in sequence. The resulting mixture, after stirring for another 1 h, was incubated at 37 ◦ C for 6 h in dark. The mixture was extracted three times with equal volume of ethyl acetate. The combined organic fractions, after drying with anhydrous sodium sulphate, were evaporated under reduced pressure to dryness at 30 ◦ C. The residue was then dissolved in 5 mL of methanol, centrifuged and taken for preparative HPLC separation. The separation, carried out on a Waters Alliance 2695 HPLC system equipped with a 2996 PDA detector (Milford, MA, USA), was monitored at 254 nm. Aliquots (100 ␮L) of the methanolic solution were applied to a preparative HPLC column (150 mm × 6.0 mm, 5 ␮m, RP-18, YMC) and eluted at a flow rate of 1.5 mL/min with the solvent mixture of 0.3% aqueous acetic acid (A) and methanol (B) using the following solvent gradient: initially at 30% B, raised to 80%. B in 7 min and then held for 3 min before re-conditioning. Fractions of dA–AAII were rechromatographed on an analytical column (Hypersil BDS C18, 250 mm × 4.6 mm, 5 ␮m) with the same solvent mixture but using the following solvent gradient: initially 30% B, raised to 80% B in 30 min before re-conditioning. The collected dA–AAII was characterized by UV absorption, fluorescence and HRMS analyses. 2.4. Animal experiments Male Sprague–Dawley rats (n = 9), weighing 180–200 g were used in this study. They were kept in a temperature- and humidity-controlled room with artificial dark/light cycles. The rats were divided into three groups and acclimatized for 5 days prior to dosing. Groups of three rats received a single oral dose of 0, 5 or 30 mg/kg body weight of AA in 1% NaHCO3 solution, respectively. Rats were sacrificed 24 h after the AA dosing by decapitation. Kidney and liver samples were collected and stored at −80 ◦ C until DNA extraction using Trizol reagent according to the procedure prescribed by the manufacturer (Invitrogen, CA, USA). 2.5. DNA digestion and adduct enrichment The DNA isolated from tissues of the AA-dosed rats were subjected to enzymatic hydrolysis as described previously [21,24]: 3.5 mL of 0.01 M Tris buffer, 5 mM of sodium chloride (pH 7.5), 150 ␮L DNase I (1 mg/mL) and 350 ␮L of 0.01 M magnesium chloride and 0.01 M Tris buffer (pH 7.0) were added to 1 mg of modified DNA and incubated at 37 ◦ C for 1 h. After addition of 4 mL of 0.2 M Tris buffer (pH 9.0) and 0.15 units of phosphodiesterase, the incubation was continued for another 48 h. After that, 110 ␮L of alkaline phosphatase (3.5 unit/mL) was added and incubated for another 24 h.

2.6. LC–ESI-MS analysis

2.7. 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–DNA adducts in the rat tissues (kidney and liver). Different amounts of the dA–AAII standard (ranged from 0.7 to 84.4 pmol on column) were spiked to blank CT–DNA digestion extract and analyzed by LC–MS. The calibration line, obtained by plotting the peak areas of the extracted ion chromatograms (XIC) vs. the amount of the standard on column, was used for quantification of AA–DNA content in the rat tissues. The concentration of the unmodified nucleosides was determined by LC–MS analysis of the diluted DNA digest, which was obtained by diluting 50 ␮L of the digested DNA samples with 450 ␮L of methanol/water (50:50, v/v). The AA–DNA adduct concentrations ([AA–DNA]) were expressed in terms of adducts per normal 109 nucleotides.

[AA–DNA] =

No. of AA–DNA adducts × 109 No. of normal nucleotides

3. Results 3.1. Characterization of 7-(deoxyadenosin-N6 -yl)-aristolactam II (dA–AAII) Reduction of AA in the presence of DNA leads to the formation of AA–DNA adducts [13] (Fig. 1). The preparation of deoxynucleoside-AA adducts from 2-deoxynucleosides and AAI or AAII using xanthine oxidase (XO) and hypoxanthine has been reported previously with the product yields for dA–AAI and dG–AAI as low as 0.1–0.4% [33,34]. Under these (enzymatic) conditions, AA was found to be either not-reacted or reduced to aristolactams. However, by using zinc-acetic acid (1%) as the activation system, the reduction of AAII could reach completion in 6 h, giving a yield of 1.8% for dA–AAII with the remaining AA reduced to aristolactams. The dA–AAII adduct was isolated from the reaction mixture by preparative HPLC and purified by analytical HPLC. The purified adduct, after freeze-drying, was dissolved in methanol and analyzed by UV absorption, fluorescence spectroscopy and HRMS. The UV spectrum of dA–AAII showing absorption maxima at 267, 282, 293 and 396 nm was identical to that reported in the literature [34].

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The fluorescence spectrum (in methanol) showed a broad emission maximum at 464 nm (ex 291 nm) and three excitation maxima at 291, 340 and 394 nm (em 464 nm). The HRMS analysis showed ion peaks at m/z 513.1533 and m/z 397.1057, corresponding to the [M+H]+ and [M−deoxyribose+H]+ ions. 3.2. Quantitative analysis of AA–DNA adducts The DNA samples that were extracted from rat kidney and liver tissues after a single oral administration of AA were digested, SPE enriched and analyzed by LC–MS. The recovery of AA–DNA adducts in the SPE enrichment process (Section 2.5) as determined by spiking 50 pmol of dA–AAII into 8 mL of blank CT–DNA digest was determined to be 96.9 ± 6.6% (n = 5). The reproducibility of the LC–MS method developed, assessed by seven replicated analysis of dA–AAII (0.7 pmol dA–AAII/injection) in blank CT–DNA digest, showed a relative standard deviation (R.S.D., standard deviation/mean × 100%) of 8.2%. The limit of detection, defined as the amount of dA–AAII that generated a response that was three times the standard deviation of the baseline noise (i.e., S/N = 3), was 0.04 pmol/injection. Three AA–DNA adducts, namely, dA–AAII (Fig. 2B and C), dA–AAI (Fig. 2F and G) and dC–AAII (Fig. 3), were detected in kidney tissue of rats dosed with 5 and 30 mg/kg of AA by LC–MS. The identification of the AA–DNA adducts in the sample extracts was based on the chromatographic retention time and HRMS data. No AA–DNA adducts were detected in the samples taken from the kidney and liver tissues of the control rats. By using the purified dA–AAII as standard, the concentrations of dA–AAII, dA–AAI and dC–AAII in the tissues of the AA-dosed rats were determined. The determination was conducted by assuming that dA–AAI and dC–AAII had the same ESI-MS response as dA–AAII. The levels of total AA–DNA adducts in the kidney tissue of rats exposed to 5 and 30 mg/kg of AA were found to be 2.5 and 11.4/109 normal nucleotides, respectively (Table 1). Although the dC–AAII adduct was detected in the tissue samples from the kidney of rats dosed with 5 mg/kg of AA with the S/N > 3, its concentration was below the limit of quantification defined at S/N > 10. The level of dA–AAII adduct found in the kidney samples was higher than that of dA–AAI and much higher than that of dC–AAII. For the rats dosed with 30 mg/kg of AA, the analysis of kidney DNA showed that the concentration of dA–AAII was 1.6 and 5.2 times of dA–AAI and dC–AAII, respectively. The dA–AAII and dA–AAI adducts were also detected and quantified in the liver tissue of rats dosed with 30 mg/kg of AA. While the dA–AAII level was significantly higher than that of dA–AAI in kidney tissue, the levels of these two DNA adducts were similar in liver tissue. The total AA–DNA adduct concentration was 3.6/109 Table 1 AA–DNA adduct concentrations in the kidney and liver tissues of rats treated with a single oral dose of AA with different dosing levels Tissue

Adduct

Aristolochic acidsa (mg/kg) 5

30

Kidney

dA–AAII dA–AAI dC–AAII

1.6 ± 0.2 0.9 ± 0.1 NQc

6.2 ± 1.0 4.0 ± 0.5 1.2 ± 0.4

Liver

dA–AAII dA–AAI

NDd ND

1.6 ± 0.2 2.0 ± 0.1

Total adduct level in kidney and liver

2.5

a b c d

b

15.0

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

normal nucleotides in the liver of rats exposed to 30 mg/kg of AA, which was three to four times lower than that in the kidney tissue. No AA–DNA adducts were detected in the liver of rats dosed with AA at 5 mg/kg. The dG–AA adducts were not detected in the DNA samples of the rats after a single oral dose of AA. dG–AAI and dG–AAII were detected in rats kidney samples after 30 mg/kg dosage of AA for three consecutive days (Fig. 4). It was however, they were not quantified because the peaks were below the limit of quantification defined at S/N > 10. 4. Discussion As nitrophenanthrene carboxylic acid alkaloids, AAI and AAII undergo nitro-reduction to form reactive aristolactam-nitrenium ions. Electrophilic attack of aristolactam-nitrenium ion via its C7 position to the exocyclic amino group in the DNA bases led to the formation of major adducts (Fig. 1) [13]. The AA–DNA adducts were identified spectroscopically as 7-(deoxyadenosin-N6 yl)-aristolactam I (dA–AAI), 7-(deoxyguanosin-N6 -yl)-aristolactam I (dG–AAI), 7-(deoxyadenosin-N6 -yl)-aristolactam II (dA–AAII) and 7-(deoxyguanosin-N6 -yl)-aristolactam II (dG–AAII). While the deoxyadenosine adducts dA–AAI and dA–AAII exhibited imino character, the deoxyguanosine adducts dG–AAI and dG–AAII displayed a secondary amine type of bonding [33,34]. Characterization and quantification of the adducts are important for elucidating the pathway of activation as well as the carcinogenic and nephrotoxic effects of AA [13]. In our previous in vitro study of the DNA adducts induced by AA, the HRMS and MS/MS capability of a QqTOF MS were demonstrated to be efficient for the characterization of AA–DNA adducts [29]. The AA–DNA adducts were detected in forestomach, glandular stomach, liver, kidney and urinary bladder of rodents [13]. Even though the same adduct pattern was found, AA only targeted kidney and forestomach for tumor induction [26]. Thus, AA exhibited a higher carcinogenicity in the kidney and forestomach [27]. In this study, the DNA adduct formation was investigated by LC–MS in both liver (non-target tissue) and kidney (target tissue) of the rats dosed with AA to better understand the genotoxicity of AA. Three AA–DNA adducts, namely, dA–AAII (Fig. 2B and C), dA–AAI (Fig. 2F and G) and dC–AAII (Fig. 3), were detected in the kidney tissue of rats dosed with 5 and 30 mg/kg of AA. Though dC–AA adducts were detected in previous in vitro studies by 32 P-postlabeling [35,36] and LC–MS [29] analyses, the detection of dC–AA in vivo has not been reported. To our best knowledge, this is the first report of dC–AA adducts detected in vivo. The detection of dC–AAII demonstrated that LC–MS might be superior to the 32 P-postlabeling assay for DNA adduct analysis because of its capability for the structure characterization of unknown DNA adducts. dA–AAII was synthesized via an in vitro reaction and used as the standard for the quantitative analysis of the tissue samples. In this study, standards of dA–AAI and dC–AAII were not available because of their low reaction yield even under the optimized synthetic protocol (Section 2.3). Because dA and dC showed similar proton affinity [37], it was assumed that dA–AAII, dA–AAI and dC–AAII had the same ESI-MS response for the quantification of dA–AAI and dC–AAII. Higher concentrations of the DNA adducts were found in the kidney tissue of rats dosed with higher level of AA (Table 1). The concentrations of dA–AAII and dA–AAI in the kidney samples of rats dosed with 30 mg/kg of AA were 3.9 and 4.4 times higher than those of the rats dosed with 5 mg/kg AA, respectively. The relative abundance of the AA–DNA adducts observed in kidney was dA–AAII > dA–AAI > dC–AAII. This pattern agrees with the reported observation in recent studies [26,27] that AAII generated a higher level of DNA adducts than AAI in the target tissue

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

(kidney). Given that the content ratio of AAI and AAII was 1:1 in the AA dosing standard, the reason of the detected different abundance of the DNA adducts might be attributed to the different repair efficiencies towards DNA adducts derived from AAI and AAII and/or the different rates of conversion of AAI and AAII to their reactive aristolactam-nitrenium ion by the nitroreductase enzymes [27]. The DNA adduct dA–AAI detected in this study was found to be at concentration of 0.9 adducts/109 normal nucleotides in kidney DNA samples of the rats that were dosed with AA (mixture of AAI and AAII) at 5 mg/kg. The result was different from that was reported Bieler et al. in which dA–AAI was detected approximately as 70 adducts/109 normal nucleotides when Wister rats were given AAI at 5 mg/kg of body weight [11]. A higher concentration of dA–AAI was also detected in the study by Fernando et al. when male Wister rats were given a single oral dose of 13.8 mmol of AAI [38]. Apart from the lowered dosage that was used in our study (2.5 mg AAI/kg), the discrepancy may have been arose by the different type of rats that were used (S.D. vs. Wister). The possibility of mutual competition of AAI and AAII in DNA binding sites or in active sites of the enzymes may be the other causal factors for the observed discrepancy. In the liver tissue samples from the rats dosed with AA 5 mg/kg, no AA–DNA adducts were detected (Table 1). However, dA–AAII

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and dA–AAI were identified in the liver samples with the dosage of 30 mg/kg. The concentration of dA–AAI in the liver tissue was 2.0/109 normal nucleotide, which was slightly higher than that of dA–AAII (1.6/109 normal nucleotide). The observation of slightly higher levels of dA–AAI compared to dA–AAII in rat liver tissue has been reported by Mei et al. [26] and Dong et al. [27]. The levels of dA–AAI and dA–AAII in kidney were found to be 2.0 and 3.9 times higher than those in the liver, respectively, which was similar to the results reported in the literature [27]. The observed difference might have been attributed by the different repair efficiencies and/or different activation rates of the enzymes, e.g., nitroreductase(s), cytochrome P450 1A1 and P450 1A2, prostaglandin H synthase in these organs [13]. dG–AAI and dG–AAII adducts were not detected in both kidney and liver tissue samples from the rats receiving one single oral dose of AA. It was reported that the concentration ratios of dG–AA:dA–AA adducts were 1:2.6–1:33 in the internal organs of AA-dosed rats and the kidney and ureteric tissue of CHN patients [11,25–27]. Lower level of dG–AA adducts compared to dA–AA adducts was also observed in the in vitro experiment [22,23,39,40], suggesting a lower dG–AA yield in the reaction between AA and DNA. The different yield might be the result of the different accessibility of the AA-reactive amino group between dG and dA in the DNA

Fig. 2. Extracted ion chromatograms of dA–AAII (m/z 513.0–513.2) obtained from LC–MS analyses of dA–AAII standard (0.7 pmol) in blank CT–DNA digestion extract (A) as well as the kidney tissue samples of rats after a single dosage of AA at 30 mg/kg (B), 5 mg/kg (C) and 0 mg/kg (D). (E), (F), (G) and (H) showed the chromatograms of dA–AAI (m/z 543.0–543.2) obtained from LC–MS analyses of the sample extract from in vitro incubation as well as the kidney tissue samples of rats dosed at 30, 5 and 0 mg/kg, respectively. High-resolution ESI-MS spectra inserted.

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Fig. 3. Extracted ion chromatograms of the dC–AAII (m/z 489.0–489.2) obtained from LC–MS analysis of the in vitro incubation sample (A) as well as the kidney tissue samples of rats dosed with AA at 30 mg/kg (B), 5 mg/kg (C) and 0 mg/kg (D). High-resolution ESI-MS spectra were inserted.

helical structure. The exocyclic amino group of dG was found in the rather narrow minor groove of DNA, whereas that of dA was in the more openly accessible major groove [41]. The more easily accessible amino group on dA might account for the higher dA–AA yield observed. The same explanation might also be applied to the detection of dC–AA adduct, where the reactive amino group is located in the major groove. To accumulate sufficient amount of dG–AA adduct for the LC–MS analysis, additional experiment using rats multiply dosed with AA at 30 mg/kg for three consecutive days was performed. Under the identical experimental conditions and instrument settings, the dG–AAI and dG–AAII adducts were detected at S/N > 3. However, the adducts were not quantified in the kidney samples (Fig. 4) because the peak was below the limit of quantification defined at S/N > 10. No dG–AA adducts were detected in the liver tissue samples. The non-detection of dG–AA adducts in the liver DNA samples could have been attributed by the relative lowered adduct concentration when compared with the kidney DNA samples. This is supported by the observation that the concentration of dA–AA adducts was three

to four times lower than that in the kidney tissue (see Section 3). The use of more sensitive/selective mass spectrometry e.g., triple quadrupole mass spectrometer may allow better identification and quantification of the dG–AA adducts. Chronic [4,42] and acute [43] nephrotoxicity have been observed in laboratory rodents upon the AA dosing. Dose-dependent renal lesion was observed in AA-dosed rats [4]. It was suggested that the AA–DNA adducts somehow trigger the fibrotic process that progressively destroys the kidney of the AAN and BEN patients [13]. The current study demonstrated the significant higher concentration of AA–DNA adducts detected in kidney than in liver tissue and the dose-dependence of AA–DNA concentration in the kidney DNA (Table 1). The obtained results supports the postulation that AA–DNA adducts might have been associated with the nephrotoxicity of AA. The amount of DNA samples required for this study is relative large when compared with that required for previous LC–ESI-MS analysis, in which a triple quadrupole MS was used [44]. It is however, the high resolution and MS/MS capability of the Qq-TOF MS

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Fig. 4. Extracted ion chromatograms of the dG–AAII (m/z 529.0–529.3) obtained from LC–MS analysis of the in vitro incubation sample (A) as well as the kidney tissue samples of rats that were multiply dosed with AA at with 30 mg/kg (B) and 0 mg/kg (C) for three consecutive days. (D), (E) and (F) showed the chromatograms of dG–AAI (m/z 559.0–559.3) for the in vitro incubation sample as well as the kidney tissue samples of rats dosed at 30 mg/kg for three consecutive days and 0 mg/kg, respectively. High-resolution ESI-MS spectra were inserted.

is excellent for structural elucidation of the DNA adduct, especially when it comes to the study of unknown/new DNA adducts. Strong fluorescence was observed for the AA–DNA adducts, similar to the nitroreduction derivatives of AA or aristolactams [3]. Therefore, HPLC with fluorescence detection might provide an alternative method for the analysis of AA–DNA adducts other than the LC–MS and radioactive labeling assays. Further investigation of rat urine and other internal organs of rats dosed with AA by HPLC with fluorescence detection might provide additional information about the carcinogenicity of AA. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements We are grateful to Dr. Jian Zhen Yu of the Department of Chemistry, Hong Kong University of Science and Technology for her suggestions in doing the quantitative analysis of the AA–DNA adducts. The supports of the Research Grant Council, University Grants Committee of Hong Kong (HKBU2459/06M), the Food and Health Bureau and Health and Health Services Research Fund (05060141) of Hong Kong and the Sino-German Corporation (GZ364) on this study are acknowledged. References [1] X.G. Zhou, C.Y. Zheng, J.Y. Sun, T.Y. You, Analysis of nephroloxic and carcinogenic aristolochic acids in Aristolochia plants by capillary electrophoresis with electrochemical detection at a carbon fiber microdisk electrode, J. Chromatogr. A 1109 (2006) 152–159.

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