Analysis of urinary 8-nitroguanine, a marker of nitrative nucleic acid damage, by high-performance liquid chromatography–electrochemical detection coupled with immunoaffinity purification: Association with cigarette smoking

Free Radical Biology & Medicine 40 (2006) 711 – 720 www.elsevier.com/locate/freeradbiomed

Original Contribution

Analysis of urinary 8-nitroguanine, a marker of nitrative nucleic acid damage, by high-performance liquid chromatography–electrochemical detection coupled with immunoaffinity purification: Association with cigarette smoking Tomohiro Sawa a,*,1, Masayuki Tatemichi b, Takaaki Akaike c, Alain Barbin a, Hiroshi Ohshima a b

a International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon Cedex 08, France Department of Hygiene and Preventive Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan c Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan

Received 20 July 2005; revised 30 August 2005; accepted 16 September 2005 Available online 21 October 2005

Abstract We have developed an analytical method to quantitate urinary 8-nitroguanine, a product of nitrative nucleic acid damage caused by reactive nitrogen species such as peroxynitrite and nitrogen dioxide. 8-Nitroguanine was purified from human urine using immunoaffinity columns with an anti-8-nitroguanine antibody, followed by quantitation by high-performance liquid chromatography-electrochemical detection. Four sequential electrodes were used to (a) oxidize interfering compounds (+250 mV), (b) reduce nitrated bases (two online electrodes at 1000 mV), and (c) quantitate reduced derivatives (+150 mV). Using this system 8-nitroxanthine can also be detected, with the detection limits being 25 and 50 fmol/ injection for 8-nitroguanine and 8-nitroxanthine, respectively. The method was used to analyze both adducts in the urine of smokers (n = 12) and nonsmokers (n = 17). We found that smokers excrete more 8-nitroguanine [median, 6.1 fmol/mg creatinine; interquartile range (IQR), 23.8] than nonsmokers (0; IQR, 0.90) ( p = 0.018), and although 8-nitroxanthine was detected in human urine, its level was not related to smoking status. This is the first report to show that 8-nitroguanine is present in human urine and the methodology developed can be used to study the pathogenic roles of this adduct in the etiology of cancers associated with cigarette smoking and inflammation. D 2005 Elsevier Inc. All rights reserved. Keywords: 8-Nitroguanine; 8-Nitroxanthine; Peroxynitrite; Nitric oxide; Cigarette smoking; DNA damage; Immunoaffinity purification; Human urine; Free radicals

8-Nitroguanine is formed by the reaction of guanine, either in free base, nucleosides and nucleotides, or in RNA and DNA, with various reactive nitrogen species (RNS) such as peroxAbbreviations: BP-6-N7-Ade, 7-(benzo[a]pyren-6-yl)adenine; BP-6-N7Gua, 7-(benzo[a]pyren-6-yl)guanine; (Ade, 1,N 6-ethenoadenine; ECD, electrochemical detector; (dAdo, 1,N 6-etheno-2V-deoxyadenosine; (dCyd, 3, N 4-etheno-2V-deoxycytidine; (Gua, ethenoguanine; (Cyt, 3,N 4-ethenocytosine; 5-HMUra, 5-hydroxymethyluracil; IQR, interquartile range; M1G, malondialdehyde-2V-deoxyguanosine; MPO, myeloperoxidase; NO, nitric oxide; 8-oxodGuo, 8-oxo-7,8-dihydro-2V-deoxyguanosine; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate-buffered saline; PhIP, 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine; RNS, reactive nitrogen species; ROS, reactive oxygen species. * Corresponding author. Fax: +81 96 362 8362. E-mail address: [email protected] (T. Sawa). 1 Current address: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.09.035

ynitrite and nitryl chloride formed from nitrite and hypochlorous acid or by the myeloperoxidase (MPO)-H2O2-nitrite system, in vitro [1 –6]. In vivo formation of 8-nitroguanine has been shown immunohistochemically under various pathological conditions, including in the lungs of mice with RNA virus-induced pneumonia [7], in the intrahepatic bile ducts of hamsters infected with liver fluke Opisthorchis viverrini [8], and in the gastric mucosa of patients with Helicobacter pyloriinduced gastritis [9]. 8-Nitroguanine in DNA is unstable, with a half-life of approximately 4 h at 37-C, pH 7.4, and spontaneously depurinates to release free 8-nitroguanine [2], resulting in the formation of mutagenic abasic sites that facilitate the generation of G to T transversions [10]. It has been recently reported that 8-nitroguanine in oligodeoxynucleotides mispairs with adenine in vitro and, thus, could directly induce G to T transversions in vivo during replication

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[11]. 8-Nitroguanine in the form of nucleosides and nucleotides has been found to act as a pro-oxidant to stimulate superoxide generation by NADPH cytochrome P450 reductase and nitric oxide (NO) synthases [7,12]. 8-Nitroguanine once formed in cells may have pathophysiological consequences because of its mutagenic and pro-oxidant properties, as well as serving as an inert footprint of biological nitration. Cigarette smoking is associated with an increased risk for human cancers at various sites. The IARC Monograph Programme has recently reported that there is ‘‘sufficient’’ evidence of carcinogenicity of cigarette smoking for more than 10 cancer sites, including cancers of the lung, upper aerodigestive tract (oral cavity, nasal cavity, nasal sinuses, pharynx, larynx, esophagus), pancreas, stomach, liver, lower urinary tract (renal pelvis and bladder), kidney, and uterine cervix and for myeloid leukemia [13]. It has been estimated that cigarette smoking is currently responsible for approximately 30% of cancer deaths in developed countries and that if current smoking patterns persist, an epidemic of cancer attributable to cigarette smoking is expected to occur in developing countries [14]. Cigarette smoke can be divided into two phases, the gas phase and particulate matter (tar), both of which contain high concentrations of reactive oxygen species (ROS) and RNS [15,16]. In cigarette tar, long-lived semiquinone radicals derived from catechol compounds have been identified [15]. This quinone-hydroquinone-semiquinone radical system can produce superoxide anion radicals in the presence of oxygen under redox cycling [15]. The gas phase of the cigarette smoke contains up to 600 Ag of NO per cigarette [16]. Importantly, the reactive species present in both phases can react with each other to produce more S potent reactive species such as NO2 and peroxynitrite [17], which can react with guanine either in free bases, nucleosides and nucleotides, or in DNA and RNA to form 8-nitroguanine. In addition, cigarette smoke causes an acute inflammatory reaction in the lung characterized by the accumulation and activation of leukocytes, which can also produce ROS and RNS in high concentrations [18,19]. Hsieh et al. reported increased levels of 8-nitroguanine in DNA extracted from peripheral blood lymphocytes of smokers compared to nonsmokers [20]. However, the experimental procedures (the volume of blood taken for extraction of DNA, conditions for electrochemical detection of 8-nitroguanine, etc.) were not detailed, nor was the stability and recovery of 8-nitroguanine in DNA during isolation from lymphocytes examined. Measurement of 8-nitroguanine in DNA is complicated, because 8-nitroguanine in DNA is unstable and, hence, is lost during extraction of DNA from cells (Yermilov et al., unpublished data). In order to study the hypothesis that cigarette smoke causes nitrative damage to nucleic acids in human, we have developed an analytical method to quantify free 8-nitroguanine and 8nitroxanthine, which copurify, in biological fluids. This method consists of two parts: immunoaffinity purification using anti-8nitroguanine antibody-bound columns and reverse-phase HPLC separation with electrochemical detection. We have used this method to determine urinary levels of both adducts in smokers and nonsmokers to examine whether cigarette smoking affects their formation in vivo.

Materials and methods Materials Guanine, 8-bromoguanine, 8-oxoguanine (2-amino-6,8dihydroxypurine), and catalase (from bovine liver, 21,000 units/mg) were obtained from Sigma Chemical Co. (St Louis, MO, USA). 8-Chloroguanine was purchased from Biolog Life Science Institute (Bremen, Germany). Mouse ascites fluid containing anti-8-nitroguanosine antibody (Mab NO2G52) was kindly provided by Dojindo Laboratories (Kumamoto, Japan). Preparation and specificity of the antibody have been described elsewhere [21]. Protein A-Sepharose CL-4B was obtained from Amersham Biosciences (Orsay, France). Peroxynitrite was prepared using a quenched-flow reactor as previously described [22]. 8-Nitroguanine and 8-nitroxanthine were synthesized by reactions of guanine or xanthine with peroxynitrite as described previously [1,5]. Concentrations of 8-nitroguanine and 8-nitroxanthine were determined photoscopically by using their molar absorption coefficients: (400 = 9144 M 1 cm 1 (pH 7.0) for 8-nitroguanine and (382 = 7673 M 1 cm 1 (pH 7.0) for 8-nitroxanthine [5]. 14C-labeled 8-nitroguanine was prepared in a manner similar to that described above, from 14C-guanine, which was obtained from acid hydrolysis of [U-14C]guanosine (Dositek, Orsay, France). All other chemicals of reagent grade were purchased from Sigma-Aldrich (Milwaukee, WI, USA) and used without further purification. Preparation and characterization of immunoaffinity columns Immunoaffinity columns for purification of 8-nitroguanine were prepared according to the literature [23]. In brief, 2.5 ml of mouse ascites containing the monoclonal anti-8-nitroguanine antibody NO2G52 was diluted into 22.5 ml of borate buffer (50 mM sodium tetraborate, 3 M sodium chloride, pH 9.0), followed by incubation with 15 ml of protein ASepharose 4B-CL at 4-C for 1 h under gentle mixing. Then, the gel was washed twice with the borate buffer to remove unbound antibody. To form covalent bonds between the gel and the antibody, the gel was treated with 20 mM dimethyl pimelimidate dissolved in the borate buffer at room temperature for 30 min. The reaction was terminated by washing the gels twice with 0.2 M ethanolamine dissolved in 0.25 M triethanolamine buffer (pH 8.0) and then three times with phosphate-buffered saline (PBS; pH7.4). Finally, the gel was poured into polystyrene minicolumns (Product No. 29920; Pierce Chemical Co., Rockford, IL, USA) in 0.5 ml aliquots and maintained in place by use of hydrophobic plastic frits. The binding characteristics of the immunoaffinity columns were examined by two methods. First, 1 ml of authentic 8-nitroguanine solution (50 nM in PBS) was loaded onto the column, followed by washing with PBS (10 ml) and H2O (10 ml) at 4-C and 85% methanol (8 ml) at room temperature. All eluates were collected and the contents of 8-nitroguanine were determined by HPLC (see below) to obtain the elution profile of 8-nitroguanine from the immunoaffinity columns. The columns were then washed

T. Sawa et al. / Free Radical Biology & Medicine 40 (2006) 711 – 720

with a further 3 ml of 85% methanol, 10 ml of H2O, and 10 ml of PBS. After this step, the columns were reusable. In a second experiment, PBS (1 ml) containing 8-[14C]nitroguanine (1000 dpm) and nonradioactive competitors (0– 100 AM) was applied to the columns. The columns were washed with 10 ml of PBS, 10 ml of H2O, and 5 ml of 85% methanol as described above. The 85% methanol fraction was collected and dried in a Savant Speed-Vac. The dried samples were dissolved in 1 ml of H2O and mixed with Picofluor (Beckman; 4 ml), and the radioactivity was determined by scintillation counting. The results were expressed as the percentage of 8-[14C]nitroguanine retained on the columns. Quantification of 8-nitroguanine by means of reverse-phase HPLC with electrochemical detectors 8-Nitroguanine was analyzed by means of reverse-phase HPLC with electrochemical detectors (ECD) as described previously [4,24] with certain modifications. An HPLC system with two Model 582 solvent delivery modules (ESA, Inc., Chelmsford, MA, USA) equipped with an Ultrasphere ODS column (4.6  250 mm and particle size 5 Am; Beckman, Fullerton, CA, USA) was used with isocratic elution conditions (20 mM citric acid-sodium acetate, pH 3.0, containing 4% acetonitrile at a flow rate of 1 ml/min) and a column temperature of 30-C. A Model 5600A CoulArray detector (ESA, Inc.) equipped with a Model 6210 four-sensor electrode (ESA, Inc.) was used to detect 8-nitroguanine electrochemically. The electrochemical potential settings were +250, 1000, 1000, and +150 mV, for the first to the fourth electrode. Under these conditions, 8-nitroguanine cannot be detected at the first electrode, whereas it can be detected at the fourth electrode, after on-electrode reduction at the second and third electrodes. The presence of nitrated bases can be confirmed by the disappearance of peaks after switching off the second and third electrodes. This HPLC program also provides a good separation of 8-nitroxanthine, a deaminated product of 8-nitroguanine, with electrochemical sensitivity similar to that for 8-nitroguanine (see below for details). Urine analysis This study was approved by the Ethical Review Committee of the International Agency for Research on Cancer. The study subjects provided a first morning urine sample in polypropylene tubes that contained sodium azide (final concentration to be approximately 5 mM) as an antibacterial agent. They also completed a consent form and a questionnaire that provided the information on age, sex, and the number of cigarette smoked per day. Urine samples were collected from smokers (n = 12) and nonsmokers (n = 17). Urine samples were stored at 20-C before analysis. 8-[14C]Nitroguanine (500 dpm) was added to 50 ml of the urine sample in order to determine the recovery efficiency of 8-nitroguanine after the purification steps. Before immunoaffinity purification, urine samples (50 ml) were deproteinated by mixing with 150 ml of ethanol, followed by storage at

713

20-C for 30 min. The insoluble materials were removed by centrifugation (2500g, 4-C, 15 min), and the supernatant was dried in a Speed-Vac. The dried residue was then dissolved in 50 ml of PBS, and 3.3 ml of this solution was applied to each of 15 immunoaffinity columns per subject. Each column was washed with 10 ml of PBS, 10 ml of H2O at 4-C, and 5 ml of 85% methanol at room temperature as described above. The 85% methanol fraction eluted from the set of 15 columns was combined and dried in a Speed-Vac. The residues were dissolved in 5 ml of PBS, and this solution was purified again on one immunoaffinity column as described. Finally, the 85% methanol fraction was dried and redissolved in 0.2 ml of HPLC buffer (see above). One aliquot (0.1 ml) was used to measure radioactivity and the second (0.05 ml) was used for HPLC analysis of 8-nitroguanine. The concentration of creatinine in urine was determined as previously described [25]. The urinary levels of 8-nitroguanine and 8-nitroxanthine were corrected by the individual recovery efficiencies (average efficacy approximately 30%) and expressed as fmol of 8-nitroadduct/mg of creatinine. The levels of nitrite and nitrate in urine samples were determined as previously described [26]. We studied the possible artifactual formation of 8-nitroguanine in urine as follows. Urine samples (1 ml), to which [14C]guanine (40,000 dpm/ml, 80 nM) was added, were incubated for 3 h at room temperature with or without further addition of the nitrating agent, peroxynitrite (10 AM final). As our urine samples contained sodium azide (¨5 mM) and one study reported nitration of tyrosine by sodium azide in the presence of catalase and H2O2 [27], we incubated urine samples containing [14C]guanine in the presence of H2O2 (0.1 mM) and catalase (0.1 mg/ml). Urine samples were purified by immunoaffinity purification and 8-[14C]nitroguanine was measured as an index of the formation of nitroadducts. For comparison, PBS containing [ 14C]guanine (40,000 dpm/ml, 80 nM) with or without nitrating agents was incubated under similar conditions. Statistical analysis All statistical tests were carried out using SPSS 10.1J (SPSS Japan, Inc., Tokyo, Japan). Differences between smokers and nonsmokers in background characteristics were compared using an unpaired t test or Fisher’s exact test. Statistical significances on the levels of creatinine, nitrite plus nitrate, and nitro-adducts in urine were examined using the Mann – Whitney U test. A p value <0.05 was considered statistically significant. Results Immunoaffinity purification of 8-nitroguanine As shown in Fig. 1A, the immunoaffinity columns used in this study could strongly adsorb 8-nitroguanine; no detectable level of 8-nitroguanine was eluted during extensive washing steps using PBS (10 ml) and H2O (10 ml) when 50 pmol was loaded onto the column. 8-Nitroguanine could be eluted

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toward the binding of 8-[14C]nitroguanine, at concentrations up to 100 AM. In contrast, addition of 1 AM 8-nitroguanine resulted in the 50% inhibition of 8-[14C]nitroguanine binding. 8-Nitroxanthine also competed with 8-[14C]nitroguanine to an extent similar to that of 8-nitroguanine. Thus, this immunoaffinity step allowed us to selectively purify and concentrate 8-nitroguanine and 8-nitroxanthine. HPLC-ECD analysis of 8-nitroguanine

Fig. 1. Characteristics of immunoaffinity columns containing an anti-8nitroguanine antibody. (A) Elution profile. One milliliter of 8-nitroguanine standard solution (50 nM in PBS) was applied to the immunoaffinity column, which was washed sequentially with PBS (10 ml), H2O (10 ml), and 85% aqueous methanol (8 ml). Each 1-ml fraction was collected, and the amount of 8-nitroguanine eluted was determined by HPLC-ECD analysis. (B) Binding specificity. 8-[14C]Nitroguanine (500 dpm) with an indicated concentration of unlabeled competitor was applied to the immunoaffinity column and then washed with PBS (10 ml), H2O (10 ml), and 85% methanol (5 ml). Radioactivity recovered into the 85% methanol fraction was counted. Abbreviations: NO2-G, 8-nitroguanine; NO2-X, 8-nitroxanthine; G, guanine; Cl-G, 8-chloroguanine; BrG, 8-bromoguanine; oxo-G, 8-oxoguanine. Data are means T SD (n = 3).

The nitro group of 8-nitroguanine can be chemically reduced by sodium dithionite to form 8-aminoguanine [1]. 8-Aminoguanine is electrochemically active and can be readily detected by ECD, so that this sodium dithionite-mediated chemical conversion of 8-nitroguanine to 8-aminoguanine has been used to quantify 8-nitroguanine [2] and 8-nitroguanosine [6]. In a preliminary study, however, it was found that sodium dithionite remaining in the sample solution interfered with the detection of low nanomolar levels of 8-nitroguanine (data not shown). 8-Nitroguanine can also be reduced electrochemically to form an electrochemically active compound, most probably 8-aminoguanine [4,24]. In this study, we used four sequential electrodes to monitor the electrochemical response of 8-nitroguanine, before and after on-electrode reduction. As shown in Fig. 2A, 8-nitroguanine was not detected by the first electrode when a voltage of +250 mV was applied. After electrochemical reduction at the second and third electrodes using a voltage of 1000 mV, 8-nitroguanine could be clearly detected by the fourth electrode at an even lower voltage (+150 mV) compared to the first electrode. If the second and third electrodes were switched off, no peaks corresponding to 8-nitroguanine and 8-nitroxanthine were detected with the fourth electrode. This electrochemical response of nitro-adducts is unique and can be used for identification, in addition to HPLC retention times. Under the present experimental conditions, 8-nitroxanthine was well separated from 8-nitroguanine and thus could be detected by the fourth electrode. The electrochemical responses of 8nitroguanine and 8-nitroxanthine increased linearly as a function of the amount injected in the range of 25 to 3200 fmol/injection (Fig. 3). The detection limits of 8-nitroguanine and 8-nitroxanthine, defined as a signal-to-noise ratio for the nitrated bases of 4:1, were approximately 25 and 50 fmol/ injection. Identification of 8-nitroguanine in human urine

from the columns with 5 ml of 85% methanol with a recovery efficiency of 90 to 100%. No chemical modifications such as oxidation or deamination of 8-nitroguanine were observed during this immunoaffinity purification step (data not shown). A competitive assay using a combination of 8-[14C]nitroguanine and nonradiolabeled competitors revealed that the immunoaffinity columns adsorb 8-nitroguanine in a very selective manner (Fig. 1B). Guanine and its 8-substituted analogs, including 8-chloroguanine, 8-bromoguanine, and 8-oxoguanine, showed no or very slight competition, if any,

Table 1 shows background characteristics of the study subjects. No statistical differences were observed in age, gender, and urinary excretion level of nitrate between smokers and nonsmokers. None of the urine samples contained nitrite at concentrations above the limit of detection of the assay used (2.5 AM) but all contained nitrate in a range between 450 and 4571 AM. The pH of urine samples was in the range of 5.1– 7.4 (smokers, 5.3 – 7.4; nonsmokers, 5.17.2). The numbers of cigarette smoked per day ranged from 2 to 30, and the mean was 15.7 cigarettes per day.

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ml urine for smokers. After correction by urinary creatinine concentration, the median urinary level of 8-nitroguanine was significantly higher in smokers [range, 0 – 151.6 fmol/mg creatinine; median, 6.1 fmol/mg of creatinine; interquartile range (IQR), 23.8] than that in nonsmokers (range, 0 –9.2; median, 0; IQR, 0.9) ( p = 0.018) (Fig. 4A). No significant difference in the level of 8-nitroxanthine was observed between smokers (range, 0– 95.4; median, 0; IQR, 7.6) and nonsmokers (range, 0 –7.8; median, 0; IQR, 0.6) ( p = 0.283) (Fig. 4B). Cigarette smokers excreted significantly higher amounts of 8-nitroguanine plus 8-nitroxanthine in urine (range, 0 –247.1; median, 10.0; IQR, 29.5) compared with nonsmokers (range, 0– 9.2; median, 0; IQR, 5.4) ( p = 0.021) (Fig. 4C). The effect of the number of cigarettes smoked per day was examined by comparing the levels of nitrated bases in smokers who smoked fewer than 20 cigarettes (n = 8) and more than 20 cigarettes (n = 4), and no significant dosedependent effect of cigarettes smoked on the urinary levels of 8-nitroguanine and 8-nitroxanthine was found. However, this is based on small numbers of heavy smokers and warrants further investigation. There were no significant differences in the urinary levels of 8-nitroguanine or 8-nitroxanthine between men (n = 17) and women (n = 12) among all participants or between male smokers (n = 8) and female smokers (n = 4). In addition, no apparent relationship between urinary levels of nitro-adducts and urinary pH or nitrate levels was observed. In order to study the possible artifactual formation of 8-nitroguanine or 8-nitroxanthine in urine samples during collection and analysis, we performed two sets of experiments. First, guanine (10 AM) was incubated with nitrite (0.1 mM) alone or nitrite (0.1 mM) and H2O2 (0.1 mM), both of which may be present in urine, in buffer solutions at pH 3.0 – 7.4 for 1 h at 37-C. Neither 8-nitroguanine nor 8-nitroxanthine was formed

Fig. 2. HPLC-ECD analysis of 8-nitroguanine and 8-nitroxanthine. (A) 8-Nitroguanine and 8-nitroxanthine standard (2.5 pmol/injection each). (B) Human urine processed by immunoaffinity columns. Electrode potentials were set at +250 mV (first electrode), 1000 mV (second and third electrodes), and +150 mV (fourth electrode).

Fig. 2B shows a representative chromatogram of human urine processed by the immunoaffinity columns and analyzed by HPLC-ECD. This chromatogram clearly demonstrates that a peak identical to 8-nitroguanine with respect to the retention time and electrochemical response can be detected in human urine after immunoaffinity purification. In addition to 8-nitroguanine, we could detect a peak eluted at 4.5 min, corresponding to the retention time of 8-nitroxanthine. (A peak detected by the first electrode with an elution time of 6.6 min is an unknown compound and was not further characterized). Eight of 12 urine samples from smokers contained detectable levels of 8-nitroguanine, compared to only 4 of 17 from nonsmokers. Urinary levels of 8-nitroguanine were in the range of 0– 9.5 fmol/ml urine for nonsmokers and 0 – 85 fmol/

Fig. 3. Calibration curves for 8-nitroguanine and 8-nitroxanthine detected by HPLC-ECD. Electrochemical detector responses were plotted as a function of analyte concentration. Data are means T SD (n = 3, error bars are smaller than the symbols).

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Table 1 Background characteristics of the participants

Age (years) [mean (SD)] Male/female Cigarettes/day [mean (SD)] Urinary pH (range) NO2 in urine (AM) NO3 in urine (AM) [median (IQR)]

Smokers (n = 12)

Nonsmokers (n = 17)

p

42.7 (11.3)

42.9 (8.6)

nsa

8/4 15.7 (9.1)

9/8 0

nsb <0.001a

5.3 – 7.4 <2.5 810 (770)

5.1 – 7.2 <2.5 914 (1379)

nsc nsc nsc

IQR, interquartile range. a Unpaired t test. b Fisher’s exact test c Mann – Whitney U test.

under these conditions. In similar experiments that used 8-nitroguanine instead of guanine, no 8-nitroxanthine formation was observed, indicating that 8-nitroguanine was not deaminated by nitrite alone or nitrite plus H2O2 (data not shown). Second, we analyzed the possible artifactual formation of 14C-labeled 8-nitroguanine during the collection and analysis of urine samples in which 14C-labeled guanine was added exogenously. As shown in Table 2, 14C-labeled 8-nitroguanine was not formed in urine samples even when they were treated with the strong nitrating agent peroxynitrite (10 AM). Our urine samples contained sodium azide in order to avoid bacterial growth and chemical nitrosation with nitrite [28]. However, one publication has reported the formation of nitrotyrosine by sodium azide in the presence of catalase and H2O2, both of which may be present in human urine [27]. Although 8-nitroguanine was formed in PBS by sodium azide in the presence of catalase and H2O2, as reported for nitrotyrosine [28], no detectable levels of nitro-adducts were formed in urine samples incubated with these reagents under similar conditions. These results suggest that human urine contains substances that inhibit the nitration reaction mediated by peroxynitrite or sodium

azide-catalase-H2O2. Thus, our study indicates that the artifactual formation of nitro-adducts during sample collection and preparation are negligible. Discussion In the present study, we have developed a sensitive method to quantify free 8-nitroguanine in biological fluids, including urine, using immunoaffinity purification with an anti-8nitroguanine antibody, followed by analysis with HPLCECD. As shown in Fig. 2B, urine extracts processed with immunoaffinity columns were very clean with respect to contamination by electrochemically active compounds. Approximately a 250-fold concentration can be achieved when 50 ml of urine is used as a starting material for immunoaffinity purification (final volume, 0.2 ml). Our method used four sequential electrodes with potential settings at +250, 1000, 1000, and +150 mV to achieve on-electrode reduction and detection of 8-nitroguanine and related compounds. Some interfering compounds, if present in the sample, could be oxidized at the first electrode. Nitrated bases were then reduced online with two electrodes under a reduction mode at 1000 mV and the reduced derivatives were analyzed quantitatively with the fourth electrode at +150 mV. This method is simple, specific, and sensitive for the analysis of 8-nitroguanine and 8-nitroxanthine, with detection limits for both compounds of approximately 25– 50 fmol/injection. If 50 ml of urine is used as starting material, the actual detection limit in urine is approximately 4 fmol/ml of urine. By using this method, we have demonstrated for the first time the presence of 8-nitroguanine and its deaminated product 8-nitroxanthine in human urine. The origin of 8nitroguanine in urine is currently unknown. However, as shown in this study, the artifactual formation of these adducts during sample collection and preparation could be excluded. Therefore, urinary 8-nitroguanine may be formed endogenously with one possible source being nitrated DNA. 8-

Fig. 4. Effects of cigarette smoking on urinary levels of (A) 8-nitroguanine, (B) 8-nitroxanthine, and (C) 8-nitroguanine plus 8-nitroxanthine. The urinary levels of 8nitroguanine and 8-nitroxanthine in smokers (n = 12) and nonsmokers (n = 17) were quantified by HPLC-ECD. Data are presented as box-and-whisker plots displaying medians and interquartile ranges. Top of each box, 75th percentile; bottom of each box, 25th percentile; solid center line, 50th percentile; top bar, maximum observation within 1.5 times the interquartile range from the end of a box; bottom bar, minimum observation. One urine sample of a smoker contained extreme values, 151.6 fmol/mg creatinine (8-nitroguanine), 95.4 fmol/mg creatinine (8-nitroxanthine), and 247.1 fmol/mg creatinine (8-nitroguanine plus 8-nitroxanthine), that are not shown.

T. Sawa et al. / Free Radical Biology & Medicine 40 (2006) 711 – 720 Table 2 Nitration of a

Sample

PBS Urine Urine Urine Urine Urine a b c d e f g h

1 2 3 4 5

14

717

C-labeled guanine added exogenously to urine pH

7.4 6.8 5.5 5.9 5.9 5.1

Cigarettes/day g

na 5 20 30 0 0

Urinary levels (fmol/mg creatinine)b

Immunocolumn-bound radioactivity (dpm/ml)c

8-Nitroguanine

Controld

na 49.9 0 9.5 9.2 0

8-Nitroxanthine na 0 10.5 0 0 6.2

<10 <10 <10 <10 <10 <10

+ Peroxynitritee h

1251 <10 <10 <10 <10 <10

+ H2O2/catalasef 121h <10 <10 <10 <10 <10

Phosphate-buffered saline (PBS) and urine samples contained 10 and ¨5 mM sodium azide, respectively. Determined by HPLC-ECD (without adding exogenous 14C-labeled guanine). 1000 dpm/ml is equivalent to 2 nM 8-nitroguanine. [14C]Guanine (40,000 dpm/ml, 80 nM) was incubated in PBS or urine for 3 h at room temperature. [14C]Guanine (40,000 dpm/ml, 80 nM) in PBS or urine was reacted with 0.01 mM peroxynitrite. [14C]Guanine (40,000 dpm/ml, 80 nM) in PBS or urine was incubated in the presence of 0.1 mM H2O2 and 0.1 mg/ml catalase for 3 h at room temperature. Not applicable. Mean of three experiments.

Nitroguanine is rapidly and spontaneously depurinated from DNA [2] and may be excreted into urine as a free base, as has been reported for several N 3- and N 7-alkylated purines [29]. It may also arise from the metabolism of nitratively damaged RNA and of nitrated nucleosides/nucleotides in the nucleotide pool, as immunohistochemical localization of 8-nitroguanine indicates that it is formed in the cytosol of inflamed tissues [7]. We have found that, in healthy nonsmokers, 13 of 17 urine samples contained undetectable levels of 8-nitroguanine, whereas the remaining 4 samples contained detectable levels, suggesting that the basal level of 8-nitroguanine in healthy nonsmokers may be close to or slightly below the detection limit of the present method (¨4 fmol/ml of urine). In the nonsmoker group, mean urinary creatinine concentration was 1.8 mg/ml of urine, so that the basal level of 8-nitroguanine is estimated to be below 2.2 fmol/mg of creatinine. Urinary 8nitroguanine levels measured in this study were in the range of 0 –9.5 fmol/ml of urine or 0– 9.2 fmol/mg of creatinine for nonsmokers and 0 –85 fmol/ml of urine or 0– 151 fmol/mg of creatinine for smokers. Comparing these data with those available in the literature for other DNA adducts, urinary levels of 8-nitroguanine were considerably lower than products such as 8-oxo-7,8-dihydro-2V-deoxyguanosine (8-oxodGuo) and 5-hydroxymethyluracil (5-HMUra). For example, urinary levels of 8-oxodGuo and 5-HMUra have been reported in the range of 1 – 3 Amol/mol of creatinine (corresponding to 8.8 –26.5 pmol/mg of creatinine) for 8-oxodGuo ([30] and references therein) and 55 –205 [31] or 28 – 165 pmol/mg of creatinine [32] for 5-HMUra. Urinary excretion of etheno DNA adducts has been measured as a marker of oxidative stress involving in lipid peroxidation-mediated DNA damage in populations who are not exposed occupationally to etheno DNA adducts forming carcinogens such as vinyl chloride [33]. The range of urinary levels has been reported for ethenoguanines ((Gua) (<300 – 8000 fmol/ml urine) [34], 3,N 4-ethenocytosine ((Cyt) (27 – 1276 fmol/mg of creatinine) [35], 3,N 4etheno-2V-deoxycytidine ((dCyd) (0 – 980 fmol/mg creatinine) [36], 1,N 6-ethenoadenine ((Ade) (0 –370 fmol/mg creatinine) [37], and 1,N 6-etheno-2V-deoxyadenosine ((dAdo) (10 –150

fmol/mg creatinine [38] or 2.3 – 105 fmol/ml urine [39]). Malondialdehyde-2V-deoxyguanosine adduct (M1G) in human urine has been measured to be in a range of 0.23 – 0.96 fmol/ml urine [40]. Urinary levels of DNA adducts induced by benzo[a]pyrene, including 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7-Gua) and 7-(benzo[a]pyren-6-yl)adenine (BP-6N7-Ade), have been measured as risk-associated biomarkers for exposure to polycyclic aromatic hydrocarbons (PAH). BP-6-N7-Gua was undetectable, but the levels in a range of 0.1– 0.6 fmol/mg creatinine of BP-6-N7-Ade were detected in urine samples from women with smoking habits [41]. Fang et al. tried to measure a 2V-deoxyguanosine adduct of the food-derived carcinogenic heterocyclic amine 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at the C 8 position in human urine, but its urinary levels were below the detection limit (<2.5 ng of adduct/L of urine, corresponding to approximately 5 fmol/ml of urine) [42]. Although the subjects investigated in these studies are different, urinary levels of adducts seem to be in the order of 5-HMUra > 8-oxodGuo >> (Gua > (dCyd, (Cyt, (Ade > 8-nitroguanine, (dAdo > BP-6-N7Ade, M1G, PhIP-deoxyguanosine, BP-6-N7-Gua. The interindividual variations in urinary 8-nitroguanine levels (undetectable to 151 fmol/mg of creatinine) suggest a variation of nitrative nucleic acid damage between individuals. An interesting finding of this study is the identification of cigarette smoking as a factor associated with increased urinary levels of 8-nitroguanine (Fig. 4). The urine samples of five smokers contained 6.1- to 69-fold higher levels of 8-nitroguanine (13.6, 13.8, 27.1, 49.9, and 151.6 fmol/mg of creatinine) than the estimated basal urinary levels of 8-nitroguanine in nonsmokers (2.2 fmol/mg of creatinine, see above). These findings clearly indicate that cigarette smoking increases in vivo formation of 8-nitroguanine in human. Enhancing effects of cigarette smoking on nitrative guanine damage may be multifactorial. Cigarette smoke contains high concentrations of NO and RNS [15 – 17] that can penetrate into cells causing guanine nitration directly. Cigarette smoke can also cause inflammatory reactions that produce high concentrations of ROS and RNS [18,19]. The importance of the inflammatory reaction in the induction of guanine nitration has

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been revealed immunohistochemically in several previous studies [7 –9]. It is also known that cigarette smoke consumes antioxidants in biological fluids such as plasma [43], resulting in cells more susceptible to RNS-induced guanine nitration. This notion is supported by the finding that plasma levels of 3-nitrotyrosine, a product of nitrative protein/amino acid damage [44,45], in smokers show an inverse correlation to plasma total antioxidant capacity [46]. Additionally, nicotine, which is abundant in cigarette smoke, can stimulate guanine nitration caused by RNS generated from nitrite plus hypochlorous acid, the MPO-H2O2-nitrite system, and activated neutrophils in vitro [47]. It was previously reported that nicotine and other tertiary amines such as trimethylamine can significantly enhance guanine base modifications including nitration, oxidation, and chlorination induced by these RNS. The effects of cigarette smoking may operate synergistically or additionally to induce high levels of nitrative nucleic acid damage. It is currently unknown whether the increased formation of 8-nitroguanine caused by cigarette smoking is relevant to human cancer development; however, several issues discussed below support this plausible association. First, biochemical analyses have suggested that 8-nitroguanine formed in DNA may be mutagenic, inducing G-to-T transversions, possibly through depurination-dependent formation of mutagenic abasic sites [2] and/or direct mispairing with adenine during replication [11]. G-to-T transversions are a predominant form of mutation detected in the p53 tumor suppressor gene in human lung cancers associated with cigarette smoking and, hence, are considered a molecular signature of cigarette smoke mutagens in smoking-associated lung cancers [48]. Although PAHs have been implicated in the induction of this type of mutation in smokers, 8-nitroguanine may be at least partly responsible for such mutations. In fact, the urinary levels of 8-nitroguanine determined in this study are comparable to or slightly higher than those reported for PAH-DNA adducts. Casale et al. have reported that the urinary level of the benzo[a]pyrene-adducted base BP-6-N7-Gua was undetectable and that of BP-6-N7-Ade was 0.1– 0.6 fmol/mg of creatinine in women who smoke cigarettes, whereas these two adducts were undetectable in the urine of nonsmoking study controls [41]. It is noteworthy that PAH adduction induces destabilization of N-glycosyl bonds, resulting in depurination of adducted bases [49], similar to that seen for 8-nitroguanine bases, and that this depurination process has been considered to be involved in the induction of mutations [50]. Oxidants and free radicals contained in cigarette smoke may cause oxidative DNA damage in humans. 8-OxodGuo has been frequently quantified as a marker for oxidative DNA damage in the urine of smokers and nonsmokers [51]. However, cigarette smoking usually results in only modest increases of 8-oxodGuo levels in urine, in amounts 9 –50% greater than in nonsmokers [52 –55], although no significant difference in urinary 8-oxodG levels between smokers and nonsmokers has also been reported in some studies [56,57]. Smoking cessation caused a decrease in the excretion of 8-oxodGuo by 21% [58]. For other DNA adducts there is limited evidence of an association between smoking status and

urinary excretion. For instance cigarette smoking had no effect on urinary levels of 5-HMUra [55] and for the etheno DNA adducts, the effects of cigarette smoking seem to vary dependent on the type of adducts [31]. Cigarette smoking is associated with a 2.8- to 5.2-fold increase in urinary levels of (Cyt [35], (dCyd [36], and (Ade [37], but it does not affect the urinary levels of (dAdo [39]. There are no reports on the urinary levels of (Gua in relation to cigarette smoking. In the present study, we have shown that urinary 8-nitroguanine levels are significantly associated with smoking habits and thus monitoring of this adduct could provide an important noninvasive quantitative assessment of nitrative nucleic acid damage associated with cigarette smoking and inflammatory disease. We observed that 4 of 17 nonsmokers also excreted detectable levels of 8-nitroguanine (1.8, 2.7, 6.2, and 9.2 fmol/mg of creatinine). The origin of 8-nitroguanine in nonsmokers is unknown. Inflammation may be responsible for this increased excretion of 8-nitroguanine. It could be possible to examine the effects of inflammation on urinary 8-nitroguanine levels by analyzing urine samples obtained from subjects infected with H. pylori and/or liver fluke O. viverrini, because these infections have been demonstrated to form 8-nitroguanine in vivo [8,9]. In the present study, we also detected 8-nitroxanthine, a deaminated product of 8-nitroguanine, in human urine. In vitro experiments have suggested that 8-nitroxanthine can be formed from reactions of (a) xanthine with RNS such as peroxynitrite and nitryl chloride and with the MPO-H2O2nitrite system [5,59] and (b) 2V-deoxyguanosine or DNA with nitryl chloride [5]. When 8-nitroguanine is orally administered to rats a fraction is metabolically converted to 8-nitroxanthine (Yermilov, Rubio, Ohshima, unpublished data). However, the exact rate of formation of this adduct in vivo remains to be determined. It should be noted, however, that urinary 8-nitroxanthine levels do not seem to be associated with smoking habits in the subjects studied. In conclusion, we have developed a sensitive assay method to quantify the levels of 8-nitroguanine in biological fluids, using immunoaffinity purification with an anti-8nitroguanine antibody, followed by quantitation with HPLCECD. This method allowed us to identify for the first time 8-nitroguanine and also 8-nitroxanthine in human urine and revealed that cigarette smoking is associated with elevated urinary levels of 8-nitroguanine. Urinary 8-nitroguanine analysis may offer a unique noninvasive technique for the measurement of endogenous occurrence of nitrative guanine damage. The method described here not only is useful for the analysis of urinary 8-nitroguanine, but also can be used for its analysis in other biological matrices, including plasma, tissue homogenate, and cell culture lysate, in order to study the association between chronic inflammation and cancer. Acknowledgments The authors thank Dr. J. Hall for critical review of the manuscript and Mrs. P. Collard for secretarial assistance.

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