Rapid Report pubs.acs.org/crt
Quantification of Aristolochic Acid-RNA Adducts in the Urine of Aristolochic Acid-Treated Rats by Liquid Chromatography−Tandem Mass Spectrometry Elvis M. K. Leung and Wan Chan* Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China S Supporting Information *
ABSTRACT: Balkan endemic nephropathy (BEN) is a peculiar renal disease affecting thousands of farmers living in the suburban areas of the Balkan countries. Emerging evidence suggested that BEN is an environmental disease caused by chronic food poisoning with aristolochic acid (AA). We have developed a sensitive liquid chromatography−tandem mass spectrometry method to detect urinary RNA-AA adducts. Results revealed high levels of RNA-AA adducts in the urine samples collected from AA-treated rats. To the best of our knowledge, this study is the first to report on the detection of urinary RNA-AA adducts. Compared with previous studies that quantified DNA-AA adducts, this method is more sensitive and user-friendly.
A
samples of the target organs by a noninvasive method as well as in obtaining ethical approval to conduct the experiment on human tissues. Therefore, noninvasive analytical methods for monitoring the risk of human exposure to AA would be extremely useful. A few studies have analyzed DNA-AA adducts in exfoliated cells in the urine of rats treated with AA.8,9 However, liters of urine samples had to be collected before a sufficient amount of cells/DNA was accumulated for the analysis. We recently reported the first noninvasive method for assessing exposure to AA by quantifying urinary DNA-AA adducts from AA-treated rats.10 With emerging evidence showing high frequencies of formation of RNA adducts in vitro and in vivo and given that RNA is less stable than DNA,11 we postulate that abundant RNA-AA adducts may be excreted in urine. Thus, quantifying urinary RNA-AA adducts may provide a more sensitive and equally noninvasive approach to monitoring exposure to AA. To test this hypothesis, we developed a highly sensitive and selective liquid chromatography−tandem mass spectrometry (LC−MS/MS) method to quantify urinary RNA-AA adducts in rat urine. To the best of our knowledge, the identification of RNA-AA adducts in urine has not been reported in the literature. The LC-MS/MS analysis of the SPE enriched urine samples collected from rats treated with AA (Figures S2B, S2D, S3B, and S3D, Supporting Information) revealed unambiguous identification of the targeted adducts by comparing chromato-
ristolochic acid (AA) are a family of nitrophenanthrene carboxylic acid derived from Aristolochia, and they were found to be potent nephrotoxins.1−3 For instance, prolonged exposure to AA-containing herbal remedies has resulted in kidney failure among Belgian women participating in a slimming regime in the 1990s.3,4 This unique type of rapid, progressive renal fibrotic disease, initially referred to as “Chinese herb nephropathy,” was later renamed aristolochic acid nephropathy (AAN).2 Significant evidence also suggested that chronic food poisoning by AA-contaminated wheat flour is one of the major reasons leading to Balkan endemic nephropathy (BEN),3 a peculiar renal disease that has been observed extensively in farmers living alongside the Danube River over the past 50 years. AA are also strongly carcinogenic and are classified as Group I human carcinogens by the International Agency for Research on Cancer (IARC).2 Specifically, tumors have been observed in multiple internal organs of AA-treated rodents, and a high frequency of AT → TA transversions was found in these tumor tissues.5 Covalently bonded DNA-AA adducts were also identified in the internal organs of AA-treated rats as well as in patients suffering from AAN or BEN.3,5,6 Owing to the strong nephrotoxicity and potent carcinogenicity of AA, analytical methods capable of monitoring human exposure to AA are needed.7 Current exposure assessments focus exclusively on analyzing tissue-isolated DNA samples for DNA-AA adducts.3,6,7 However, one of the major obstacles in conducting large-scale risk assessment based on DNA-AA adducts, e.g., in the Balkan Peninsula, is the accessibility to relevant biopsy tissue samples. Difficulties exist in collecting © 2015 American Chemical Society
Received: January 13, 2015 Published: February 25, 2015 567
DOI: 10.1021/acs.chemrestox.5b00021 Chem. Res. Toxicol. 2015, 28, 567−569
Chemical Research in Toxicology
Rapid Report
easily than those of A-AA adducts-containing RNA. Interestingly, despite the dramatically higher levels of G-AAII than GAAI adducts in the internal organs of AA-treated rats,11 these adducts were determined to be at similar concentrations in the urine samples collected from the AA-treated rats. Compared with the concentrations of DNA-AA adducts in urine samples collected from AA-treated rats,10 all of the corresponding RNA-AA adducts were detected at concentrations roughly 2-fold higher, with the exception of A-AAII, which was detected at levels similar to those of dA-AAII. Potential reasons for this observation include the higher frequencies of RNA adduct formation and greater stability of the DNA-AA lesions in the genome. Given that RNA is more reactive toward electrophilic attack by the nitrenium ion intermediate from AA metabolism, RNA adducts of AA are thus formed at frequencies significantly greater than those of the DNA adducts in the internal organs of AA-treated rats.11 Apart from the higher formation rate, the RNA adducts of AA degrade more easily when compared with the DNA-AA adducts. While RNA containing the AA-modified ribonucleosides are routinely being degraded by RNase and eliminated via urination, the DNA adducts of AA, dA-AA in particular, have a long lasting persistence.6 We understand that in the present study, rats were exposed to dosages of AA larger than those determined in humans.14 Improvement in analytical sensitivity would be needed to meet the challenge of analyzing RNA-AA adducts in human urine samples. While we used 3 mL of rat urine for a single experiment, enhancement in analytical sensitivity could be achieved by using a larger urine volume. With the high hydrophobicity of the RNA-AA adducts, it is also envisaged that the incorporation of a liquid/liquid extraction process prior to the SPE cleanup may assist in reducing the matrix effects during electrospray ionization, thereby increasing the sensitivity of the LC-MS/MS analysis. The key advantage of our method is the lack of invasive procedures for sample collection, making it feasible to use it on a large-scale risk assessment for AA exposure. A minor drawback is that urinary RNA-AA adducts could only be identified for roughly one month after the AA exposure. On the contrary, DNA-AA adducts persist for years postexposure. It is expected that an increased analytical sensitivity would facilitate the analysis of RNA-AA adducts in urine samples collected after a longer period of exposure, complementing the analysis of DNA-AA adducts in tissue-isolated DNA samples. In summary, the results of this study suggest that urinary RNA adducts of AA may serve as effective biomarkers and that the developed LC−MS/MS method could provide a sensitive and noninvasive means for the determination of human exposure to AA by quantifying urinary RNA adducts. Furthermore, the quantification of urinary RNA−carcinogen adducts may prove useful in assessing environmental exposure to other chemical carcinogens, such as aflatoxin and polycyclic aromatic hydrocarbons.15,16
graphic motilities and MS/MS properties with those of synthetic RNA-AA adduct standards. To the best of our knowledge, this study is the first to report on the identification of RNA-AA adducts in urine samples. By using matrix matched calibration curves that were established for A-AAI and G-AAI, we quantified the urinary RNA-AA adduct levels. Concentration profiles of A-AAI, A-AAII, G-AAI, and G-AAII in the urine samples from AA-treated rats are depicted in Figure 1.
Figure 1. Urinary excretion profiles of (A) A-AA and (B) G-AA in AA treated rats. Rats were given a single, oral dose of AA at 30 mg/kg body wt at the time indicated with ↑, and 24 h urine samples at days 1, 2, 3, 7, 14, and 26 postdosing were collected. The data represent the mean ± SD for five independent rats.
As shown in Figure 1, RNA-AA adducts were rapidly formed upon reaction of the activated intermediates with the purine bases.2 The AA-bonded RNAs were promptly degraded by the hydrolytic action of cytoplasmic RNase and then excreted from the body via urination. RNA-AA adducts were detected at their highest concentrations in the urine samples collected 24 h after dosing the rats with AA. Specifically, A-AAI and A-AAII were detected at 10.2 ± 0.6 ng/mL and 21.1 ± 0.6 ng/mL, respectively. G-AAI and G-AAII were detected at concentrations significantly higher than that of the A-AA adducts with their respective concentrations detected at 47.7 ± 8.8 ng/mL and 48.3 ± 0.1 ng/mL. In general, the urinary adduct levels declined linearly and rapidly in the first 3 days postdosing, which is probably due to the combined effect of rapid AA metabolism and the short half-life of RNA.12,13 This was followed by a week of a more gradual decrease in adduct concentration. In this study, the urinary A-AAII concentration is roughly 2fold higher than that of the A-AAI adduct, which is consistent with a previous report that demonstrated higher levels of AAAII than A-AAI in internal organs of AA-treated rats.5,11 The greater chemical reactivity of AAII toward RNA maybe one of the potential reasons explaining the observed higher level of AAAII.11 A higher enzymatic selectivity of RNase for AAII adducts may also have contributed to this observation. The higher levels of AAII adducts is consistent with a previous report that demonstrated higher levels of A-AAII than A-AAI in internal organs of AA-treated rats.5,11 In accordance with the literature showing higher levels of guanosine adducts than those of adenosine adducts in RNA,11 G-AAI and G-AAII adducts were detected at concentrations roughly 2-fold higher than those of adenosine adducts. The greater nucleophilicity, thus the greater reaction efficiency of guanosine toward the electrophilic attack from the nitrenium ion intermediate, may explain the higher levels of guanosine adducts than those of adenosine adducts. It is also possible that the G-AA adducts-containing RNA were hydrolyzed more
■
ASSOCIATED CONTENT
S Supporting Information *
Materials and methods; characterization of RNA-AA adduct reference standards by MS analysis; and method calibration and validation. This material is available free of charge via the Internet at http://pubs.acs.org. 568
DOI: 10.1021/acs.chemrestox.5b00021 Chem. Res. Toxicol. 2015, 28, 567−569
Chemical Research in Toxicology
■
Rapid Report
performance liquid chromatography-triple quadrupole mass spectrometry. J. Chromatogr., B. 879, 153−158. (10) Leung, E. M., and Chan, W. (2014) Noninvasive measurement of aristolochic acid-DNA adducts in urine samples from aristolochic acid-treated rats by liquid chromatography coupled tandem mass spectrometry: Evidence for DNA repair by nucleotide-excision repair mechanisms. Mutat. Res. 766−767, 1−6. (11) Leung, E. M., and Chan, W. (2015) Comparison of DNA and RNA adduct formation: Significantly higher levels of RNA than DNA modifications in the internal organs of aristolochic acids-dosed rats. Chem. Res. Toxicol. 28, 248−255. (12) Chen, C. Y., Ezzeddine, N., and Shyu, A. B. (2008) Messenger RNA half-life measurements in mammalian cells. Methods Enzymol. 448, 335−357. (13) Chan, W., Cui, L., Xu, G., and Cai, Z. (2006) Study of the phase I and phase II metabolism of nephrotoxin aristolochic acid by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 20, 1755−1760. (14) Lai, M. N., Wang, S. M., Chen, P. C., Chen, Y. Y., and Wang, J. D. (2010) Population-based case-control study of Chinese herbal products containing aristolochic acid and urinary track cancer risk. J. Natl. Cancer Inst. 102, 179−186. (15) Bennett, R. A., Essigmann, J. M., and Wogan, G. N. (1981) Excretion of an aflatoxin-guanine adduct in the urine of aflatoxin B1treated rats. Cancer Res. 41, 650−654. (16) Shuker, D. E., and Farmer, P. B. (1992) Relevance of urinary DNA adducts as markers of carcinogen exposure. Chem. Res. Toxicol. 5, 450−460.
AUTHOR INFORMATION
Corresponding Author
*Tel: (852) 2358-7370. E-mail: [email protected]. Funding
This research was supported by the Research Grant Council of Hong Kong (ECS 609913). Wan Chan expresses his sincere thanks to the Hong Kong University of Science and Technology for Startup Funding (grant R9310). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We extend our gratitude to AB Sciex for providing the LC− MS/MS system for this research.
■
ABBREVIATIONS AA, aristolochic acid; LC−MS/MS, liquid chromatography− tandem mass spectrometry; HPLC, high-performance liquid chromatography; AAN, aristolochic acid nephropathy; BEN, Balkan endemic nephropathy; A-AAI, 7-(adenosine-N6-yl)aristolactam I; A-AAII, 7-(adenosine-N6-yl)-aristolactam II; GAAI, 7-(guanosine-N2-yl)-aristolactam I; G-AAII, 7-(guanosineN2-yl)-aristolactam II
■
REFERENCES
(1) Chan, W., Lee, K. C., Liu, N., and Cai, Z. (2007) A sensitivity enhanced high-performance liquid chromatography fluorescence method for the detection of nephrotoxic and carcinogenic aristolochic acid in herbal medicines. J. Chromatogr., A 1164, 113−119. (2) Arlt, V. M., Stiborova, M., and Schmeiser, H. H. (2002) Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis 17, 265−277. (3) Grollman, A. P., Shibutani, S., Moriya, M., Miller, F., Wu, L., Moll, U., Suzuki, N., Fernandes, A., Rosenquist, T., Medverec, Z., Jakovina, K., Brdar, B., Slade, N., Turesky, R. J., Goodenough, A. K., Rieger, R., Vukelic, M., and Jelakovic, B. (2007) Aristolochic acid and the etiology of endemic (Balkan) nephropathy. Proc. Natl. Acad. Sci. U.S.A. 104, 12129−12134. (4) Vanherweghem, J. L., Depierreux, M., Tielemans, C., Abramowicz, D., Dratwa, M., Jadoul, M., Richard, C., Vandervelde, D., Verbeelen, D., Vanhaelen-Fastre, R., and Vanhaelen, M. (1993) Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet 341, 387−391. (5) Arlt, V. M., Wiessler, M., and Schmeiser, H. H. (2000) Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene. Carcinogenesis 21, 235−242. (6) Bieler, C. A., Stiborova, M., Wiessler, M., Cosyns, J. P., van Ypersele de Strihou, C., and Schmeiser, H. H. (1997) 32P-postlabelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy. Carcinogenesis 18, 1063−1067. (7) Yun, B. H., Rosenquist, T. A., Sidorenko, V., Iden, C. R., Chen, C. H., Pu, Y. S., Bonala, R., Johnson, F., Dickman, K. G., Grollman, A. P., and Turesky, R. J. (2012) Biomonitoring of aristolactam-DNA adducts in human tissues using ultra-performance liquid chromatography/iontrap mass spectrometry. Chem. Res. Toxicol. 25, 1119−1131. (8) Fernando, R. C., Schmeiser, H. H., Nicklas, W., and Wiessler, M. (1992) Detection and quantitation of dG-AAI and dA-AAI adducts by 32P-postlabeling methods in urothelium and exfoliated cells in urine of rats treated with aristolochic acid I. Carcinogenesis 13, 1835−1839. (9) Guo, L., Wu, H., Yue, H., Lin, S., Lai, Y., and Cai, Z. (2011) A novel and specific method for the determination of aristolochic acidderived DNA adducts in exfoliated urothelial cells by using ultra 569
DOI: 10.1021/acs.chemrestox.5b00021 Chem. Res. Toxicol. 2015, 28, 567−569
Quantification of Aristolochic Acid-RNA Adducts in the Urine of Aristolochic Acid-Treated Rats by Liquid Chromatography−Tandem Mass Spectrometry Elvis M. K. Leung and Wan Chan* Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China S Supporting Information *
ABSTRACT: Balkan endemic nephropathy (BEN) is a peculiar renal disease affecting thousands of farmers living in the suburban areas of the Balkan countries. Emerging evidence suggested that BEN is an environmental disease caused by chronic food poisoning with aristolochic acid (AA). We have developed a sensitive liquid chromatography−tandem mass spectrometry method to detect urinary RNA-AA adducts. Results revealed high levels of RNA-AA adducts in the urine samples collected from AA-treated rats. To the best of our knowledge, this study is the first to report on the detection of urinary RNA-AA adducts. Compared with previous studies that quantified DNA-AA adducts, this method is more sensitive and user-friendly.
A
samples of the target organs by a noninvasive method as well as in obtaining ethical approval to conduct the experiment on human tissues. Therefore, noninvasive analytical methods for monitoring the risk of human exposure to AA would be extremely useful. A few studies have analyzed DNA-AA adducts in exfoliated cells in the urine of rats treated with AA.8,9 However, liters of urine samples had to be collected before a sufficient amount of cells/DNA was accumulated for the analysis. We recently reported the first noninvasive method for assessing exposure to AA by quantifying urinary DNA-AA adducts from AA-treated rats.10 With emerging evidence showing high frequencies of formation of RNA adducts in vitro and in vivo and given that RNA is less stable than DNA,11 we postulate that abundant RNA-AA adducts may be excreted in urine. Thus, quantifying urinary RNA-AA adducts may provide a more sensitive and equally noninvasive approach to monitoring exposure to AA. To test this hypothesis, we developed a highly sensitive and selective liquid chromatography−tandem mass spectrometry (LC−MS/MS) method to quantify urinary RNA-AA adducts in rat urine. To the best of our knowledge, the identification of RNA-AA adducts in urine has not been reported in the literature. The LC-MS/MS analysis of the SPE enriched urine samples collected from rats treated with AA (Figures S2B, S2D, S3B, and S3D, Supporting Information) revealed unambiguous identification of the targeted adducts by comparing chromato-
ristolochic acid (AA) are a family of nitrophenanthrene carboxylic acid derived from Aristolochia, and they were found to be potent nephrotoxins.1−3 For instance, prolonged exposure to AA-containing herbal remedies has resulted in kidney failure among Belgian women participating in a slimming regime in the 1990s.3,4 This unique type of rapid, progressive renal fibrotic disease, initially referred to as “Chinese herb nephropathy,” was later renamed aristolochic acid nephropathy (AAN).2 Significant evidence also suggested that chronic food poisoning by AA-contaminated wheat flour is one of the major reasons leading to Balkan endemic nephropathy (BEN),3 a peculiar renal disease that has been observed extensively in farmers living alongside the Danube River over the past 50 years. AA are also strongly carcinogenic and are classified as Group I human carcinogens by the International Agency for Research on Cancer (IARC).2 Specifically, tumors have been observed in multiple internal organs of AA-treated rodents, and a high frequency of AT → TA transversions was found in these tumor tissues.5 Covalently bonded DNA-AA adducts were also identified in the internal organs of AA-treated rats as well as in patients suffering from AAN or BEN.3,5,6 Owing to the strong nephrotoxicity and potent carcinogenicity of AA, analytical methods capable of monitoring human exposure to AA are needed.7 Current exposure assessments focus exclusively on analyzing tissue-isolated DNA samples for DNA-AA adducts.3,6,7 However, one of the major obstacles in conducting large-scale risk assessment based on DNA-AA adducts, e.g., in the Balkan Peninsula, is the accessibility to relevant biopsy tissue samples. Difficulties exist in collecting © 2015 American Chemical Society
Received: January 13, 2015 Published: February 25, 2015 567
DOI: 10.1021/acs.chemrestox.5b00021 Chem. Res. Toxicol. 2015, 28, 567−569
Chemical Research in Toxicology
Rapid Report
easily than those of A-AA adducts-containing RNA. Interestingly, despite the dramatically higher levels of G-AAII than GAAI adducts in the internal organs of AA-treated rats,11 these adducts were determined to be at similar concentrations in the urine samples collected from the AA-treated rats. Compared with the concentrations of DNA-AA adducts in urine samples collected from AA-treated rats,10 all of the corresponding RNA-AA adducts were detected at concentrations roughly 2-fold higher, with the exception of A-AAII, which was detected at levels similar to those of dA-AAII. Potential reasons for this observation include the higher frequencies of RNA adduct formation and greater stability of the DNA-AA lesions in the genome. Given that RNA is more reactive toward electrophilic attack by the nitrenium ion intermediate from AA metabolism, RNA adducts of AA are thus formed at frequencies significantly greater than those of the DNA adducts in the internal organs of AA-treated rats.11 Apart from the higher formation rate, the RNA adducts of AA degrade more easily when compared with the DNA-AA adducts. While RNA containing the AA-modified ribonucleosides are routinely being degraded by RNase and eliminated via urination, the DNA adducts of AA, dA-AA in particular, have a long lasting persistence.6 We understand that in the present study, rats were exposed to dosages of AA larger than those determined in humans.14 Improvement in analytical sensitivity would be needed to meet the challenge of analyzing RNA-AA adducts in human urine samples. While we used 3 mL of rat urine for a single experiment, enhancement in analytical sensitivity could be achieved by using a larger urine volume. With the high hydrophobicity of the RNA-AA adducts, it is also envisaged that the incorporation of a liquid/liquid extraction process prior to the SPE cleanup may assist in reducing the matrix effects during electrospray ionization, thereby increasing the sensitivity of the LC-MS/MS analysis. The key advantage of our method is the lack of invasive procedures for sample collection, making it feasible to use it on a large-scale risk assessment for AA exposure. A minor drawback is that urinary RNA-AA adducts could only be identified for roughly one month after the AA exposure. On the contrary, DNA-AA adducts persist for years postexposure. It is expected that an increased analytical sensitivity would facilitate the analysis of RNA-AA adducts in urine samples collected after a longer period of exposure, complementing the analysis of DNA-AA adducts in tissue-isolated DNA samples. In summary, the results of this study suggest that urinary RNA adducts of AA may serve as effective biomarkers and that the developed LC−MS/MS method could provide a sensitive and noninvasive means for the determination of human exposure to AA by quantifying urinary RNA adducts. Furthermore, the quantification of urinary RNA−carcinogen adducts may prove useful in assessing environmental exposure to other chemical carcinogens, such as aflatoxin and polycyclic aromatic hydrocarbons.15,16
graphic motilities and MS/MS properties with those of synthetic RNA-AA adduct standards. To the best of our knowledge, this study is the first to report on the identification of RNA-AA adducts in urine samples. By using matrix matched calibration curves that were established for A-AAI and G-AAI, we quantified the urinary RNA-AA adduct levels. Concentration profiles of A-AAI, A-AAII, G-AAI, and G-AAII in the urine samples from AA-treated rats are depicted in Figure 1.
Figure 1. Urinary excretion profiles of (A) A-AA and (B) G-AA in AA treated rats. Rats were given a single, oral dose of AA at 30 mg/kg body wt at the time indicated with ↑, and 24 h urine samples at days 1, 2, 3, 7, 14, and 26 postdosing were collected. The data represent the mean ± SD for five independent rats.
As shown in Figure 1, RNA-AA adducts were rapidly formed upon reaction of the activated intermediates with the purine bases.2 The AA-bonded RNAs were promptly degraded by the hydrolytic action of cytoplasmic RNase and then excreted from the body via urination. RNA-AA adducts were detected at their highest concentrations in the urine samples collected 24 h after dosing the rats with AA. Specifically, A-AAI and A-AAII were detected at 10.2 ± 0.6 ng/mL and 21.1 ± 0.6 ng/mL, respectively. G-AAI and G-AAII were detected at concentrations significantly higher than that of the A-AA adducts with their respective concentrations detected at 47.7 ± 8.8 ng/mL and 48.3 ± 0.1 ng/mL. In general, the urinary adduct levels declined linearly and rapidly in the first 3 days postdosing, which is probably due to the combined effect of rapid AA metabolism and the short half-life of RNA.12,13 This was followed by a week of a more gradual decrease in adduct concentration. In this study, the urinary A-AAII concentration is roughly 2fold higher than that of the A-AAI adduct, which is consistent with a previous report that demonstrated higher levels of AAAII than A-AAI in internal organs of AA-treated rats.5,11 The greater chemical reactivity of AAII toward RNA maybe one of the potential reasons explaining the observed higher level of AAAII.11 A higher enzymatic selectivity of RNase for AAII adducts may also have contributed to this observation. The higher levels of AAII adducts is consistent with a previous report that demonstrated higher levels of A-AAII than A-AAI in internal organs of AA-treated rats.5,11 In accordance with the literature showing higher levels of guanosine adducts than those of adenosine adducts in RNA,11 G-AAI and G-AAII adducts were detected at concentrations roughly 2-fold higher than those of adenosine adducts. The greater nucleophilicity, thus the greater reaction efficiency of guanosine toward the electrophilic attack from the nitrenium ion intermediate, may explain the higher levels of guanosine adducts than those of adenosine adducts. It is also possible that the G-AA adducts-containing RNA were hydrolyzed more
■
ASSOCIATED CONTENT
S Supporting Information *
Materials and methods; characterization of RNA-AA adduct reference standards by MS analysis; and method calibration and validation. This material is available free of charge via the Internet at http://pubs.acs.org. 568
DOI: 10.1021/acs.chemrestox.5b00021 Chem. Res. Toxicol. 2015, 28, 567−569
Chemical Research in Toxicology
■
Rapid Report
performance liquid chromatography-triple quadrupole mass spectrometry. J. Chromatogr., B. 879, 153−158. (10) Leung, E. M., and Chan, W. (2014) Noninvasive measurement of aristolochic acid-DNA adducts in urine samples from aristolochic acid-treated rats by liquid chromatography coupled tandem mass spectrometry: Evidence for DNA repair by nucleotide-excision repair mechanisms. Mutat. Res. 766−767, 1−6. (11) Leung, E. M., and Chan, W. (2015) Comparison of DNA and RNA adduct formation: Significantly higher levels of RNA than DNA modifications in the internal organs of aristolochic acids-dosed rats. Chem. Res. Toxicol. 28, 248−255. (12) Chen, C. Y., Ezzeddine, N., and Shyu, A. B. (2008) Messenger RNA half-life measurements in mammalian cells. Methods Enzymol. 448, 335−357. (13) Chan, W., Cui, L., Xu, G., and Cai, Z. (2006) Study of the phase I and phase II metabolism of nephrotoxin aristolochic acid by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 20, 1755−1760. (14) Lai, M. N., Wang, S. M., Chen, P. C., Chen, Y. Y., and Wang, J. D. (2010) Population-based case-control study of Chinese herbal products containing aristolochic acid and urinary track cancer risk. J. Natl. Cancer Inst. 102, 179−186. (15) Bennett, R. A., Essigmann, J. M., and Wogan, G. N. (1981) Excretion of an aflatoxin-guanine adduct in the urine of aflatoxin B1treated rats. Cancer Res. 41, 650−654. (16) Shuker, D. E., and Farmer, P. B. (1992) Relevance of urinary DNA adducts as markers of carcinogen exposure. Chem. Res. Toxicol. 5, 450−460.
AUTHOR INFORMATION
Corresponding Author
*Tel: (852) 2358-7370. E-mail: [email protected]. Funding
This research was supported by the Research Grant Council of Hong Kong (ECS 609913). Wan Chan expresses his sincere thanks to the Hong Kong University of Science and Technology for Startup Funding (grant R9310). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We extend our gratitude to AB Sciex for providing the LC− MS/MS system for this research.
■
ABBREVIATIONS AA, aristolochic acid; LC−MS/MS, liquid chromatography− tandem mass spectrometry; HPLC, high-performance liquid chromatography; AAN, aristolochic acid nephropathy; BEN, Balkan endemic nephropathy; A-AAI, 7-(adenosine-N6-yl)aristolactam I; A-AAII, 7-(adenosine-N6-yl)-aristolactam II; GAAI, 7-(guanosine-N2-yl)-aristolactam I; G-AAII, 7-(guanosineN2-yl)-aristolactam II
■
REFERENCES
(1) Chan, W., Lee, K. C., Liu, N., and Cai, Z. (2007) A sensitivity enhanced high-performance liquid chromatography fluorescence method for the detection of nephrotoxic and carcinogenic aristolochic acid in herbal medicines. J. Chromatogr., A 1164, 113−119. (2) Arlt, V. M., Stiborova, M., and Schmeiser, H. H. (2002) Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis 17, 265−277. (3) Grollman, A. P., Shibutani, S., Moriya, M., Miller, F., Wu, L., Moll, U., Suzuki, N., Fernandes, A., Rosenquist, T., Medverec, Z., Jakovina, K., Brdar, B., Slade, N., Turesky, R. J., Goodenough, A. K., Rieger, R., Vukelic, M., and Jelakovic, B. (2007) Aristolochic acid and the etiology of endemic (Balkan) nephropathy. Proc. Natl. Acad. Sci. U.S.A. 104, 12129−12134. (4) Vanherweghem, J. L., Depierreux, M., Tielemans, C., Abramowicz, D., Dratwa, M., Jadoul, M., Richard, C., Vandervelde, D., Verbeelen, D., Vanhaelen-Fastre, R., and Vanhaelen, M. (1993) Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet 341, 387−391. (5) Arlt, V. M., Wiessler, M., and Schmeiser, H. H. (2000) Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene. Carcinogenesis 21, 235−242. (6) Bieler, C. A., Stiborova, M., Wiessler, M., Cosyns, J. P., van Ypersele de Strihou, C., and Schmeiser, H. H. (1997) 32P-postlabelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy. Carcinogenesis 18, 1063−1067. (7) Yun, B. H., Rosenquist, T. A., Sidorenko, V., Iden, C. R., Chen, C. H., Pu, Y. S., Bonala, R., Johnson, F., Dickman, K. G., Grollman, A. P., and Turesky, R. J. (2012) Biomonitoring of aristolactam-DNA adducts in human tissues using ultra-performance liquid chromatography/iontrap mass spectrometry. Chem. Res. Toxicol. 25, 1119−1131. (8) Fernando, R. C., Schmeiser, H. H., Nicklas, W., and Wiessler, M. (1992) Detection and quantitation of dG-AAI and dA-AAI adducts by 32P-postlabeling methods in urothelium and exfoliated cells in urine of rats treated with aristolochic acid I. Carcinogenesis 13, 1835−1839. (9) Guo, L., Wu, H., Yue, H., Lin, S., Lai, Y., and Cai, Z. (2011) A novel and specific method for the determination of aristolochic acidderived DNA adducts in exfoliated urothelial cells by using ultra 569
DOI: 10.1021/acs.chemrestox.5b00021 Chem. Res. Toxicol. 2015, 28, 567−569
