Quantitation of the DNA Adduct of Semicarbazide in Organs of

Wang , Y., Jester , E. L. E., El-Said , K. R., Abraham , A., Hooe-Rollman , J., and .... E. (2005) Semicarbazide: occurrence in food products and stat...
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Quantitation of the DNA Adduct of Semicarbazide in Organs of Semicarbazide-Treated Rats by Isotope-Dilution Liquid ChromatographyTandem Mass Spectrometry: A Comparative Study with the RNA Adduct Yinan Wang, Tin-Yan Wong, and Wan Chan Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00232 • Publication Date (Web): 10 Aug 2016 Downloaded from http://pubs.acs.org on August 16, 2016

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Quantitation of the DNA Adduct of Semicarbazide in Organs of Semicarbazide-Treated Rats by Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry: A Comparative Study with the RNA Adduct

Yinan Wang, Tin-Yan Wong, and Wan Chan* Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

* Correspondence author. Address: The Hong Kong University of Science and Technology, Department of Chemistry, Room 4520, Academic Building, Clear Water Bay, Kowloon, Hong Kong, HK 00000; Phone: +852 2358-7370; Fax: +852 2358-1594; E-mail: [email protected].

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Table of Contents Graphic

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Abstract Semicarbazide is a wide-spread food contaminant that is produced by multiple pathways. However, the toxicity of semicarbazide to human health remains unclear. Using a highly accurate and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry method, we identified and quantitated in this study for the first time the DNA and RNA adduct of semicarbazide in DNA/RNA isolated from the internal organs of semicarbazide-exposed rats. The analysis revealed a dose-dependent formation of the adducts in the internal organs of the semicarbazide-dosed rats and with the highest adduct levels identified in the stomach and small intestine. Furthermore, results showed significantly higher levels of the RNA adduct (4.1-7.0 times) than that of the DNA adducts. By analyzing DNA/RNA samples isolated from rat organs in semicarbazide-dosed rats at different time points post-dosing, the adduct stability in vivo was also investigated. These findings suggest that semicarbazide could have exerted its toxicity by affecting both the transcription and translation processes of the cell.

Keywords: Semicarbazide, Genotoxicity, Abasic site, DNA adduct, RNA adduct

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Introduction Semicarbazide is a hydrazine-like food contaminant that has been detected in a wide variety of food products such as meat, honey and bread, arising from multiple pathways. For example, semicarbazide is one of the metabolic products of the antibiotic nitrofuranzone, and semicarbazide was found to bind with proteins in aquatic organisms, poultry, and livestock exposed to the antibiotic.1-3 Semicarbazide was also detected in hypochlorite-treated, nitrogen-rich food products.4,5 Moreover, semicarbazide is a thermal degradation product of azodicarbonamide,6,7 which is a blowing agent used extensively in plastic and bread productions.8,9 We recently also detected semicarbazide in commercially available bread samples.3 Therefore, humans are constantly being exposed to semicarbazide through food intake.

Despite decades of research, the biological implications of semicarbazide to humans remained controversial.10-12 For example, a recent study concluded that semicarbazide was not carcinogenic in either male or female Wistar Hannover GALAS rats when administered in the diet for 2 years.13 However, mice administered high doses of semicarbazide apparently developed lung and blood vessel tumors.14

It is well-known that a key step in chemical carcinogenesis involves the 4

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formation of covalent adducts between DNA and genotoxins.15,16 In this regard, we recently reported the first identification of a covalently bonded semicarbazide-DNA adduct in vitro and in bacteria where semicarbazide reacted with the apurinic/apyrimidinic (AP) sites of DNA forming semicarbazone adducts.17 The AP sites are among the most common DNA lesions that arise from base excision repair (BER) of modified nucleotide bases and by spontaneous depurination under physiological conditions,18,19 thus we believe that the formation of the semicarbazide-DNA adducts could possibly be linked to the observed genotoxicity of semicarbazide.

In this study, we explored beyond our previous findings to determine the internal organ-specific distribution of semicarbazide-DNA/RNA adducts in rats that were exposed to semicarbazide, quantitating the adducts through an isotope-dilution liquid chromatography coupled tandem mass spectrometry (LC−MS/MS) technique in DNA/RNA samples extracted from their heart, lung, stomach, small intestine, large intestine, liver, and kidney tissues.

Our study revealed a dose-dependent formation of the DNA and RNA adducts of semicarbazide in the stomach, small intestine, large intestine, liver, and kidney tissues 5

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of semicarbazide-treated rats, but neither of these adducts could be detected in the lung and heart tissues. To our understanding, this is the first report on detecting DNA and RNA adducts of semicarbazide in vivo. The highest level of adducts found in our study were from the stomach and small intestine, and we also established some understanding on the in vivo stability of semicarbazide-DNA and semicarbazide-RNA adducts in these internal organs. We believe these results can help to better understand the observed toxicity and health implications of semicarbazide.

Materials and Methods Chemicals and Reagents. 2’-Deoxy-D-ribose (dR), D-ribose (R), semicarbazide, 13

C-1,2-15N2-semicarbazide, proteinase K, RNase A, alkaline phosphatase, DNase I,

and nuclease P1 were purchased from Sigma-Aldrich (St. Louis, MO). Snake venom phosphodiesterase was obtained from US Biological (Swampscott, MA). Absolute ethanol and HPLC grade acetonitrile were purchased from Tedia (Fairfield, OH). Deionized water was further purified by a Milli-Q Ultrapure water system (Billerica, MA) and was used in all experiments.

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Unlabeled DNA adducts of semicarbazide (semicarbazide-dR) and its isotope-labeled internal standard (13C-1,2-15N2-semicarbazide-dR) were synthesized previously by reacting dR with an excess of semicarbazide or 13

C-1,2-15N2-semicarbazide.17 Using a similar strategy, the RNA adducts of

semicarbazide (semicarbazide-R) and its isotope-labeled internal standard (13C-1,2-15N2- semicarbazide-R) were synthesized by reacting R with unlabeled or isotope-labeled semicarbazide. The unlabeled RNA adduct of semicarbazide and its isotope-labeled internal standard, after being characterizing by high-performance liquid chromatography (Figure S1), high-accuracy mass spectrometry, and tandem mass spectrometry (Figure S2), were used as standards for quantifying the amount of RNA adduct of semicarbazide in the extracted RNA from semicarbazide-dosed rats.

Animal Experiment. Male Sprague-Dawley rats were obtained from the Animal and Plant Care Facility, HKUST. The protocol for animal experiments was approved by the Committee on Research Practice, HKUST and all experiments were performed in accordance with the Animal Ordinance established by the Department of Health, HKSAR. In brief, Sprague-Dawley rats (∼200 g, n=15) were divided randomly into three groups with food and water provided ad libitum. The high-dosage (n=5) and low-dosage (n=5) group received oral gavage of 80 and 40 mg/kg of semicarbazide in 7

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1 mL of potassium phosphate buffer (50 mM, pH 7.4) for five consecutive days; the control group (n=5) received an equal volume of the dosing vehicle. One day after the last dose, the rats were sacrificed by decapitation, and the lung, heart, stomach, small intestine, large intestine, kidney, and liver were harvested to investigate the organ-specific distribution and dose-dependent formation of the adducts.

Using a similar approach, the rats (n=30) for investigating the stability of the semicarbazide-dR adducts were dosed with 80 mg/kg of semicarbazide for five consecutive days. At 1, 4, 7, 14, 28, and 49 days after the semicarbazide dosing, five rats from each group were randomly selected and sacrificed by decapitation. Stomach, small intestine, large intestine, kidney, and liver tissues were collected immediately, rinsed with KCl solution, and stored at −80 °C until DNA/RNA extraction.

DNA/RNA Isolation and Digestion. DNA was isolated from the collected tissue samples using a high-salt method described previously.20,21 Tissue RNA was extracted separately using the Trizol reagent (Life Technologies, Carlsbad, CA) according to the procedures recommended by the manufacturer. The isolated DNA/RNA samples, after being quantitated by UV absorption spectroscopy (260 nm), were fortified with isotope labeled internal standard and hydrolyzed enzymatically as described 8

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before.22,23 The hydrolysates, after passing through an Omega Nanosep spin filter (MWCO 3K, PALL) to remove the enzymes, were analyzed by the isotope-dilution LC−MS/MS method.

LC−MS/MS Analysis. Analysis of the semicarbazide-dR adduct was performed using our previously developed LC−MS/MS method.17 In brief, ribonucleosides in 10 µL of the hydrolysate was resolved with a Waters XBridge Amide column (50 mm × 3.0 mm, 3.5 µm particle size) eluted with a combined solvent of (A) 0.1% (v/v) formic acid in water and (B) 0.1% (v/v) formic acid in acetonitrile at a flow rate of 0.2 ml/min. Gradient elution was programmed as follows: 0−2 min, 100% B; 2−4 min, a linear gradient decreasing from 100−1% B; 4−6 min, 1% B; 6−15 min, 100% B.

The HPLC column was coupled to an AB Sciex 4000 QTRAP mass spectrometer operated at multiple-reaction monitoring (MRM) mode. Positive electrospray ionization source voltages and source gas parameters were optimized as follows: declustering potential, 32 V; entrance potential, 9 V; and ion spray voltage, 5000 V. The curtain gas (CUR), collision gas (CAD), ion source gas I (GSI), and ion source gas II (GSII), were set at 30, 5, 20 and 40, respectively. Temperature of the nebulizer gas (GSII) was set at 400 °C. The collision energy for collision-induced dissociation 9

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was set at 12.

MRM transitions of m/z 192→76 and 195→79 were used as quantifying transitions for quantitative analysis of the unlabeled and labeled semicarbazide-dR adduct, respectively. The corresponding MRM transitions of m/z 192→117 and 195→117 were used as qualifying transitions for the unlabeled and labeled semicarbazide-dR adduct. The dwell time for each transition was set at 100 ms. Using a similar LC-MS/MS method with MRM transition of m/z 208→76, the RNA adduct of semicarbazide was also quantitated using isotope labeled RNA adduct as internal standard (m/z 211→79).

Half-Life Calculation. The half-life (t1/2) of the RNA and DNA adducts of semicarbazide in internal organs of semicarbazide-dosed rats were calculated using a first order exponential fit of the data obtained from LC-MS/MS analysis of the DNA/RNA samples isolated from rats’ organs.24 Specifically, the ln[S]/[S0] values were plotted against time t, where [S] and [S0] represents the amount of adduct at the time t and on the first day, respectively.

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Results and Discussion Quantitation of Semicarbazide-DNA Adducts in Tissues of Semicarbazide-dosed Rats. The tissue samples, e.g. lung, heart, stomach, small intestine, liver, kidney, and large intestine, harvested from rats that were sacrificed one day post-dosing were used to investigate the organ specific distribution and the dose-dependent formation of semicarbazide-dR adduct. To this end, the tissue-isolated DNA samples were fortified with isotope labeled semicarbazide-dR internal standard, enzyme digested, and analyzed by LC–MS/MS using the methods described above. Depicted in Figure 2 are typical chromatograms obtained from LC–MS/MS analysis of synthetic semicarbazide-dR standard and the semicarbazide-dR adduct in stomach DNA isolated from a rat dosed with 80 mg/kg of semicarbazide. The analyses of tissue-isolated DNA samples revealed excellent agreement of chromatographic retention time and the peak area ratio of the quantifying transition to qualifying transition with that of the synthetic semicarbazide-dR standard, which unambiguously demonstrated the detection of the semicarbazide-DNA adduct in the DNA samples isolated from the stomach.

Using a similar approach, the semicarbazide-dR adduct was detected and quantitated in all the tissue-isolated DNA samples obtained from rats that were dosed 11

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with both 40 and 80 mg/kg of semicarbazide (Table 1). In both groups of rats, the highest adduct concentration was detected in DNA samples isolated from the small intestine. No semicarbazide-dR adduct was detected in the DNA samples isolated from the tissues of the control rats. Furthermore, the semicarbazide-dR adduct was not detected in either the lung or heart-isolated DNA samples, probably because they were further away from the gastrointestinal tract where the absorption of semicarbazide takes place. Among the tested tissue-isolated DNA samples that showed positive identification of the semicarbazide-dR adduct, the large intestine and kidney were similar but had the lowest adduct concentrations.

Furthermore, the analysis revealed a dose-dependent formation of the DNA adduct in the stomach, small intestine, liver, kidney, and large intestine samples in the low and high-dosage groups of rats (Table 1). The detection of semicarbazide-dR adduct in DNA samples isolated from tissues of the gastrointestinal tract, i.e. stomach, small intestine, and large intestine indicated that semicarbazide is a direct-acting genotoxin and may exert its genotoxicity by forming covalently bonded DNA adducts without hepatic metabolic activation.

The results also demonstrated a general decreasing trend of adduct levels as the 12

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drug is passing through the gastrointestinal tract (Table 1), i.e. from the small intestine to the large intestine. However, a slightly higher adduct level was observed in the small intestine than that in the stomach which could be attributed to the role of small intestines as the major absorption organ where more semicarbazide could be uptaken into the cells. Thus, a higher cellular concentration of semicarbazide would be available for reacting with DNA in the cells of the small intestine than that of the stomach. Furthermore, we observed a significantly lower reaction yield of the formation of semicarbazide-dR adduct under acidic conditions (pH 3.0) than that in media with higher pH (pH 5.8 and 7.4; Figure S3). Since the pH value increases down the digestive tract, from ~3.2 in the stomach to ~6.6 in the large intestine,25 it is reasonably that a lower adduct concentration was formed in the stomach than that in the small intestine because of the more acidic gastric environment.

Persistence of Semicarbazide-DNA Adducts in Selected Tissue of Rats. After identifying and quantitating the semicarbazide-dR adduct in the internal organs of semicarbazide-dosed rats, the study was extended to investigate the stability of semicarbazide-dR adduct in stomach, small intestine, liver, kidney, and large intestine for up to 49 days after the oral doses of semicarbazide to the rats. Data showed a rapid 13

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decrease of semicarbazide-dR adduct level during the first 7 days, during which ~50 % of the adduct observed at day 1 were removed, and the adduct concentrations in the selected tissue-isolated DNA continued to decrease. At 28 days post-dosing, the adduct concentrations in large intestine and kidney fell to below the minimum detection limit of the LC-MS/MS method (1 adducts per 106 nt), whereas 20% of the adducts observed at day 1 remained in the stomach, small intestine, and liver (Figure 3). Nevertheless, the analysis revealed a similar removal kinetics of the adduct in all the organs analyzed (t1/2 = ~6 days, Table 1). These observations suggest that the disappearance of the adducts may be the result of a combined effect of DNA repair and cell turnover.

Quantitation of the RNA Adduct of Semicarbazide in Tissues of Semicarbazide Dosed Rats. We previously identified that carcinogenic aristolochic acids form adducts with RNA at significantly higher levels than that of DNA.26 To investigate the feasibility of semicarbazide in forming covalently bonded adduct with RNA, we analyzed RNA samples isolated from tissues of the semicarbazide-treated rats for the presence of semicarbazide-RNA adduct using the LC-MS/MS method described above.

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Similar to that observed in the DNA adduct analysis, LC-MS/MS analysis revealed confident identification of semicarbazide-RNA adduct in stomach, small intestine, liver, kidney, and large intestine RNA isolated from semicarbazide-exposed rats. Figure 2 shows typical chromatograms obtained from LC-MS/MS analysis of the synthetic semicarbazide-RNA standard and semicarbazide-RNA adduct in stomach tissue RNA harvested from rats dosed with semicarbazide at 80 mg/kg. Furthermore, data demonstrated a similar pattern of RNA adduct distribution as that observed in the DNA adduct analysis (Table 1), with the highest adduct levels being observed in the stomach and small intestine.

Interestingly, the analysis revealed a significantly higher level of adduct of RNA than that of DNA in the tissue samples, where the concentration of semicarbazide adduct of RNA is 4.1-7.0 times higher than that of DNA (Table 1), which is similar to that observed in our previous study with aristolochic acids.26 A significantly higher level of adducts in RNA than DNA was also observed for other nucleic acid-reactive compounds such as aflatoxin and benzo[a]pyrene.27,28

Results also showed the half-life of the RNA adduct (from 2.0-3.7 days) is generally 50% shorter than that of the DNA adduct (from 4.6-6.9 days). This result is 15

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in reasonable agreement with the half-life of the long-lived RNAs (38-79 hrs for tRNA and rRNA),29 which implies the RNA adduct may get removed as the RNA molecules themselves are degraded.

Conclusion We report in this study the first identification of in vivo formed DNA and RNA adducts formed by semicarbazide, one of the most commonly found food contaminants, in the internal organs of semicarbazide-dosed rats. Using LC-MS/MS method of high sensitivity and selectivity, we quantitated the adduct levels in DNA/RNA samples extracted from the stomach, small intestine, liver, kidney, and large intestine tissues from semicarbazide-dosed rats. Data showed stomach and small intestine are the organs with the highest levels of accumulated semicarbazide-DNA and semicarbazide-RNA adducts. Furthermore, the analyses revealed a significantly higher level of RNA adduct compared to DNA adduct. It is believed that the results from this study will help to better understand the molecular mechanism underlying the genotoxicity of semicarbazide.

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Conflict of Interest Statement The authors declare that there are no conflicts of interest.

Acknowledgments We also thank AB Sciex for providing the LC–MS/MS system for this research.

Funding Sources This work was supported by the Research Grant Council of Hong Kong (ECS 609913). W. Chan thanks The Hong Kong University of Science and Technology for a Startup Funding (Grant R9310).

Author Contributions W. Chan and Y.W. designed research; Y.W. and T.-Y.W. performed research; W. Chan and Y.W. analyzed data; and W. Chan wrote the paper.

Abbreviations AP site, apurinic/apyrimidinic site; BER, base excision repair; dR, 2’-Deoxy-D-ribose; LC–MS/MS, liquid chromatography-tandem mass spectrometry; MRM; multiple-reaction monitoring; R, D-ribose. 17

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Supporting Information Available Chromatograms obtained from HPLC analysis of the purified standard of the RNA adduct of semicarbazide. High-accuracy MS spectra from ESI-MS analyses of the unlabeled semicarbazide−ribose adduct and isotope-labeled semicarbazide−ribose internal standard. Reaction efficiency of semicarbazine with 2’-deoxyribose in different pH conditions. This material is available free of charge via the Internet at http://pubs.acs.org

References (1) Wang, Y., Jester, E. L. E., El-Said, K. R., Abraham, A., Hooe-Rollman, J., and Plakas, S. M. (2010) Cyano metabolite as a biomarker of nitrofurazone in channel catfish. J. Agric. Food Chem. 58, 313-316. (2) Du, N. N., Chen, M. M., Sheng, L. Q., Chen, S. S., Xu, H. J., Liu, Z. D., Song, C. F., and Qiao, R. (2014) Determination of nitrofuran metabolites in shrimp by high performance liquid chromatography with fluorescence detection and liquid chromatography–tandem mass spectrometry using a new derivatization reagent. J. Chromatogr. A 1327, 90-96.

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(3) Wang, Y., and Chan, W. (2016) Automated in-injector derivatization combined with high-performance liquid chromatography–fluorescence detection for the determination of semicarbazide in fish and bread samples. J. Agric. Food Chem. 64, 2802-2808. (4) Hoenicke, K., Gatermann, R., Hartig, L., Mandix, M., and Otte, S. (2004) Formation of semicarbazide (SEM) in food by hypochlorite treatment: is SEM a specific marker for nitrofurazone abuse? Food Addit. Contam. 21, 526-537. (5) de la Calle, M. B., and Anklam, E. (2005) Semicarbazide: occurrence in food products and state-of-the-art in analytical methods used for its determination. Anal. Bioanal. Chem. 382, 968-977. (6) Noonan, G. O., Warner, C. R., Hsu, W., Begley, T. H., Perfetti, G. A., and Diachenko, G. W. (2005) The determination of semicarbazide (N-aminourea) in commercial bread products by liquid chromatography−mass spectrometry. J. Agric. Food Chem. 53, 4680-4685. (7) Ye, J., Wang, X. H., Sang, Y. X., and Liu, Q. (2011) Assessment of the determination of azodicarbonamide and its decomposition product semicarbazide: investigation of variation in flour and flour products. J. Agric. Food Chem. 59, 9313-9318.

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(8) Becalski, A., Lau, B. P. Y., Lewis, D., and Seaman, S. W. (2004) Semicarbazide formation in azodicarbonamide-treated flour: a model study. J. Agric. Food Chem. 52, 5730-5734. (9) Noonan, G. O., Begley, T. H., and Diachenko, G. W. (2008) Semicarbazide formation in flour and bread. J. Agric. Food Chem. 56, 2064-2067. (10) Abramsson-Zetterberg, L., and Svensson, K. (2005) Semicarbazide is not genotoxic in the flow cytometry-based micronucleus assay in vivo. Toxicol. Lett. 155, 211-217. (11) Cabrita, A. M. S., Farinha, R., Ramos, A., Silva, F. C., and Patricio, J. (2007) Effects of semicarbazide exposure on endocrine pancreas morphology. Toxicol. Lett. 172, S201. (12) Maranghi, F., Tassinari, R., and Marcoccia, D. (2010) The food contaminant semicarbazide acts as an endocrine disrupter: evidence from an integrated in vivo/in vitro approach. Chem-Biol. Interact. 183, 40-48. (13) Takahashi, M., Yoshida, M., Inoue, K., Morikawa, T., Nishikawa, A., and Ogawa, K. (2014) Chronic toxicity and carcinogenicity of semicarbazide hydrochloride in Wistar Hannover GALAS rats. Food Chem. Toxicol. 73, 84-94. (14) Toth, B., Shimizu, H., and Erickson, J. (1975) Carbamylhydrazine hydrochloride as a lung and blood vessel tumour inducer in Swiss mice. Eur. J. Cancer 11, 17-22. 20

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(15) Faucet, V., Pfohl-Leszkowicz, A., Dai, J., Castegnaro, M., and Manderville, R. A. (2004) Evidence for covalent DNA adduction by ochratoxin A following chronic exposure to rat and subacute exposure to pig. Chem. Res. Toxicol. 17, 1289-1296. (16) Manderville, R. A. (2005) A case for the genotoxicity of ochratoxin A by bioactivation and covalent DNA adduction. Chem. Res. Toxicol. 18, 1091-1097. (17) Wang, Y., Chan, H. W., and Chan, W. (2016) Facile formation of a DNA adduct of semicarbazide on reaction with apurinic/apyrimidinic sites in DNA. Chem. Res. Toxicol. 29, 834-840. (18) Azqueta, A., and Collins, A. R. (2013) The essential comet assay: a comprehensive guide to measuring DNA damage and repair. Arch. Toxicol. 87, 949-968. (19) Jumpathong, W., Chan, W., Taghizadeh, K., Babu, I. R., and Dedon, P. C. (2015) Metabolic fate of endogenous molecular damage: urinary glutathione conjugates of DNA-derived base propenals as markers of inflammation. P. Natl. Acad. Sci. U. S. A. 112, E4845-E4853. (20) Miller, S. A., Dykes, D. D., and Polesky, H. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, S1215.

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(21) Wang, J., Yuan, B., Guerrero, C., Bahde, R., Gupta, S., and Wang, Y. (2011) Quantification of oxidative DNA lesions in tissues of Long-Evans Cinnamon rats by capillary high-performance liquid chromatography−tandem mass spectrometry coupled with stable isotope-dilution method. Anal. Chem. 83, 2201-2209. (22) Taghizadeh, K., McFaline, J. L., Pang, B., Sullivan, M., Dong, M., Plummer, E., and Dedon, P. C. (2008) Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry. Nat. Protoc. 3, 1287-1298. (23) Li, J., Leung, E. M. K., Choi, M. M. F., and Chan, W. (2013) Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC−MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA. Anal. Bioanal. Chem. 405, 4059-4066. (24) Küpfer, P. A., and Leumann, C. J. (2007) The chemical stability of abasic RNA compared to abasic DNA. Nucleic Acids Res. 35, 58-68. (25) McConnell, E. L., Basit, A. W., and Murdan, S. (2008) Measurements of rat and mouse gastrointestinal pH, fluid and lymphoid tissue, and implications for in-vivo experiments. J. Pharm. Pharmacol. 60, 63-70.

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(26) 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 acid-dosed rats. Chem. Res. Toxicol. 28, 248-255. (27) Swenson, D. H., Lin, J. K., Miller, E. C., and Miller, J. A. (1977) Aflatoxin B1-2, 3-oxide as a probable intermediate in the covalent binding of aflatoxins B1 and B2 to rat liver DNA and ribosomal RNA in vivo. Cancer Res. 37, 172-181. (28) Ivanovic, V., Geacintov, N. E., Yamasaki, H., and Weinstein, I. B. (1978) DNA and RNA adducts formed in hamster embryo cell cultures exposed to benzo[a]pyrene. Biochemistry 17, 1597-1603. (29) Karnahl, U., and Wasternack, C. (1992) Half-life of cytoplasmic rRNA and tRNA, of plastid rRNA and of uridine nucleotides in heterotrophically and photoorganotrophically grown cells of Euglena gracilis and its apoplastic mutant W3BUL. Int. J. Biochem. 24, 493-497.

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Table 1 Concentration of DNA and RNA Adducts of Semicarbazide (adducts/106 normal nucleotide) in DNA/RNA in Tissues of Rats Treated with Different Doses of Semicarbazide.

Semicarbazide (mg/kg) Adduct

40

80

t1/2, days b

DNA

16±4 a

38±5

6.7

RNA

105±18

267±22

3.5

Small intestine

DNA RNA

24±4 127±30

51±5 288±13

6.9 3.6

Large intestine

DNA RNA

9±1 56±4

24±3 147±16

6.2 2.0

Liver

DNA RNA

13±3 55±5

32±6 151±9

6.7 3.7

Kidney

DNA

8±2

23±3

4.6

RNA

50±4

140±12

2.1

DNA

ND

ND

-

RNA

ND

ND

-

DNA

ND

ND

-

RNA

ND

ND

-

Stomach

Lung

Heart a

6

Mean ± standard deviation for adducts/10 normal nucleotide (n = 5). No semicarbazide-DNA or RNA adduct were detected in the control group. b Half-life calculated using the data obtained from the persistent study with rats dosed with 80 mg/kg or semicarbazide.

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Figure Legend

Figure 1. Formation of semicarbazide from nitrofurazone metabolism and from thermal degradation of azodicarbonamide; together with its reaction with the ring-opened form of apurinic/apyrimidinic sites in DNA/RNA to form a covalently bonded adducts.

Figure 2. Typical chromatograms obtained from LC–MS/MS analysis of semicarbazide-dR (A) standard, (B) in DNA in stomach; and semicarbazide-R (C) standard, (D) in RNA in stomach of semicarbazide-treated rats. The excellent agreement of retention time and the peak area ratio with that of the standard indicate confident identification of the semicarbazide-DNA and semicarbazide-RNA adducts in stomach and small intestine.

Figure 3. Time course of the (A) DNA and (B) RNA adduct levels of semicarbazide in the stomach, small intestine, liver, large intestine, and kidney of the semicarbazide-dosed rats. Male S.D. rats were administered orally with five oral doses of semicarbazide (80 mg/kg body wt per day). Animals were killed 1, 4, 7, 14, 28, and 49 days post-treatment. Neither DNA nor RNA adduct was detected in DNA/RNA isolated from samples collected at 49 days post-dosing.

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Figure 1

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Figure 2

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Figure 3

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