Quantification of DNA and Protein Adducts of 1-Nitropyrene

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Quantification of DNA and Protein Adducts of 1-Nitropyrene: Significantly Higher Levels of Protein than DNA Adducts in the Internal Organs of 1-Nitropyrene Exposed Rats Wan Chan, Sum-Kok WONG, and Weiwei Li Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00035 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Chemical Research in Toxicology

Quantification of DNA and Protein Adducts of 1-Nitropyrene: Significantly Higher Levels of Protein than DNA Adducts in the Internal Organs of 1Nitropyrene Exposed Rats

Wan Chan1,2,3,*, Sum-Kok Wong3, and Weiwei Li1

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Department of Chemistry, 2 Division of Environment and Sustainability, and 3

Environmental Science Programs, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

* Corresponding author (Tel: +852 2358-7370; Fax: +852-2358-1594; E-mail: [email protected])

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Table of Contents Graphic

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ABSTRACT 1-Nitropyrene (1NP) level is closely associated with the mutagenicity of diesel exhaust and is being used as the marker molecule for diesel exhaust. Thus, quantitation of exposure to 1NP may provide an efficient method for biomonitoring human exposure to diesel exhaust and risk assessment. Using ultra-performance liquid chromatography coupled with fluorescence or tandem mass spectrometric detection methods, we quantitated and compared in this study the DNA and protein adducts of 1NP in internal organs of 1NP-exposed rats. While previous studies using radioactivity-based detection methods were descriptive in nature and focused on the mutation-associated genetic materials, the results of our quantitative analysis showed for the first time a significantly higher concentration of the protein adduct than the DNA adduct in the tissue samples. The data also revealed higher in vivo stability of the protein adduct than that of the DNA adduct. Our results provide the solid evidence demonstrating that the protein adduct might be a more sensitive dosimeter for 1-NP, and thus diesel exhaust exposure.

Keywords: 1-Nitropyrene, Diesel exhaust, Biomarker, DNA adduct, Protein adduct

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INTRODUCTION Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are a family of potent mutagens that are produced during incomplete combustion of fossil fuels.1,2 Emerging evidence suggests that they are also formed in photo-catalyzed reactions of PAHs with NOx in the ambient environment.3 Together with PAHs, nitro-PAHs are widespread in food and environmental samples.4,5 Among the different nitro-PAHs in engine exhaust, 1-nitropyrene (1NP) is one of the most abundant nitro-PAHs.6 The 1NP content in polluted air samples correlate closely with their potential to induce mutations, thus 1NP was chosen as a marker molecule to evaluate the mutagenicity of diesel exhaust.7,8 Although measuring the amount of 1NP in food or in air samples could provide an indirect means to monitor human exposure risk, it does not take into account the toxicity of the pollutant substances, which is manifested in their exposure pathway-dependent metabolisms. By quantifying these 1NP derived biomarker molecules, it may be possible to firmly establish the link between environmental exposure to diesel exhaust and human disease. However, similar to many other nitroPAHs, methods for biomonitoring human exposure to 1NP still remained unavailable.

There is significant evidence demonstrating that 1NP is carcinogenic to laboratory rodents and that 1NP has also been classified by the IARC as probably carcinogenic to humans.9 Similar to many other nitroaromatic compounds, the genotoxicity of 1NP is manifested mainly during its hepatic metabolism in a nitroreduction process,10-12 4


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where it is converted into 1-aminopyrene (1AP). During this process, highly reactive intermediates are generated and they readily form covalently bound DNA and protein adducts in cells to exert its toxicity (Figure 1).11,17 DNA, hemoglobin, and albumin adducts have all been detected in tissue samples taken from1NP-exposed rats.13,14 Quantifying these adducts could provide a convenient method for biomonitoring of human exposure to 1NP from breathing diesel exhaust and consuming contaminated food products.

Chemical modification of DNA is one of the crucial events in chemical carcinogenesis.15,16 Thus, quantification of covalently-bound DNA adducts is widely used to assess human exposure to carcinogens.17-19 However, in certain cases protein adducts could be sensitive dosimeters because unlike DNA adducts, most of the protein adducts are not repaired by enzymes and they could accumulate during the lifespan of the protein.20-23 For example, protein adducts of aflatoxin B1 and 4aminobiphenyl have been used as biomarkers in the risk assessment of liver and bladder cancers, respectively.24-26 Previous studies assessing human exposure to diesel exhaust has focused mainly on measuring the concentration of 1NP in airborne particulates,27 studies and reports on quantifying protein adducts (by radioactivity) or DNA adducts (by 32P-post-labelling analysis) are limited in number and descriptive in nature at this time. The present study is focused on the development and application of chemical approaches to quantify the DNA and protein adducts formed after 5



exposure to 1NP (Figure 1).28-30 Using our developed methods of ultra-highperformance liquid chromatography coupled with highly sensitive and selective fluorescence (UPLC−FLD) or tandem mass spectrometry (UPLC−MS/MS), we quantitated and compared the protein and DNA adducts formed after in vitro and in vivo 1NP exposure.

The aim of this study was twofold. The first was to develop analytical methods to biomonitor the hydrolysis products of the 1NP-protein, 1AP and the 1NP-DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP).13,28 Highly sensitive UPLC−FLD and UPLC−MS/MS techniques, which were proven to be suitable for 1AP and DNA adduct analysis, respectively, were developed and used in these studies. The second aim was to define the formation of these adducts in vitro and in vivo and to evaluate their suitability for use as marker signatures for 1NP and thus diesel exhaust exposure. While previous studies of the protein and DNA adducts of 1NP have been descriptive in nature, attempts to quantitate the protein and DNA adducts have revealed that the protein adduct is formed in dramatically higher abundance and is more stable in the internal organs of 1NP-treated rats compared to DNA adducts. Our results led to the conclusion that the 1NP-protein adduct represents a better biomarker for assessing the risk of exposure to 1NP through dietary intake and diesel exhaust.

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EXPERIMENTAL Chemicals and Reagents. 1-Aminopyrene, 1-nitropyrene, 2’-deoxyguanosine monohydrate (dG), bovine serum albumin (BSA), calf thymus DNA (CT-DNA), glutathione (GSH), alkaline phosphatase, DNase I, and nuclease P1 were acquired from Sigma (St. Louis, MO). Snake venom phosphodiesterase was purchased from US Biological (Swampscott, MA). The reference standard of dG-C8-AP was synthesized using the method reported previously and purified by HPLC.31,32 N-(2’Deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP) was obtained from TRC (Toronto, Canada). Synthetic peptides (TyrArgAsnMetValHisLeuIleGluSerGlyTrp, TrpSerGlnAsnIleLysAspAlaPheGlyThrPro) were acquired from GL Biochem (Shanghai, China). HPLC grade acetonitrile and methanol were purchased from Honeywell Burdick and Jackson (Muskegon, MI). Deionized water was further purified by a Milli-Q ultrapure water purification system (Billerica, MA) and used during the entire study.

Instrumentation. UV absorbance measurements were recorded on a Varian Cary 50 UV-vis absorption spectrophotometer (Walnut Creek, CA). HPLC purification of the reference standard was performed on an Agilent 1260 Infinity LC system (Palo Alto, CA) equipped with a UV detector. High-accuracy mass spectrometry and tandem mass spectrometry (MS/MS) analyses were performed on a hybrid quadrupole timeof-flight mass spectrometer (Xevo G2 Q-TOF, Waters, Milford, MA). UPLC−FLD 7



analyses were performed on a Thermo Dionex Ultimate 3000 UPLC system (Waltham, MA) coupled to a programmable fluorescence detector. UPLC−MS/MS analyses were performed on a Shimadzu Nexera X2 (Kyoto, Japan) coupled to an AB Sciex 4000 QTRAP tandem mass spectrometer (Foster City, CA).

Synthesis and Purification of dG-C8-AP. The reference standard of the DNA adduct of 1NP, dG-C8-AP was synthesized using the method described previously.31,32 In brief, 1.8 mg of dG in 0.9 mL of phosphate buffer (50 mM, pH=5.8) was mixed with 0.2 mg of 1NP in 0.1 mL of acetonitrile and 10 mg of zinc dust. The mixture was vortex mixed and incubated at 37 °C for 16 h. After incubating overnight, the supernatant was extracted with ethyl acetate, the organic extracts were dried under nitrogen, and the residue was redissolved in acetonitrile before being purified by HPLC. The purified dG-C8-AP adduct after characterization by UV absorption, high-accuracy MS, MS/MS, and pseudo MS3 analyses (Figure S1), was accurately weighted, dissolved in acetonitrile, and stored at -20 oC freezer until used.

In vitro Experiment. Protein adduct. In vitro experiment for protein analysis was conducted by incubating BSA or GSH (900 µL; 2 mg/mL in 50 mM phosphate buffer, pH 5.8) with 1NP (100 µL; 0.02, 0.1, 0.2, 0.5, 1, and 2 mg/mL in acetonitrile) at 37 °C for 16 h using zinc dust (10 mg) as the activator. After incubating for overnight, the supernatant was separated from the zinc dust by centrifugation, and the protein in 8


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the supernatant was precipitated by adding four volumes of ice-cold acetone. The protein pellet was washed three times with acetone and redissoved in water before the protein content was quantitated by UV absorption spectrophotometry. Using a previously reported alkaline hydrolysis method,28 with modifications, the protein samples were hydrolyzed to liberate the protein-bound 1AP for UPLC−FLD analysis.

DNA adduct. In vitro experiment for DNA adduct analysis was performed using a similar approach with the same amount of CT-DNA (900 µL of 2 mg/mL CT-DNA in 50 mM phosphate buffer, pH=5.8), zinc dust (10 mg), and 1NP (100 µL; 0.02, 0.1, 0.2, 0.5, 1, and 2 mg/mL) under identical reaction conditions. The 1NP exposed DNA was precipitated using ice-cold ethanol, washed three times with 75 % ethanol, before being redissolved in water and quantitated by UV absorption spectrophotometry. Then, 100 µg of the DNA samples were hydrolyzed using a method reported previously,29 and 10 µL of dG-C8-4-ABP (0.2 µg/mL) was added as an internal standard for UPLC−MS/MS analysis of dG-C8-AP.

In Vivo Experiment. Animal experiment. Female 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 regulations by the Animal Ordinance established by the Department of Health, HKSAR. Fifteen rats (∼200 g) 9



were divided randomly into three groups with food and water provided ad libitum. While the high-dosage (n = 5) and low-dosage (n = 5) rats received an oral gavage of 100 mg/kg and 30 mg/kg of 1NP in 1 mL of peanut oil for five consecutive days, respectively, the control rats (n = 5) received an equal volume of the dosing vehicle. At 24 h after the last dose, the rats were sacrificed by decapitation, and the liver， heart, lung, stomach, large intestine, small intestine and kidney were harvested to investigate the organ-specific distribution and dose-dependent formation of the DNA and protein adducts.

Rats (n = 40) for investigating the in vivo stability of the adducts in internal organs of 1NP-exposed rats that were dosed with 100 mg/kg/day of 1NP for five consecutive days. At days 7, 14, 28, 56, and 84 post-dosing, five rats were randomly selected and sacrificed by decapitation. The internal organs (liver，heart, lung, stomach, large intestine, small intestine and kidney) were harvested, and the tissue protein and DNA were isolated for analysis using the methods described below.

Protein Isolation and Hydrolysis. Whole protein from rat tissues was isolated using the method described previously.26 In brief, 50 mg of minced tissue was suspended in 5 mL of lysis buffer (25 M Tris, 10 % glycerol, pH=8) and homogenized using a tissue tearor. The samples were then centrifuged at 18,000 g at 4 °C for 50 min. Tissue protein in the supernatant was then collected by precipitation in 4 volumes of 10


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ice-cold acetone. The isolated protein, after washing three times using acetone, was dissolved in water and quantified by UV detection. The protein samples were then hydrolyzed using a previously developed alkaline hydrolysis method,28 with modifications. Briefly, 100 µg of protein was dissolved in 200 µL of 0.5 M NaOH and set at room temperature for 6 h. The hydrolyzed samples were extracted three times using 600 µL of ethyl acetate, the organic extracts were combined, dried under nitrogen gas, and reconstituted in methanol for UPLC−FLD analysis.

DNA Isolation and Digestion. DNA from rat tissues was extracted using QIAGEN genomic DNA isolation columns (Valencia, CA).33 In brief, 100 mg of tissue was suspended in 9.5 mL lysis buffer and homogenized using a tissue tearor. After incubation for 2 h, the homogenized samples were loaded onto the columns and the DNA was isolated using the method suggested by the manufacturer. The isolated DNA samples were then washed with 75 % ethanol, redissolved in water, and quantified by UV before 100 µg of the isolated DNA was hydrolyzed using a previously described procedure for digesting chemically modified DNA.29 The hydrolyzed samples were then extracted three times using 300 µL of ethyl acetate, the organic extracts were combined and dried under nitrogen before being reconstituted in 100 µL of methanol, followed by the addition of 10 µL of dG-C8-4-ABP (0.2 µg/mL) as an internal standard for UPLC−MS/MS analysis.

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Instrumental Analysis. UPLC−FLD analysis of protein adduct. 1AP, the hydrolysis product of the protein adduct of 1NP, was quantitated using a previously developed HPLC−FLD method.34 A 100 mm × 3.0 mm i.d., 2.5 μm, Waters XSelect CSH C18 column (Waters, Milford, MA) was used for the chromatographic separation. The column was eluted at a constant flow rate of 0.4 mL/min using a gradient elution program with water and acetonitrile as the mobile phase. The excitation and emission wavelengths of the fluorescence detector were set at 240 nm and 435 nm, respectively.

UPLC−MS/MS analysis of DNA adduct. dG-C8-AP, the hydrolysis product of the DNA adduct of 1NP was analyzed using a QTRAP LC−MS/MS system with dG-C84-ABP as an internal standard. A 100 mm × 2.1 mm i.d., 1.8 μm, Eclipse Plus C18 RRHD column (Agilent, Palo Alto, CA) was used for the chromatographic separation. The mobile phase used was 0.1 % formic acid in water and methanol at a constant flow rate of 0.3 mL/min. The mass spectrometer was operated in multiple reaction monitoring mode using the optimized MS parameters. The transitions of m/z 483 → m/z 367 and m/z 435 → m/z 319 were used to monitor dG-C8-AP and the internal standard, respectively.

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RESULTS AND DISCUSSION Development and Validation of a UPLC−FLD Method for the Quantification of Protein Adduct of 1-Nitropyrene. Results from previous studies have showed that reactive intermediates generated from hepatic metabolism of 1NP binds to protein and DNA, forming covalently-bonded protein and DNA adducts (Figure 1).35 Results from our analysis also indicated that Cys is the major target site for 1NP in protein (Figure S2). It is believed that the nitroso intermediate generated from reductive metabolism of nitroaromatic compounds reacts with the SH group of cysteine in proteins to produce a sulfinamide adduct (Figure 1),35 which could be released from the protein by mild acid/alkaline hydrolysis to liberate the associated 1AP for indirect analysis of the protein adduct of 1NP.28,35 While previous studies were descriptive in nature, we developed and validated an analytical method using 1NP modified BSA and GSH, respectively.

The first step in method development involved optimizing the hydrolysis parameters for the release of 1AP from the 1NP-modified BSA for UPLC−FLD analysis. Because it was reported that 1AP can be released from the 1NP-modified protein by alkaline hydrolysis,28,35 we evaluated the hydrolysis efficiency in various amounts of sodium hydroxide, reaction temperature, and reaction time. Result showed incubating the 1NP-treated BSA at room temperature for 6 h in 0.5 M NaOH allow efficient release

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of 1AP from the protein for UPLC−FLD analysis (Figure 2). These conditions were used for all the subsequent protein hydrolysis.

We then determine the overall efficiency of the hydrolysis and extraction steps using a 1NP-modified tripeptide that was isolated in >99% purity by HPLC purification of the products of a reaction of GSH with the 1NP in the presence of Zn/H+ (Figure S2), as described in Experimental section. To this end, 98.2 pmol of the purified standard was hydrolysed to release 1AP and UPLC−FLD analysis of ethyl acetate extracts, as described above. The yields of 1AP was found to be 47.3 ± 2.4% of the theoretical value, which represents the overall efficiency for the analytical method. Measurement of 1AP was thus corrected by a factor of 2.1 to arrive at the quantity of protein adduct.

We then determined the method accuracy and precision by spiking different amounts of the GSH adduct (at 0.9 and 13.2 pmol) into 100 µg of blank BSA samples, hydrolyzed, extracted using ethyl acetate, and analyzing by UPLC−FLD. The overall performance of the analytical method, calculated by dividing the quantities of 1AP measured in the UPLC−FLD analysis by the quantities of GSH adduct added, was found to range from 91.2 to 93.5%, indicating that the developed method was highly quantitative. The precision of the analytical method was determined to be with interand intra-day variations small than 10%, indicating the method is highly reproducible. The results of method accuracy and precision are shown in Table S1. 14


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Quantitation of Protein and DNA Adducts in 1NP-treated Protein and DNA. The validated method combining alkaline hydrolysis and UPLC−FLD analysis described above was used to quantitate the protein adduct in cell-free protein exposed to 1NP. The results revealed that 1NP exposure produced a dose-dependent formation of protein adducts in the 1NP-treated protein samples. As shown in Figure 3A, purified protein exposed to 1NP produced 7.4 adducts per 106 amino acids at every mM concentration of 1NP used. Given that the unbound 1AP from the nitroreduction of 1NP was removed in the washing process using acetone (Figure S3), it is believed that the detected 1AP was released from the alkaline hydrolysis, which reflected the amount of covalently bonded protein adduct. Figure 4B shows a typical chromatogram obtained from UPLC−FLD analysis of a hydrolysate of 1NP-treated protein sample.

Via a similar reductive activation mechanism, the mutagenic dG-C8-AP adduct was also formed upon reacting the N-hydroxy-1-aminopyrene intermediate from 1NP metabolism with the C8 carbon on dG of DNA (Figure 1).13,29 Because the DNA adduct of 1NP, dG-C8-AP, exhibits no fluorescence property, the hydrolysis product from the enzymatic hydrolysis of the 1NP exposed DNA was quantitated by UPLC−MS/MS. Using a previously described protocol for digesting DNA containing 15



bulky lesions in combination with an optimized UPLC−MS/MS method,29,33 as described in the Materials and Methods section, we analyzed dG-C8-AP in 1NPtreated DNA samples. Figure 4D shows a typical chromatogram obtained from UPLC−MS/MS analysis of dG-C8-AP in an DNA hydrolysate of 1NP-treated DNA sample. As elucidated from the indistinguishable chromatographic migration time with a peak from that of the reference standard, the present analysis confirmed that dG-C8-AP was formed upon incubating 1NP with purified DNA. Similar to that observed in the protein study, the results indicated that 1NP exposure induced the dose-dependent formation of dG-C8-AP and produced 103.3 dG-C8-AP adducts per 106 normal nucleotides at every mM concentration of 1NP used (Figure 3B).

Although both the protein and DNA adducts were formed by reaction with intermediates in the nitroreduction of 1NP (Figure 1), the results showed a ~15-fold higher level of the DNA adduct than of the protein adduct (Figure 3). Possible reasons for the observed discrepancy include the relative lower chemical stability of the protein adduct than of the DNA adduct formed. It was reported that although the protein adduct is usually stable in vivo, it is acid/alkaline labile and could be easily hydrolyzed to the respective arylamine in vitro.36,37 For example, it was reported that over 70 % of the protein-bound aromatic amines are hydrolyzable.38,39 It is possible that some of the protein adducts formed by 1NP were lost during the sample clean-up

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process. Thus, a lower yield of the protein adduct than of the DNA adduct was observed in the in vitro study.

Quantitation of Protein and DNA Adducts in Internal Organs of 1NP-exposed Rats. The feasibility of detecting DNA and protein adducts of 1NP in purified DNA and protein was then extended to quantitate the adducts in the tissue-isolated DNA and protein samples obtained from rats that had been dosed with 30 and 100 mg/kg 1NP. The analyses detected both the DNA and protein adducts in all the analyzed organs (Figures 5A and 5B), with the highest levels being observed in the liver, followed by the large intestine, which was in good agreement with the previous observation that tumors were observed in the liver of 1NP-exposed rats.40-42 The high levels of adducts in these two organs could be explained by the regional high enzymatic (reductase) activity. Because the liver is the organ responsible for xenobiotic metabolism and the large intestine is housed with anaerobic microbes that produce a high concentration of bacterial nitroreductase for the nitroreduction of 1NP,43,44 it is reasonable that adducts were formed at high levels in these two organs.

The results of the analysis are shown in Figure 5, which again showed a dosedependent formation of the adducts in the quantitated protein samples. Nevertheless, the DNA adduct was detected only in the DNA samples obtained from the high-dosed rats (at tens per 109 nt) and not in rats that were dosed with 1NP at 30 mg/kg, very 17



likely due to the low adduct level in the tissue-isolated DNA samples that was below the detection limit of the analysis (0.01 adducts/106 nt). No DNA or protein adducts were detected in the tissue-isolated DNA and protein samples in the control rats.

Interestingly, despite being detected at concentrations 15 times lower than that of the DNA adduct in the in vitro study (Figure 3), the protein adduct was detected at a concentration that was dramatically higher (~ 270 times) than that of the DNA adduct in all the tissue-isolated protein samples (Figures 5A and 5B). This could have been attributed to the higher in vivo stability of the protein adduct than that of the DNA adduct. While chemically modified DNA lesions are subjected to the cell’s DNA repair machinery, protein adduct is not repaired and are accumulated during the lifespan of the protein.45 Thus, high concentrations of protein adduct were formed in the internal organs of 1NP-exposed rats. The results from this study shed light on the use of protein adduct as a more sensitive marker signature in risk assessment of the exposure to diesel exhaust-associated 1NP.

Comparative Stability of the DNA and Protein Adducts of 1NP Formed in Vivo. After identifying and quantifying the DNA and protein adducts in the internal organs of 1NP-exposed rats, the study was extended to investigate the stability of the adducts in the stomach, liver, kidney, small intestine, large intestine, lung, and heart for up to 18


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84 days after oral gavages of 1NP to the rats at 100 mg/kg. The data showed a rapid decrease in both DNA and protein adduct levels during the first 7 days (Figure 6), during which over 95 % of the adducts observed on the first day post-dosing were removed, and the DNA adduct levels in all the tissue-isolated DNA samples were below the detection limit of the LC-MS/MS method (0.01 adducts per 106 nt). Nevertheless, the protein adduct remained detectable even after 84 days post-dosing, probably because the non-hydrolysable fraction of all the protein adducts had an average half-life of ∼9 days in the different organs. Because the analytical methods for analyzing the DNA and protein adducts have similar detection limits (0.01 adducts per 106 nt and 0.02 adducts per amino acids, respectively), the results again suggest that the protein adduct may act as a better biomarker than the DNA adduct for assessing exposure to 1NP.

CONCLUSION In this study, we performed the first comparative analysis of the DNA and protein adducts formed by reacting DNA and protein with mutagenic 1NP, which is the most predominant nitro-PAH found in diesel exhaust and in a wide variety of food products. The results indicated a significantly higher (~ 270 times) level of protein adduct than DNA adduct in the internal organs of 1NP-exposed rats. Furthermore, the protein adduct was found to be more stable than the DNA adduct and can be detected in the tissue protein after ~3 months post-dosing, indicating that the protein adduct 19



may be a more sensitive dosimeter than the DNA adduct for biomonitoring the exposure to 1NP. Because 1NP is one of the most abundant nitro-PAHs emitted from combustion sources and the mutagenicity of diesel exhaust was observed to be closely associated with the concentration 1NP, it is believed that quantitating the protein adduct of 1NP may serve as a sensitive biomarker for assessing the risk of human exposure to genotoxic carcinogens in diesel exhaust.

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Author Contributions W. Chan designed the research; S.-K. Wong, K. Deng, and W. Li performed the research; S.-K. Wong, K. Deng, W. Li, and W. Chan analyzed the data; and W. Chan wrote the paper. We thank Ms. Kailin Deng for her assistant with the sample preparation.

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

Funding Sources This work was supported by the Research Grant Council of Hong Kong (GRF 16303117). W. Chan thanks The Hong Kong University of Science and Technology for an Innovative Exploratory Grant (Grant IEG17SC04).

Abbreviations 1AP, 1-aminopyrene; 1NP, 1-nitropyrene; 4-ABP, 4-aminobiphenyl; BSA, bovine serum albumin; CT-DNA, calf thymus DNA; dG-C8-AP, N-(deoxyguanosin-8-yl)-1aminopyrene; nitro-PAHs, nitrated polycyclic aromatic hydrocarbons; UPLC−FLD, ultra-high performance liquid chromatography-fluorescence spectrophotometry; 21



UPLC–MS/MS, ultra-high performance liquid chromatography-tandem mass spectrometry.

Supporting Information Available Accuracy and precision of the method for protein analysis. Chromatogram obtained from HPLC analysis of the purified standard of the DNA adduct of 1-nitropyrene, together with the spectra acquired by UV absorption spectrophotometry and tandem mass spectrometry. Chromatogram obtained from HPLC analysis of the reaction mixture after incubating GSH with the 1NP in the presence of Zn/H+, together with the MS and

MS/MS spectra of the 1NP-GSH adduct. Effect of the successive acetone washing on level of non-bounded 1-nitropyrene metabolite in protein samples. This material is available free of charge via the Internet at http://pubs.acs.org

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41. Imaida, K., Hirose, M., Tay, L., Lee, M. S., Wang, C. Y., and King, C. M. (1991) Comparative carcinogenicities of 1-nitropyrene, 2-nitropyrene, and 4-nitropyrene and structurally related-compounds in the female CD rat. Cancer Res. 51, 2902-2907. 42. Gyongyi, Z., Nadasi, E., Varga, C., Kiss, I., and Ember, I. (2001) "Long-term" effects of 1-nitropyrene on oncogene and tumor, suppressor gene expression. Anticancer Res. 21, 3937-3940. 43. Howard, P. C., Beland, F. A., and Cerniglia, C. E. (1983) Reduction of the carcinogen 1-nitropyrene to 1-aminopyrene by rat intestinal bacteria. Carcinogenesis, 4, 985-990. 44. Kinouchi, T., Nishifuji, K., and Ohnishi, Y. (1987) In vitro intestinal microfloramediated metabolism of biliary metabolites from 1-nitropyrene-treated rats. Microbiol. Immunol. 31, 1145-1159. 45. Elbayoumy, K., Johnson, B., Roy, A. K., Upadhyaya, P., Partian, S., and Hecht, S. S. (1994) Development of methods to monitor exposure to 1-nitropyrene. Environ. Health Perspect. 102, 31-37.

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Figure 1

Figure 1. Metabolic activation and DNA/protein adduct formation of 1-nitropyrene.

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Figure 2

Figure 2. (a) Optimization of the hydrolysis temperature. 1-Nitropyrene-modified BSA was dissolved in water with 0.5 M of NaOH, and the solutions were heated at different temperature for 6 h. (b) Optimization of the concentration of NaOH for the hydrolysis of the protein adduct of 1-nitropyrene. 1-Nitropyrene-modified BSA was 32


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dissolved in water with different concentrations of NaOH, and the hydrolysis reactions were continued at 25 oC for 6 h. (c) Optimization of the hydrolysis time. Protein was dissolved in 0.5 M NaOH, and the reactions were carried out at 25 oC for time different periods of time. The values were normalized to the highest detector response and represent the means ± SD from three independent measurements.

Figure 3

Figure 3. Formation of (A) protein and (B) DNA adduct in physiological relevant conditions (50 mM phosphate buffer, pH 5.8; 37 oC) upon exposing purified bovine serum albumin and DNA to different amount of 1-nitropyrene. 33



Figure 4

Figure 4. Typical chromatograms obtained from UPLC−FLD analysis of (A) 1aminopyrene standard (1 µg/L) and (B) 1-aminopyrene released from alkaline hydrolysis of liver protein isolated from 1-nitropyrene exposed rats, together with that from UPLC−MS/MS analysis of the (C) synthetic reference standard of N(deoxyguanosin-8-yl)-1-aminopyrene (40 µg/L) and (D) the DNA adduct released from enzyme hydrolysis of liver isolated DNA sample.

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Figure 5

Figure 5. Formation of (A) DNA and (B) protein adduct of 1-nitropyrene in the stomach, small intestine, large intestine, liver, kidney, lung, and heart of the rats that were orally gavage with 1-nitropyrene with five oral doses of 1-nitropyrene (100 mg/kg/day) and scarified at 24 h post-treatment. Together with the protein adduct in 35



the rats that were gavage with 1-nitropyrene at 30 mg/kg/day for 5 consecutive days (C). No DNA adducts were detected in the low dosed group.

Figure 6

Figure 6. Time course and half-life (t1/2) of the protein adduct of 1-nitropyrene in the stomach, small intestine, large intestine, liver, kidney, lung, and heart of the 1nitropyrene-exposed rats. Rats were administered orally with five oral doses of 1nitropyrene (100 mg/kg/day) and were scarified 7, 14, 28, 56, and 84 days posttreatment. The t1/2 were calculated using a first order exponential fit of the data obtained.

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Quantification of DNA and Protein Adducts of 1-Nitropyrene

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