High-Field Asymmetric Waveform Ion Mobility Spectrometry Analysis of Carcinogenic Aromatic Amines in Tobacco Smoke with an Orbitrap Tribrid Mass Spectrometer.

Smoking is a risk factor for bladder cancer (BC), although the specific chemicals responsible for BC remain uncertain. Considerable research has focused on aromatic amines (AAs), including o-toluidine (o-tol), o-anisidine (o-anis), 2-naphthylamine (2-NA), and 4-aminobiphenyl (4-ABP), which are linked to human BC based on elevated BC incidence in occupationally exposed factory workers. These AAs arise at nanogram levels per combusted cigarette. The unambiguous identification of AAs, particularly low-molecular-weight monocyclic AAs in tobacco smoke extracts, by liquid chromatography-mass spectrometry (LC-MS) is challenging due to their poor performance on reversed-phase columns and co-elution with isobaric interferences from the complex tobacco smoke matrix. We employed a tandem liquid-liquid and solid-phase extraction method to isolate AAs from the basic fraction of tobacco smoke condensate (TSC) and utilized high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to high-resolution accurate mass (HRAM) Orbitrap LC-MS2 to assay AAs in TSC. The employment of FAIMS greatly reduced sample complexity by removing precursor co-isolation interfering species at the MS1 scan stage, resulting in dramatically improved signal-to-noise of the precursor ions and cleaner, high-quality MS2 spectra for unambiguous identification and quantification of AAs in TSC. We demonstrate the power of LC/FAIMS/MS2 by characterizing and quantifying two low-molecular-weight carcinogenic AAs, o-tol and o-anis, in TSC, using stable isotopically labeled internal standards. These results demonstrate the power of FAIMS in trace-level analyses of AA carcinogens in the complex tobacco smoke matrix.

Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS2) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O6-carboxymethyl-2'-deoxyguanosine, O6-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 108 bases using 3 µg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-ß-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

DNA Damage and Oxidative Stress of Tobacco Smoke Condensate in Human Bladder Epithelial Cells.

Smoking is a major risk factor for bladder cancer (BC), with up to 50% of BC cases being attributed to smoking. There are 70 known carcinogens in tobacco smoke; however, the principal chemicals responsible for BC remain uncertain. The aromatic amines 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are implicated in BC pathogenesis of smokers on the basis of the elevated BC risk in factory workers exposed to these chemicals. However, 4-ABP and 2-NA only occur at several nanograms per cigarette and may be insufficient to induce BC. In contrast, other genotoxicants, including acrolein, occur at 1000-fold or higher levels in tobacco smoke. There is limited data on the toxicological effects of tobacco smoke in human bladder cells. We have assessed the cytotoxicity, oxidative stress, and DNA damage of tobacco smoke condensate (TSC) in human RT4 bladder cells. TSC was fractionated by liquid-liquid extraction into an acid-neutral fraction (NF), containing polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, phenols, and aldehydes, and a basic fraction (BF) containing aromatic amines, heterocyclic aromatic amines, and N-nitroso compounds. The TSC and NF induced a time- and concentration-dependent cytotoxicity associated with oxidative stress, lipid peroxide formation, glutathione (GSH) depletion, and apurinic/apyrimidinic (AP) site formation, while the BF showed weak effects. LC/MS-based metabolomic approaches showed that TSC and NF altered GSH biosynthesis pathways and induced more than 40 GSH conjugates. GSH conjugates of several hydroquinones were among the most abundant conjugates. RT4 cell treatment with synthetic hydroquinones and cresol mixtures at levels present in tobacco smoke accounted for most of the TSC-induced cytotoxicity and the AP sites formed. GSH conjugates of acrolein, methyl vinyl ketone, and crotonaldehyde levels also increased owing to TSC-induced oxidative stress. Thus, TSC is a potent toxicant and DNA-damaging agent, inducing deleterious effects in human bladder cells at concentrations of <1% of a cigarette in cell culture media.

Effect of Cytochrome P450 Reductase Deficiency on 2-Amino-9H-pyrido[2,3-b]indole Metabolism and DNA Adduct Formation in Liver and Extrahepatic Tissues of Mice.

2-Amino-9H-pyrido[2,3-b]indole (AαC), a carcinogen formed during the combustion of tobacco and cooking of meat, undergoes cytochrome P450 (P450) metabolism to form the DNA adduct N-(deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-AαC). We evaluated the roles of P450 expressed in the liver and intestine to bioactivate AαC by employing male B6 wild-type (WT) mice, liver-specific P450 reductase (Cpr)-null (LCN) mice, and intestinal epithelium-specific Cpr-null (IECN) mice. Pharmacokinetic parameters were determined for AαC, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (AαC-3-OSO3H), and N(2)-(ß-1-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N(2)-Glu) with animals dosed by gavage with AαC (13.6 mg/kg). The uptake of AαC was rapid with no difference in the plasma half-lives (t1/2) of AαC, AαC-3-OSO3H, and AαC-N(2)-Glu among mouse models. The maximal plasma concentrations (Cmax) and the areas under concentration-time curve (AUC0-24h) of AαC and AαC-N(2)-Glu were 4-24-fold higher in LCN than in WT mice, but they were not different between WT and IECN mice. These findings are consistent with the ablation of hepatic P450 activity in LCN mice. However, the Cmax and AUC0-24h of AαC-3-OSO3H in plasma were not substantially different among the mouse models. Similar pharmacokinetic parameters were obtained with WT and LCN mice treated with a lower AαC dose (1.36 mg kg(-1)). dG-C8-AαC was detected at similar levels in the livers of all three mouse models at the high AαC dose; levels of dG-C8-AαC in colon, bladder, and lung were greater in LCN than in WT mice and were the same in colon of IECN and WT mice. At the low AαC dose, dG-C8-AαC occurred at ∼ 40% lower levels in liver of LCN mouse than in WT mouse liver, but adduct levels remained higher in extrahepatic tissues of LCN mice. Therefore, hepatic P450 plays an important role in detoxication of AαC, but other hepatic or extrahepatic enzymes contribute to the bioactivation of AαC. P450s expressed in the intestine do not appreciably contribute to bioactivation of AαC in mice.

Measurement of the Heterocyclic Amines 2-Amino-9H-pyrido[2,3-b]indole and 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Urine: Effects of Cigarette Smoking.

2-Amino-9H-pyrido[2,3-b]indole (AαC) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic heterocyclic aromatic amines (HAAs) formed during the combustion of tobacco and during the high-temperature cooking of meats. Human enzymes biotransform AαC and PhIP into reactive metabolites, which can bind to DNA and lead to mutations. We sought to understand the relative contribution of smoking and diet to the exposure of AαC and PhIP, by determining levels of AαC, its ring-oxidized conjugate 2-amino-9H-pyrido[2,3-b]indole-3-yl sulfate (AαC-3-OSO3H), and PhIP in urine of smokers on a free-choice diet before and after a six week tobacco smoking cessation study. AαC and AαC-3-OSO3H were detected in more than 90% of the urine samples of all subjects during the smoking phase. The geometric mean levels of urinary AαC during the smoking and cessation phases were 24.3 pg/mg creatinine and 3.2 pg/mg creatinine, and the geometric mean levels of AαC-3-OSO3H were 47.3 pg/mg creatinine and 3.7 pg/mg creatinine. These decreases in the mean levels of AαC and AαC-3-OSO3H were, respectively, 87% and 92%, after the cessation of tobacco (P < 0.0007). However, PhIP was detected in <10% of the urine samples, and the exposure to PhIP was not correlated to smoking. Epidemiological studies have reported that smoking is a risk factor for cancer of the liver and gastrointestinal tract. It is noteworthy that AαC is a hepatocellular carcinogen and induces aberrant crypt foci, early biomarkers of colon cancer, in rodents. Our urinary biomarker data demonstrate that tobacco smoking is a significant source of AαC exposure. Further studies are warranted to examine the potential role of AαC as a risk factor for hepatocellular and gastrointestinal cancer in smokers.

Comparison of the intrinsic dynamics of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617-650) [1]. AARSs are grouped into two broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617-650) [1]. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia-c and IIa-c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes--(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.