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Quantitation of DNA Adducts in Target and Non-Target Organs of Aristolochic Acid I-Exposed Rats by LC-MS/MS Coupled with Stable Isotope-Dilution Method: Correlating DNA Adduct Levels with Organotropic Activities Yushuo LIU, Chi Kong Chan, Long Jin, Sum-Kok Wong, and Wan Chan Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00359 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019
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Chemical Research in Toxicology
Quantitation of DNA Adducts in Target and Non-Target Organs of Aristolochic Acid I-Exposed Rats by LC−MS/MS Coupled with Stable Isotope-Dilution Method: Correlating DNA Adduct Levels with Organotropic Activities Yushuo Liu,†,§ Chi-Kong Chan,‡,§ Long Jin,‡ Sum-Kok Wong,† and Wan Chan*,†,‡ †
Division of Environment & Sustainability and ‡Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong ABSTRACT: Chronic exposure to aristolochic acids (AAs) from Aristolochia plants is one of the major causes of nephropathy and cancer of the kidney and forestomach. However, the organotropic activities of AAs remain poorly understood. In this study, using LC−MS/MS coupled with a stable isotope-dilution method, we rigorously quantitated for the first time the organ-specific dosageand time-dependent formation of DNA-AA adducts in the tumor target and non-target organs of AA-I-treated rats. The results support the proposal that the DNA adduct level is a major contributor to the observed organotropic activities of AAs.
Aristolochic acids (AAs) are highly potent human carcinogens produced naturally by Aristolochia plants, with aristolochic acid I (AA-I) being the major and most genotoxic component (Figure 1).1 Species of the Aristolochia family were used as traditional Chinese medicines for multiple therapeutic purposes until AAs and AAs-containing herbs were observed to be highly carcinogenic and nephrotoxic in AAs-exposed laboratory rodents and humans.2,3 AAs and plants containing them have been classified by the International Agency for Research on Cancer as Group I carcinogens.1 Currently, herbs containing AAs are banned from use in the production of herbal medicines in countries all over the world.
Figure 1. Postulated carcinogenic mechanism of aristolochic acid I. Besides targeting the kidney and inducing rapidly progressive renal fibrosis, it has been observed that AAs exert organotropic genotoxicity.2,3 Specifically, previous studies have shown that AAs target the forestomach, kidney, and urinary bladder to induce carcinoma formation in rats. Despite decades of research, the molecular mechanism underlying both the nephrotoxicity and carcinogenicity of AAs remain to be explored. However, it has been speculated that DNA adduct formation is
responsible for both the kidney fibrotic process and the carcinogenicity of AAs (Figure 1).2,3 Thus, quantitating the levels of DNA adducts in different organs would provide a dosimetry to better understand AA-mediated carcinogenicity and assess the cancer risk associated with AA exposure. However, most previous studies of DNA-AA adducts were conducted using 32Ppost-labeling analyses and were only descriptive in nature.2-7 Liquid chromatography-tandem mass spectrometry (LC−MS/MS), despite being one of the most sensitive and reliable methods for bioanalysis, has only begun to be used for exploring the quantitative analysis of DNA-AA adducts.6,8,9 In addition, quantitation of DNA-AA adducts has been limited to the kidney and liver.8 In this study, our previously developed LC−MS/MS was coupled with a stable isotope-dilution method,9 and we determined for the first time the organ-specific distribution of DNA-AA adducts in the target (forestomach and kidney) and non-target (glandular stomach, small intestine, large intestine, liver, lung, heart, and spleen) organs of male Sprague-Dawley rats treated with a single oral dose of AA-I (10 mg/kg body wt; n = 5) which was previously demonstrated to induce a detectable level of DNA-AA adducts for analysis. To our surprise, we detected and quantitated 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI) in all collected tissue samples, and at concentrations ranging from 1.3 to 69.7 adducts per 107 nt (Figure 2A). Notably, the detection of DNA-AA adducts in the heart, spleen, small intestine, and large intestine has not been reported in the literature, to the best of our knowledge. The results indicate that AAs exert their genotoxicity extensively in most of the organs of the exposed rats. The highest adduct frequency (69.7 ± 9.2 adducts per 107 nt) was detected in the forestomach, which is one of the target organs for AA-mediated carcinogenesis. This observation is in line with prior knowledge that DNA-AA adduct formation is a
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Figure 2. Levels of dA-AAI in organs of rats treated with AA-I at (A) 10 and (B) at 30 mg/kg body wt, together with (C) dG-AAI concentrations in organs of rats given AA-I at 30 mg/kg body wt. Rats were given a single oral dose of AA-I and were sacrificed 24 h postdosing. The data represent mean ± SD for five independent rats. key event in the carcinogenesis of AA in rats.3,4 The forestomach is the holding chamber for food, and therefore exposed for a longer term and to a higher AA concentration. Notably, the stomach is highly acidic, which may facilitate cellular absorption of the neutral forms of the weakly acidic AAs, perhaps by increased partitioning into the lipophilic cell membranes of epithelial cells, as observed in the uptake of AA by root epithelia cells in plants.10 We believe that the combined effect of higher exposure levels, longer retention time and higher absorption efficiency of AAs into the adjacent epithelial cells of the forestomach resulted in elevated levels of DNA adducts formed in the region. This hypothesis is also supported by the decreasing adduct concentrations down the digestive tract, i.e., from forestomach to glandular stomach to small intestine to large intestine, where both the AA concentrations and the acidity decrease gradually. Interestingly, the kidney is a target organ of AA-mediated carcinogenesis, but the levels of dA-AAI adduct in kidney-isolated DNA were detected at ~6 adducts per 107 nt, which is 10fold lower than that observed in the forestomach (Figure 2A). A similar concentration was also detected in non-target tissues, such as the liver and glandular stomach. Nevertheless, these results are in good agreement with the previous observation that there is no clear correlation of DNA adduct levels with the organotropic tumor-inducing property of AA.2 It is likely that other toxicological mechanisms such as oxidative stress may also contribute to the observed carcinogenicity of AA in kidney.11 7-(deoxyguanosin-N2-yl)-aristolactam I (dG-AAI), another adduct formed by reacting the aristolactam-nitriumion with DNA was detected in DNA isolated from both the forestomach and kidney of the AA exposed rats (Figure S1), but at concentrations below the method quantitation limit (0.5 adducts per 107 nt per 45 μg of DNA). It was thus not quantitated. A general dosage-dependent increase in the levels of dA-AAI was observed when the dosage of AA-I was increased from 10 (Figure 2A) to 30 mg/kg/day (Figure 2B). Nevertheless, the adduct pattern was similar, and the highest adduct level was also observed in the forestomach (120.6 ± 27.6 adducts per 107 nt). Similar to that observed in the low-dose group, the adduct levels in the kidney, liver, and glandular stomach were detected at 5to 10-fold lower concentrations than in the forestomach. The increased dosage also induced higher levels of dG-AAI formation, which facilitated its detection. LC−MS/MS analysis of
dG-AAI in tissue-isolated DNA samples from rats treated with high dose of AA-I revealed a similar adduct pattern to that observed in the analysis of the dA-AAI adduct, but at 2- to 9-fold lower concentrations and with dG-AAI detected at concentrations ranging from 1.7 to 47.8 adducts per 107 nt. This observation is in line with a previous report that the adduct levels of dG-AAI are significantly lower than that of dA-AAI in DNA isolated from rat tissues,2-5 likely due to its lower persistence and lower initial DNA binding efficiency. Furthermore, the highest adduct level was again observed in the forestomach. Our study reveals that regardless of the dosage of AA-I administrated to rats, 2’-deoxyadenosine is the major target of DNA adduct formation in all tested organs, pointing to an important role of 2’-deoxyadenosine modification for the chemical carcinogenesis of AA-I. This observation is in agreement with the mutation spectrum of AAs, in which a high frequency of A→T transversion were observed in both in vitro primer extension studies and in urothelial carcinoma of the upper urinary tract in patients suffering from aristolochic acid nephropathy (AAN).3,6 This suggests a causative role of the dA-AAI adducts in AAinduced mutagenesis and carcinogenesis. To investigate the potential contribution of adduct stability to the observed organotropic activity of AA, the in vivo stability of the DNA adducts was also investigated by analyzing dA-AAI and dG-AAI in DNA isolated from the tissue of rats given a single oral dose of AA-I at 30 mg/kg body wt and scarified at 1, 2, 3, 8, 14, 21, 28, and 60 days postdosing. Analysis of the adduct kinetics revealed an interesting phenomenon that the adduct levels kept increasing from day 1 to day 2 (day 3 for dGAAI) after dosing in all tested organs, probably due to the continuous absorption, distribution, and metabolism of AA in the organs and leading to elevated levels of DNA-AA adduct formation that out-paced the rate at which they were repaired. In addition, the largest increase was observed in the renal cortex, with 6- and 8-fold increases in dA-AAI and dG-AAI (Figure 3), respectively, and with the adduct frequency approaching that of the forestomach at day 1. The data, in conjunction with the organotropic distribution of the DNA-AA adducts shown in Figure 2, explains the observation that the kidney and forestomach are the tumor target organs of AAs and the site of induced mutagenesis, carcinogenesis, and renal fibrosis, also confirmed is that the organotropic tumor-inducing property of AA is closely correlated with DNA adduct amounts. As shown in Figure 3, the adduct levels in the foresto-
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Chemical Research in Toxicology Funding Sources We thank the TUYF Charitable Trust for supporting this study.
Notes The authors declare no competing financial interest.
ABBREVIATIONS Figure 3. Time course and half-life (t1/2) of (A) dA-AAI and (B) dG-AAI adduct levels in the kidney, liver, and stomach of the rat exposed to a single oral dose of 30 mg/kg of AA-I. The data represent mean ± SD for three independent rats. mach and renal cortex peaked at different post exposure times, which could only be observed with a time-course experiment. A sharp decrease in adduct levels (over 50 % of the observed maxima) in all the tissues was observed in the first 8 days and the rapid removal of adducts in the stomach were probably influenced by cell-kinetics turnover. The average half-life of dAAAI were ~19.7 days and ~16.8 days in the target and non-target tissues, respectively, whereas dG-AAI had average half-life of ~2.8 days and ~1.7 days. The higher persistence of dA-AAI than dG-AAI in vivo may further contribute to the high frequency of A→T transversion and reinforce the role of dA-AAI in the mutagenesis. Moreover, the adducts in target tissues were observed to be more stable than in non-target tissues, which could be a result of AA-I binding to specific genomic sites in the target organs that are more resistant to repair.3 In summary, we applied LC−MS/MS coupled with a stable isotope-dilution method, to rigorously determine for the first time the organ specific-distribution and in vivo stability of AAI-induced DNA-AA adducts in the tumor target and non-target organs of AA-I exposed rats. The results reveal for the first time that the organotropic genotoxicity of AA is positively correlated with DNA-AA adduct concentrations in organs. In addition, the results indicate significantly higher concentrations and higher persistence of dA-AAI than dG-AAI, which explains the high levels of AT→TA transversion mutation observed in the urothelial carcinoma of the upper urinary tract in patients suffering from AAN. We believe that the results can help us to better understand the observed organotrophic activity of AA.
ASSOCIATED CONTENT Supporting Information Experimental section and chromatograms from LC−MS/MS analysis of DNA-AA adducts in renal cortex of rats treated with 10 and 30 mg/kg of AA-I. Calibration curves of adducts used for quantitation in rat tissues. Log scale of time course of adduct levels in tissues of rat administrated with 30mg/kg of AA-I. The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION Corresponding Author * Tel: (852) 2358-7370. E-mail:
[email protected].
Author Contributions §Y.
Liu and C.-K. Chan contributed equally to this work.
AAs, aristolochic acids; AA-I, aristolochic acid I; AAN, aristolochic acid nephropathy; dA-AAI, 7-(deoxyadenosin-N6-yl)-aristolactam I; dG-AAI, 7-deoxyguanosin-N2-yl)-aristolactam I; LC−MS/MS, Liquid chromatography-tandem mass spectrometry.
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