DNA adduct formation in the lungs and brain of rats ... - ACS Publications

Article Cite This: Chem. Res. Toxicol. 2018, 31, 332−339

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DNA Adduct Formation in the Lungs and Brain of Rats Exposed to Low Concentrations of [13C2]‑Acetaldehyde Angélica B. Sanchez,†,§,⊥ Camila C. M. Garcia,†,§,⊥ Florêncio P. Freitas,†,∥ Guilherme L. Batista,‡ Fernando S. Lopes,‡ Victor H. Carvalho,† Graziella E. Ronsein,† Ivano G. R. Gutz,‡ Paolo Di Mascio,† and Marisa H. G. Medeiros*,† †

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP 05508-020, Brazil Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP 05508-020, Brazil

‡

S Supporting Information *

ABSTRACT: Air pollution is a major environmental risk for human health. Acetaldehyde is present in tobacco smoke and vehicle exhaust. In this study, we show that [13C2]acetaldehyde induces DNA modification with the formation of isotopically labeled 1,N2propano-2′-deoxyguanosine adducts in the brain and lungs of rats exposed to concentrations of acetaldehyde found in the atmosphere of megacities. The adduct, with the addition of two molecules of isotopically labeled acetaldehyde [13C4]-1,N2-propanodGuo, was detected in the lung and brain tissues of exposed rats by micro-HPLC/MS/MS. Structural confirmation of the products was unequivocally performed by nano-LC/ESI+HRMS3 analyses. DNA modifications induced by acetaldehyde have been regarded as a key factor in the mechanism of mutagenesis and may be involved in the cancer risks associated with air pollution.

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subsequent reaction of N2-ethylidene-dGuo with a second molecule of acetaldehyde leads to the formation of the (6S,8S) and (6R,8R) diastereoisomers of 1,N2-propano-2′-deoxyguanosine (1,N2-propano-dGuo) adducts (B, Scheme 1).9 The formation of 1,N2-propano-dGuo is catalyzed by polyamines and histones.10 The 1,N2-propano-dGuo adduct is also formed by the reaction of crotonaldehyde with 2′-deoxyguanosine (D, Scheme 1).9 Crotonaldehyde is an industrial chemical aldehyde and environmental pollutant. Endogenously, crotonaldehyde can be formed by the lipid peroxidation process and as a Nnitrosopyrrolidine metabolite.11 In DNA, the 1,N2-propano-dGuo adduct is in equilibrium between the closed and open forms. Double-stranded DNA favors the open form, whereas in single-stranded DNA, the closed form is predominant.12 1,N2-propano-dGuo is mutagenic; it leads to G→T transversions and inhibits DNA replication.13 Several repair mechanisms have been associated with the removal of exocyclic DNA adducts.14 It has been shown that homologous recombination and NER (nucleotide excision repair) are involved in the repair of DNA adducts formed by acetaldehyde.15 The levels of 1,N2-propano-dGuo were significantly higher in urinary samples from residents of São Paulo City, a polluted region, compared with samples from residents of São João da Boa Vista, an unpolluted region. This result suggests a possible correlation between elevated 1,N2-propano-dGuo levels in

INTRODUCTION Outdoor air pollution is classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (IARC Group 1).1 Acetaldehyde, a known mutagenic and potential human carcinogenic compound, is widespread in the environment and has been detected in food, drinks, cigarettes, and fuel smokes.2 Chronic inhalation of acetaldehyde at high concentrations causes the increased incidence of nasal tumors in rats.3 Previously, it had been shown that the most mutagenic extracts from PM10 (particulate matter with aerodynamic diameters of less than 10 μm), collected from São Paulo City, contained aldehydes, ketones, carboxylic acids, and quinolones.4 The fleet of vehicles from São Paulo uses a variety of fuels as diesel, neat ethanol, and gasohol (mixture of gasoline and anhydrous ethanol).4 A study based on 3-D atmospheric models shows the overall increase in the concentration of acetaldehyde in the coming years due to the increasing use of fuels, such as ethanol and gasoline.5 Acetaldehyde can also be formed endogenously during ethanol metabolism6 and in small amounts during the catabolism of threonine.7 High acetaldehyde levels have been associated with a higher risk of esophageal cancer after alcohol consumption in deficient aldehyde dehydrogenase patients.8 The formation of acetaldehyde-DNA adducts has been considered as an important factor in the mechanism of mutagenesis and carcinogenesis.9 Acetaldehyde reacts with 2′-deoxyguanosine (dGuo) in DNA to primarily form N2-ethylidene-2′-deoxyguanosine (N2-ethylidene-dGuo), an unstable Schiff base (A, Scheme 1). The © 2018 American Chemical Society

Received: January 30, 2018 Published: April 30, 2018 332

DOI: 10.1021/acs.chemrestox.8b00016 Chem. Res. Toxicol. 2018, 31, 332−339

Article

Chemical Research in Toxicology Scheme 1. Formation of (A) [13C2]-1,N2-Ethylidene-dGuo from the Addition of One Molecule of Exogenous [13C2]Acetaldehyde, (B) [13C4]-1,N2-Propano-dGuo from the Addition of Two Exogenous Molecules of [13C2]Acetaldehyde, (C) [13C2]-1,N2-Propano-dGuo from the Addition of One Exogenous Molecule of [13C2]Acetaldehyde Followed by the Addition of One Molecule of Endogenous Acetaldehyde, and (D) 1,N2-Propano-dGuo from Endogenous Crotonaldehyde

principles for animal experimentation adopted by the Brazilian College. Rats Exposure. Fifteen adult male Wistar rats at 18 weeks of age were divided into three groups with five animals housed in separate cages, namely the control group, breathing ambient air without any treatment; the blank group, breathing purified air (0 ppbv, parts per billion by volume of acetaldehyde); and the doped group, breathing purified air plus 10.5 ppbv of [13C2]-acetaldehyde. The three groups of animals were preconditioned for 10 days in the cages before the 50 days of the full-time inhalation experiment, conducted in a quiet, partially windowed room (no direct sunlight incident on the cages) and presenting average day/night temperatures of 28/20 °C. Water and food were provided ad libitum, and two weekly exchanges of cage, feed, and water were performed. The water was filtered and autoclaved, the food, provided by the company Quimtia (Canguiri, Paraná, Brazil), was irradiated. Ventilife mini-isolator rat cages (Alesco, Monte Mor, SP, Brazil) (L × W × H in cm: 48.7 × 33.1 × 21.4) were adapted for the experiments by providing them with three connectors for gas inlet/outlet and a gastight lid composed of a rubber gasket glued over the upper edge of the cage and a rectangular glass plate held in place by six spring clamps. The light and dark cycles were preserved by the transparency of the cage and lid. A Mega Jet CMJ-130 diaphragm pump (Ferrari, Cotia, SP, Brazil) provided the main air flow that was purified by passing it through a cyclone for particle removal, a water condensation flask, a box filter packed with Purafil Select CP Blend (Purafil, GA, USA), and a HEPA particle filter. The CP Blend consisted of porous pellets of activated carbon mixed with alumina pellets impregnated with potassium permanganate, which was very effective for the removal of air pollutants, including low-molecular-weight aldehydes. A constant gas flow of 12 L·min−1 of purified air was blown continuously through two of the cages, but the dopant, [13C2]acetaldehyde, was added only to the input flow of one cage. A permeation tube with [13C2]-acetaldehyde was assembled in a thermostatic chamber (34 ± 1 °C) to serve as a reliable long-term dopant source. The constant emission rate was checked weekly by weighing the mass loss of the permeation tube. The emitted [13C2]acetaldehyde was continuously purged from the chamber into a dilution sector by a purified air flow of 0.1 L·min−1 sustained by an aquarium air pump. With the help of hydrodynamic resistors (PTFE coils) connected in parallel to the outlet of the chamber, a controlled fraction of the total emission of dopant was introduced into the main air stream of 12 L·min−1, lending to the doped cage and resulting in a 10.5 ± 2.3 ppbv (14.06 ± 3.1 μg·m−3) concentration of [13C2]acetaldehyde. The animals in the cages with closed lids were exposed to ambient air for approximately 10 min every 2 days during cage cleaning and food and water supply. As a protective measure for power outages or pump failure, a sensor was configured to respond to air pressure drops by opening a clamp valve from a cylinder with synthetic air to vent the cages at 2 L·min−1, while also activating an alarm and dialing programmed phone numbers. The exhaust line of the cages and from the excess dopant emission was maintained at a slightly negative pressure. At the end of the 50-day experiment, the control, blank, and exposed rats were anesthetized with 0.9 mL/kg xylazine (10%) and 0.56 mL/kg ketamine (2%) for organ withdrawal (liver, lung, and brain). Next, the organs were immediately frozen in liquid nitrogen and were stored in a freezer at −80 °C (Figure S1). DNA Extraction and Enzymatic Hydrolysis. Tubes containing lung and brain homogenates were centrifuged at 1500 g for 10 min, and each pellet was resuspended in 3 mL of a lysis solution (1% (w/v) Triton X-100, 320 mM sucrose, 5 mM MgCl2, 10 mM tris-HCl, pH 7.5). This step was repeated twice. After centrifugation at 1500 g for 10 min, the nuclei pellets were resuspended in 3 mL of 10 mM trisHCl buffer at pH 8.0 containing 5 mM EDTA and 0.15 mM desferroxamine. The enzymes RNase A (30 μL of a 10 g/L solution in 10 mM sodium acetate/acetic acid buffer, pH 5.2, heated for 15 min at 100 °C) and RNase T1 (4 μL of a 20 U/μL solution in 10 mM tris-

urine and air pollution.16 DNA repair, apoptosis, and deoxynucleoside pools could be the sources of nucleoside adducts in urine.17 This result provided evidence that elevated levels of 1,N2-propano-dGuo in urinary samples may be correlated with urban air pollution.16 Interestingly, 1,N2propano-dGuo was also quantified in human placental and leukocyte DNA from healthy volunteers.18 Here, the formation of 1,N2-propano-dGuo by inhalation of environmentally relevant doses of [13C2]-acetaldehyde was addressed in order to elucidate the mechanisms by which this adduct is formed and to contribute to a better understanding of the mutagenic effects associated with acetaldehyde exposure.

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EXPERIMENTAL METHODS

Chemicals. All chemicals were acquired with the highest commercially available purity. Acetonitrile, methanol, ammonium formate, and formic acid at mass spectrometry grade were acquired from Merck (Darmstadt, Germany). [15N5]-2′-Deoxyguanosine and [13C2]-acetaldehyde were acquired from Cambridge Isotope Laboratories (Andover, MA, USA). All other chemicals were acquired from Sigma (St. Louis, MO, USA). Water was purified using a Milli-Q system (Merck-Millipore, Darmstadt, Germany). Ethics Committee on Animal Experimentation. All the animal treatments were approved by the ethics committee on animal experimentation at the Institute of Chemistry, University of São Paulo (CECUA 26/2016), and are in accordance with the ethical 333


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μm (Eksient, Dublin, CA), in a gradient method using 10 mM ammonium formate and 10 mM acetonitrile and a flow rate of 25 L/ min. The adducts were analyzed by electrospray ionization (ESI) in the positive mode, and detection was made using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer (API 6500, Sciex). The m/z 338→222 (1,N2-propano-dGuo), 342→226 ([13C4]1,N2-propano-dGuo), and 347→231 ([13C4,15N5] 1,N2-propano-dGuo) transitions were monitored as quantification transitions with a dwell time of 100 ms. The m/z 222→178 (1,N2-propanodGuo), 226→180 ([13C4 ]1,N 2-propano-dGuo), and 231→187 ([13C4,15N5]1,N2-propano-dGuo) transitions were monitored as qualification transitions. The turbo ion spray voltage was kept at 5500 V, the curtain gas at 20 psi, and the nebulizer and auxiliary gases at 50 psi. The temperature was set at 400 °C, and the pressure of nitrogen in the collision cell was adjusted to high. A standard curve of 1,N2-propano-dGuo and [13C4]1,N2-propano-dGuo with [13C4,15N5] 1,N2-propano-dGuo as the internal standard was performed in triplicate for the quantification of the adducts. A signal-to-noise ratio of S/N ≥ 3 was used as the detection criteria for the adducts, and the quantification criteria was S/N ≥ 7. To demonstrate the reproducibility and repeatability of the method, three samples of 1 mg of calf thymus DNA, contaminated with 6 fmol of 1,N2-propano-dGuo and 5 fmol of internal standard, were hydrolyzed and injected in two subsequent days (Table S1). Nano-LC/ESI-HRMS3 Analysis of the Endogenous and Exogenous 1,N2-Propano-dGuo Adducts. An Easy-nLC 1200 system (Thermo Fisher Scientific Corp., Waltham, MA, USA) was coupled to an Orbitrap Fusion Lumos instrument equipped with a nanospray source (Thermo Fisher Scientific Corp., Waltham, MA, USA). Nano-LC solvents were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B), and the flow rate was 300 nL/ min. Samples (3 μL) were injected onto a trapping column (Acclaim PepMap 0.075 mm, 2 cm, C18, 3 μm, 100 A; Thermo) in line with a Nano-LC column (Acclaim PepMap RSLC (0.050 mm, 15 cm, C18, 2 μm, 100 A; Thermo). The sample was loaded in the trap column and washed with 20 μL of solvent A at a constant pressure (500 bar). After that, the sample was eluted to the column using a flow of 300 nL/min and was maintained at 2% B for 14 min. The organic phase content was linearly increased to 13% B in 6 min and further to 95% B in 15 min. Under these conditions, the two 1,N2-propano-dGuo isomers eluted as sharp peaks at 25.40 and 26.10 min. Nano-LC/ESI+-HRMS3 analyses were conducted in the Nano ESI+ mode. The instrument settings included the spray voltage at 1950 kV, capillary temperature at 300 °C, and S-Lens RF level at 30%. MS3 analyses were performed by isolating [M + H]+ ions of 1,N2-propanodGuo (m/z 338.146 and 342.159) and the m/z 347.137 for the internal standard in the quadrupole (isolation width of 1.6 m/z) and fragmenting them using high-collision dissociation (HCD) with a normalized collision energy set at 30%. The resulting MS/MS fragment ions corresponding to the loss of the deoxyribose [M+H2-D-erythro-pentose]+ (m/z 222.098, 226.112, and 231.089) were subjected to further fragmentation in the collision-induced dissociation (CID) cell using helium as the collision gas, a normalized collision energy of 30%, and an isolation width of 1.6 m/z. The resulting MS3 fragment ions were detected in the mass range of m/z 100−400, using the Orbitrap mass analyzer at a resolution of 30 000. To increase the sensitivity of the less abundant adducts, different injection times and AGC targets were used for each adduct. Thus, MS3 analysis for the MS1/MS2 pairs from 338/222 and 347/231 were obtained using an AGC target of 5e4 and maximum injection times (ms) of 54, 118, and 118, respectively. The pair 342/226 used an AGC target of 1e5 and a maximum injection time of 246 ms. A full-scan event was also performed over the mass range of m/z 100−400 at a resolution of 30 000 to monitor any co-eluting matrix components.

HCl buffer, pH 7.4, containing 1 mM EDTA and 2.5 mM desferroxamine) were added together with 150 μL of a 10% (w/v) solution of SDS, and the reaction mixture was incubated at 37 °C for 1 h. After this period, 60 μL of proteinase K (20 g/L) was added to the cells, followed by additional incubation at 37 °C for 1 h. After centrifugation at 5000 g for 15 min, the liquid phase was collected, and 0.6 mL of 5 M NaCl was added and rested on an ice bath for 20 min. The samples were centrifuged at 5000 g for 15 min, the liquid phase was collected, followed by the addition of 4 mL of isopropanol. The content in the tube was well mixed by inversion until a whitish precipitate appeared. The precipitate was collected by centrifugation at 5000 g for 15 min and was washed with 1 mL of 60% (v/v) isopropanol followed by 1 mL of 70% (v/v) ethanol. After additional centrifugation at 5000 g for 15 min, the DNA pellet was solubilized in a 0.1 mM solution of desferroxamine mesylate. The DNA concentration was measured spectrophotometrically at 260 nm, and its purity was assessed by ensuring A260/A280 ≥ 1.7. For the hydrolysis of 1 mg of DNA, 20 μL of 1 M sodium acetate buffer pH 5.0 and 10 U of nuclease P1 were added to the sample, which was incubated at 37 °C for 30 min. Thereafter, 40 μL of 1 M tris-HCl buffer at pH 7.4 and 40 μL of 500 mM phosphatase buffer containing 100 mM tris-acetate and 100 mM magnesium acetate were added followed by the addition of 30 U of alkaline phosphatase. The reaction mixture was incubated at 37 °C for 1 h. The internal standard, [13C4,15N5] 1,N2-propano-dGuo (5 fmol), was also added to the DNA sample before the first incubation period. The final volume of the sample was adjusted to 500 μL. Thereafter, the enzymes were removed from the hydrolysis solution by filtration with a Millipore Ultrafree Centrifugal Unit with a pore size of 0.1 μm. The concentration of dGuo in each sample was analyzed by an HPLC system consisting of a Shimadzu UFLC−Prominence system (Kyoto, Japan) with a DAD detector operating at 260 nm. The dGuo were separated from the other deoxyribonucleosides by a Luna C18 column, with 250 mm × 4.6 mm, 5 μm id., Phenomenex (Torrance, California, USA), using an isocratic method of 8% of methanol at a flow rate of 0.7 mL/min. A standard curve was performed to quantitate the dGuo. Enrichment and Purification of the 1,N2-Propano-dGuo Adducts. The hydrolyzed samples were enriched by solid-phase extraction using a Phenomenex StrataX C18-coated column. The columns were preconditioned with 5 mL of methanol followed by 5 mL of water, and then the samples were loaded and washed with 5 mL of water, 5 mL of 7% methanol, and 5 mL of 10% methanol. The adducts were extracted with 2 mL of a solution of 25% methanol. The fraction containing the 1,N2-propano-dGuo adducts were dried in a speed vacuum and were resuspended in 100 μL of water for the purification by HPLC. The purification system consisted of a Shimadzu UFLC− Prominence system (Kyoto, Japan) with a DAD detector operating at 260 nm. The adducts were separated from the deoxyribonucleosides by a Luna C18 column, with 250 mm × 4.6 mm, 5 μm id., Phenomenex (Torrance, California, USA). An isocratic method of 8% of methanol at a flow rate of 0.8 mL/min was used, and the effluent solution was collected from 11 to 20 min, lyophilized, and resuspended in 10 μL of water. Synthesis of 1,N2-Propano-dGuo, [15N5]1,N2-Propano-dGuo, and [13C4,15N5]1,N2-Propano-dGuo Standards. A total of 25 μmol of 2′-deoxyguanosine (or [15N5]-2′-deoxyguanosine) was dissolved in 2 mL of 100 mM phosphate buffer at pH 8.0, and 1 mmol of acetaldehyde (or [13C2]-acetaldehyde) and 0.05 mmol of lysine were dissolved in 2 mL of 100 mM phosphate buffer at pH 8.0. This solution was mixed at 500 rpm at 37 °C for 24 h. The products were subsequently purified by HPLC. Analysis by Micro-LC-ESI/MS/MS of 1,N2-Propano-dGuo in Rats Exposed to Isotopically Labeled Acetaldehyde. The detection method of the 1,N2-Propano-dGuo adducts was developed using a microLC Eksigent system consisting of a TAG/PAL autoinjector cooled to 10 °C, a column oven at 30 °C, and the software Analyst 1.4.2. The adducts were initially separated using a HALO Phenyl Hexyl analytical column, 150 mm × 0.5 mm id and 2.7

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RESULTS AND DISCUSSION In the present study, 15 adult male rats at 18 weeks of age were divided into 3 groups of 5 animals, one control group breathing 334


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Figure 1. Representative chromatograms of selected reaction monitoring (SRM) of the quantification transition (blue line) and confirmation transition (red line) of the micro-HPLC-ESI+-MS/MS analysis of 1,N2-propano-dGuo (endogenous), [13C4]-1,N2-propano-dGuo (exogenous), and [13C4,15N5]-1,N2-propano-dGuo (internal standard) in the lungs of rats exposed to (A) ambient air, (B) 0 ppbv, and (C) 10 ppbv of inhaled 13C2acetaldehyde.

group exposed to 10 ppbv of [13C2]-acetaldehyde (C). Two transitions were monitored in the SRM mode analysis to confirm the identity of the adduct, the product [13C4]-1,N2propano-dGuo was identified with m/z 342→226 transition and was confirmed with the additional m/z 342→180 transition. The signal-to-noise ratio (S/N) for the exogenous adduct was high enough (S/N ≥ 3) for its detection in four of five samples from each tissue (lungs and brain) but below the quantification criteria (S/N ≥ 7). The [13C2]-1,N2-propanodGuo, the adduct with the addition of one molecule of isotopically labeled acetaldehyde and one unlabeled molecule formed endogenously, was detected in a percentage similar to the natural abundance of the isotope, calculated theoretically (envipat web 2.2)20 and experimentally (Figure S2). The endogenous levels of the adduct in the tissues were quantified in the DNA of rats exposed to [13C2]-acetaldehyde; however, due to the small sample size, a high inter-individual variation was observed (Table 1). Endogenous 1,N2-propano-dGuo formation has been regarded as a product of crotonaldehyde, generated endogenously by the oxidative degradation of unsaturated lipids and as a metabolite of N-nitrosopyrrolidine.11,20 Acetaldehyde is endogenously formed by the metabolic oxidation of ethanol through hepatic NAD-dependent alcohol dehydrogenases in the liver and during threonine catabolism.21−23 Basal levels of (6S,8S)-1,N2-propano-dGuo and (6R,8R)-1,N2-propano-dGuo were 3.5 ± 0.2 and 2.4 ± 0.1 adducts per 108 dGuo, respectively, in untreated cultured MRC5 cells.24 Pan et al., using a methodology based on SPEHPLC and 32P-postlabeling detection, reported basal levels of

ambient air without any treatment and the other groups breathing purified air with no exposure of [13C2]-acetaldehyde or exposed with 10 ppbv (14 μg·m−3) of [13C2]-acetaldehyde full time during the 50 days (Figure S1). The concentrations of 10 ppbv of acetaldehyde or greater are not unusual in large cities.19 The unequivocal formation of labeled 1,N2-propanodGuo in DNA was verified in the lung and brain tissues of exposed animals by micro-HPLC/MS/MS on a triple-quadrupole mass spectrometer using a highly sensitive methodology. The addition of [13C4,15N5]-1,N2-propano-dGuo, the isotopically labeled internal standard prior to DNA hydrolysis, improves the confidence level of this method because it allows for the correction of any loss of the analyte during the procedure. Representative chromatograms of samples from the lungs and brain are shown in Figures 1 and 2, respectively. Two isomers of 1,N2-propano-dGuo, corresponding to the specific transitions of m/z 338→222 (quantification transition) and m/ z 222→178 (confirmation transition), are observed in the control group breathing ambient air (A), in the group breathing purified air (B), and in the group exposed to 10 ppb of [13C2]acetaldehyde (C), showing the formation of the endogenous adduct in all of the conditions. These transitions present the same retention time as the internal standard [15N5, 13C4]-1,N2propano-dGuo (m/z 347→231 quantification transition, m/z 231→183 confirmation transition). Interestingly, the [13C4]1,N2-propano-dGuo, the exogenous adduct with the addition of two molecules of isotopically labeled acetaldehyde, was detected in the lung and brain tissues of rats only in the 335


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Figure 2. Representative chromatograms of selected reaction monitoring (SRM) of the quantification transition (blue line) and confirmation transition (red line) of the micro-HPLC-ESI+-MS/MS analysis of 1,N2-propano-dGuo (endogenous), [13C4]-1,N2-propano-dGuo (exogenous), and [13C4,15N5]-1,N2-propano-dGuo (internal standard) in the brains of rats exposed to (A) ambient air, (B) 0 ppbv, and (C) 10 ppbv of inhaled 13C2acetaldehyde.

Table 1. Endogenous 1,N2-Propano-dGuo Levels in the Brain, Lungs, and Liver of Rats Treated with Ambient Air, 0 ppbv, and 10 ppbv of Labelled Acetaldehyde (n = 5)

Structural confirmation of the products was additionally performed by nano-LC/ESI+-HRMS3 analyses on an Orbitrap Fusion Lumos mass spectrometer using a pool of five samples from lung tissues. Figure 3 shows the MS3 spectra acquired selecting the loss of deoxyribose in the MS2 of (A) 1,N2propano-dGuo (endogenous), m/z 338 [M + H]+→222 [M + H-deoxyribose]+, generating the protonated fragments of m/z 204 [M + H-deoxyribose-H2O]+, m/z 178 [M + Hdeoxyribose-CH3CHO]+, and m/z 152 [M + H-deoxyriboseCH3CHO-CHCH]+; (B) [13C4]-1,N2-propano-dGuo (exogenous), m/z 342 [M + H]+→226 [M + H-deoxyribose]+, generating the protonated fragments of m/z 206 [M + Hdeoxyribose-H 2 O] + , m/z 180 [M + H-deoxyribose-13CH313CHO] +, and m/z 152 [M + H-deoxyribose-13CH313CHO − 13CH13CH]+; and (C) [15N5, 13C4]-1,N2propano-dGuo (internal standard), m/z 347 [M + H]+→231 [M + H-deoxyribose]+, generating the protonated fragments of m/z 213 [M + H-deoxyribose-H2O] +, m/z 185 [M + Hdeoxyribose-13CH313CHO]+, and m/z 157 [M + H-deoxyribose-13CH313CHO-13CH13CH] +, confirming, unequivocally, the identity of the products. A calibration curve was performed to check the linearity of the method, and the limit of detection was determined as 50 amol for the [13C4]-1,N2-propano-dGuo adduct (Figures S3 and S4). Acetaldehyde is found in the environment, including fuel combustion.27 It is a known cytotoxic and mutagenic compound and is carcinogenic in experimental animals.28

endogenous (1, N2-propano-dGuo/108 dGuo) mean

SD

control 0 ppb 10 ppb

20.06 17.32 15.22

21.97 4.61 17.39


18.85 23.58 42.79

19.97 12.72 5.97


0.92 0.82 0.81

0.06 0.41 0.17

Brain

Lungs

Liver

1,N2-propano-dGuo (25.9 ± 7.8 adducts/109 dGuo) in the liver DNA of Long-Evans rats.25 Accurate determinations of 1,N2propano-2′-deoxyguanosine levels in DNA extracts of IMR-90 human cultured cells (3.43 ± 0.33 1,N2-propano-dGuo/108 dGuo) and Wistar rat tissue (liver, 4.61 ± 0.69 1,N2-propanodGuo/108 dGuo; brain, 5.66 ± 3.70 1,N2-propano-dGuo/108 dGuo; and lung, 2.25 ± 1.72 1,N2-propano-dGuo/108 dGuo) were performed by HPLC-MS methodology.26 336


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Figure 3. Nano-HPLC-nano-ESI+-HRMS3 extracted chromatogram analysis and its respective MS3 spectra of (A) m/z = 222, 1,N2-propano-dGuo; (B) m/z = 226, [13C4]-1,N2-propano-dGuo; and (C) m/z = 231, [13C4,15N5]-1,N2-propano-dGuo (internal standard) in a pool of lung samples of rats exposed to 10 ppbv of [13C2]-acetaldehyde by inhalation (dR = 2-deoxyribose).

lungs than in the liver.39 Exposure to cigarette smoke increases the levels of the 1,N2- propano-dGuo adduct in human oral tissues.40 The presence of the 1,N2-propano-dGuo adduct in human tissues is mainly credited to the reaction of dGuo with the crotonaldehyde, since its formation from acetaldehyde requires two successive reactions of acetaldehyde. In a previous work, we showed the unequivocal formation of 1,N2-propanodGuo by acetaldehyde in culture cells via treatment with [13C2]acetaldehyde.41 In the present work, using a ultrasensitive methodology based on micro-HPLC/MS/MS, the [13C4]-1,N2-propanodGuo, the adduct with the addition of two molecules of isotopically labeled acetaldehyde, was detected in the lung and brain tissues of rats breathing purified air doped with 10 ppbv of [13C2]-acetaldehyde full time during the 50 days. Structural confirmation of the product was unequivocally performed by nano-LC/ESI+-HRMS3 analyses. In conclusion, the detection of the labeled 1,N2-propanodGuo in the brain and lung DNA of rats exposed to acetaldehyde shows, unequivocally, that acetaldehyde in the concentrations found in the atmosphere of megacities binds to DNA and forms a mutagenic adduct that may be involved in the cancer risks associated with air pollution exposure.

Air pollution has been associated with increased mortality among various age groups.29 The mixtures of gasoline and ethanol generate various organic components during the combustion process, and higher ambient acetaldehyde values were measured in locations exposed to bioethanol-fueled buses.30 Recently, a study determined frequently found carcinogenic air toxins or hazardous air pollutants (HAPs) combinations across the United States and analyzed the health impacts of developing cancer due to exposure to these HAPs. It was found that the frequent air toxins with a cancer risk greater than one in a million were formaldehyde, carbon tetrachloride, acetaldehyde, and benzene.31 Studies with inhaled acetaldehyde in different animal models have been reported since 1900, when Lewin showed anesthetic properties of aldehyde in animals.32 Subsequently, many studies have shown carcinogenic effects in different models breathing very high concentrations of acetaldehyde.32,33 In a 52 weeks study using aldehyde concentrations ranging from 0 to 5000 ppm for 6 h per day, 5 days a week, a degeneration of the olfactory epithelium was observed in concentrations greater than 400 ppm of acetaldehyde. At higher acetaldehyde concentration effects, ranging from hyper- and metaplasia of cells of the olfactory epithelium, a decrease in lymphocytes and urine production, an increase in neutrophils, and a growth retardation to the development of cancer (squamous cell and adenocarcinomas) were reported.3,34−36 The formation of aldehyde-DNA adducts are an important factor in the mechanism of mutagenesis and carcinogenesis.37,38 The 1,N2-propano-dGuo adduct has been found in various human tissues, but it was detected more frequently in human

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00016. 337


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Additional results; system for long-term inhalation exposure of Wistar Rats to [ 13 C 2 ]-acetaldehyde; representative chromatograms of selected reaction monitoring (SRM) of the micro-HPLC-ESI+-MS/MS analysis; nano-HPLC-nano-ESI+-HRMS3 analysis of [13 C 4]-1,N2 -propano-dGuo; nano-HPLC-nano-ESI +HRMS3 calibration curve for [13C4]-1,N2-propanodGuo; micro-HPLC-ESI+-MS/MS calibration curves for 1,N2-propano-dGuo and [13C4]-1,N2-propano-dGuo; and quantification of 1,N2propano-dGuo in 1 mg of calf thymus DNA (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: (55) 11 30912153; E-mail: [email protected]. ORCID

Ivano G. R. Gutz: 0000-0001-9759-7920 Marisa H. G. Medeiros: 0000-0002-5438-1174 Present Addresses

⊥ Center for Research in Biological Sciences & Department of Biological Sciences, Institute of Physical and Biological Sciences, Federal University of Ouro Preto, Brazil. ∥ Institute of Developmental Genetics, Helmholtz Zentrum München, Neuherberg, Germany.

Author Contributions §

A.B.S. and C.C.M.G. contributed equally to this work.

Funding

Supported by CEPID-Redoxoma (FAPESP: Proc. 2013/079378), NAP-Redoxoma (PRPUSP: Proc. 2011.1.9352.1.8), and CNPq (Proc. 302351/2011-6; 301404/2016-0; 161308/20112; 301307/2013-0; and 159068/2014-2). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS 1,N -propano-dGuo, α-methyl-γ-hydroxy-1,N2-propano-2′-deoxyguanosine; dGuo, 2′-deoxyguanosine; SPE, solid phase extraction; HPLC/MS-MS, high-performance liquid chromatography/tandem mass spectrometry; nano-LC/ESI+-HRMS3, nanoflow high- performance liquid chromatography−electrospray ionization high-resolution tandem mass spectrometry in the positive mode; NER, nucleotide excision repair

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DNA adduct formation in the lungs and brain of rats ... - ACS Publications

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