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

Article abstract of DOI:10.1021/acs.chemrestox.8b00035

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 the 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 solid evidence that demonstrates that the protein adduct might be a more-sensitive dosimeter for 1-NP and, thus, diesel-exhaust exposure.

Full text of DOI:10.1021/acs.chemrestox.8b00035

Subscriber access provided by Kaohsiung Medical University
Article
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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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
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Wan Chan1,2,3,*, Sum-Kok Wong , and Weiwei Li1
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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:
chanwan@ust.hk)
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ABSTRACT
1
-Nitropyrene (1NP) level is closely associated with the mutagenicity of diesel
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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
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mutagens that are produced during incomplete combustion of fossil fuels. Emerging
evidence suggests that they are also formed in photo-catalyzed reactions of PAHs
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with NOx in the ambient environment. Together with PAHs, nitro-PAHs are
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widespread in food and environmental samples. Among the different nitro-PAHs in
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engine exhaust, 1-nitropyrene (1NP) is one of the most abundant nitro-PAHs. 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
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diesel exhaust. 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 nitro-
PAHs, 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
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to humans. Similar to many other nitroaromatic compounds, the genotoxicity of 1NP
is manifested mainly during its hepatic metabolism in a nitroreduction process,10-12
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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
11,17
adducts in cells to exert its toxicity (Figure 1). DNA, hemoglobin, and albumin
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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
20-23
lifespan of the protein.
For example, protein adducts of aflatoxin B1 and 4-
aminobiphenyl 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
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particulates, studies and reports on quantifying protein adducts (by radioactivity) or
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DNA adducts (by P-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
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exposure to 1NP (Figure 1).28-30 Using our developed methods of ultra-high-
performance 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.
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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
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AP 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
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NP 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
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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 time-
of-flight mass spectrometer (Xevo G2 Q-TOF, Waters, Milford, MA). UPLC−FLD
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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).
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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
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absorption, high-accuracy MS, MS/MS, and pseudo MS analyses (Figure S1), was
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accurately weighted, dissolved in acetonitrile, and stored at -20 C 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
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7 °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
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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
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previously reported alkaline hydrolysis method, 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
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0 mM phosphate buffer, pH=5.8), zinc dust (10 mg), and 1NP (100 µL; 0.02, 0.1,
.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
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previously, 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)
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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
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00 mg/kg and 30 mg/kg of 1NP in 1 mL of peanut oil for five consecutive days,
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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
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NP-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
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the method described previously. In brief, 50 mg of minced tissue was suspended in
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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
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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
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hydrolyzed using a previously developed alkaline hydrolysis method, with
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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
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genomic DNA isolation columns (Valencia, CA). 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
29
previously described procedure for digesting chemically modified DNA. 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
1
00 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
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HPLC−FLD method. A 100 mm × 3.0 mm i.d., 2.5 μm, Waters XSelect CSH C18
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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-C8-
4
-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
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DNA, forming covalently-bonded protein and DNA adducts (Figure 1). 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
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proteins to produce a sulfinamide adduct (Figure 1), 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
1
NP-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.
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We then determine the overall efficiency of the hydrolysis and extraction steps using a
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NP-modified tripeptide that was isolated in >99% purity by HPLC purification of the
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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 inter-
and intra-day variations small than 10%, indicating the method is highly reproducible.
The results of method accuracy and precision are shown in Table S1.
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Quantitation of Protein and DNA Adducts in 1NP-treated Protein and DNA.
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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,
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purified protein exposed to 1NP produced 7.4 adducts per 10 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
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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 1NP-
treated 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
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06 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.
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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
1
NP. 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
43,44
1
NP,
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 dose-
dependent 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
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rats (at tens per 10 nt) and not in rats that were dosed with 1NP at 30 mg/kg, very
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likely due to the low adduct level in the tissue-isolated DNA samples that was below
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the detection limit of the analysis (0.01 adducts/10 nt). No DNA or protein adducts
were detected in the tissue-isolated DNA and protein samples in the control rats.
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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
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lifespan of the protein. 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
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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
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below the detection limit of the LC-MS/MS method (0.01 adducts per 10 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
6
per 10 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
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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.
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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
1
6303117). W. Chan thanks The Hong Kong University of Science and Technology
for an Innovative Exploratory Grant (Grant IEG17SC04).
Abbreviations
1
AP, 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)-1-
aminopyrene; nitro-PAHs, nitrated polycyclic aromatic hydrocarbons; UPLC−FLD,
ultra-high performance liquid chromatography-fluorescence spectrophotometry;
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UPLC–MS/MS, ultra-high performance liquid chromatography-tandem mass
spectrometry.
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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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REFERENCES
1
. Chatel, A., Faucet-Marquis, V., Pfohl-Leszkowicz, A., Gourlay-France, C., and
Vincent-Hubert, F. (2014) DNA adduct formation and induction of detoxification
1
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