Mass Spectrometry-Based Method to Measure Aflatoxin B1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 µg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.

Screening DNA Damage in the Rat Kidney and Liver by Untargeted DNA Adductomics.

Air pollution, tobacco smoke, and red meat are associated with renal cell cancer (RCC) risk in the United States and Western Europe; however, the chemicals that form DNA adducts and initiate RCC are mainly unknown. Aristolochia herbaceous plants are used for medicinal purposes in Asia and worldwide. They are a significant risk factor for upper tract urothelial carcinoma (UTUC) and RCC to a lesser extent. The aristolochic acid (AA) 8-methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I), a component of Aristolochia herbs, contributes to UTUC in Asian cohorts and in Croatia, where AA-I exposure occurs from ingesting contaminated wheat flour. The DNA adduct of AA-I, 7-(2'-deoxyadenosin-N6-yl)-aristolactam I, is often detected in patients with UTUC, and its characteristic A:T-to-T:A mutational signature occurs in oncogenes and tumor suppressor genes in AA-associated UTUC. Identifying DNA adducts in the renal parenchyma and pelvis caused by other chemicals is crucial to gaining insights into unknown RCC and UTUC etiologies. We employed untargeted screening with wide-selected ion monitoring tandem mass spectrometry (wide-SIM/MS2) with nanoflow liquid chromatography/Orbitrap mass spectrometry to detect DNA adducts formed in rat kidneys and liver from a mixture of 13 environmental, tobacco, and dietary carcinogens that may contribute to RCC. Twenty DNA adducts were detected. DNA adducts of 3-nitrobenzanthrone (3-NBA), an atmospheric pollutant, and AA-I were the most abundant. The nitrophenanthrene moieties of 3-NBA and AA-I undergo reduction to their N-hydroxy intermediates to form 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts. We also discovered a 2'-deoxycytidine AA-I adduct and dA and dG adducts of 10-methoxy-6-nitro-phenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-III), an AA-I isomer and minor component of the herbal extract assayed, signifying AA-III is a potent kidney DNA-damaging agent. The roles of AA-III, other nitrophenanthrenes, and nitroarenes in renal DNA damage and human RCC warrant further study. Wide-SIM/MS2 is a powerful scanning technology in DNA adduct discovery and cancer etiology characterization.

Mass Spectral Library for DNA Adductomics.

Endogenous electrophiles, ionizing and non-ionizing radiation, and hazardous chemicals present in the environment and diet can damage DNA by forming covalent adducts. DNA adducts can form in critical cancer driver genes and, if not repaired, may induce mutations during cell division, potentially leading to the onset of cancer. The detection and quantification of specific DNA adducts are some of the first steps in studying their role in carcinogenesis, the physiological conditions that lead to their production, and the risk assessment of exposure to specific genotoxic chemicals. Hundreds of different DNA adducts have been reported in the literature, and there is a critical need to establish a DNA adduct mass spectral database to facilitate the detection of previously observed DNA adducts and characterize newly discovered DNA adducts. We have collected synthetic DNA adduct standards from the research community, acquired MSn (n = 2, 3) fragmentation spectra using Orbitrap and Quadrupole-Time-of-Flight (Q-TOF) MS instrumentation, processed the spectral data and incorporated it into the MassBank of North America (MoNA) database, and created a DNA adduct portal Web site (https://sites.google.com/umn.edu/dnaadductportal) to serve as a central location for the DNA adduct mass spectra and metadata, including the spectral database downloadable in different formats. This spectral library should prove to be a valuable resource for the DNA adductomics community, accelerating research and improving our understanding of the role of DNA adducts in disease.

Tissue Organoid Cultures Metabolize Dietary Carcinogens Proficiently and Are Effective Models for DNA Adduct Formation.

Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce in vitro the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B1 (AFB1), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB1; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB1, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.

Nuclear DNA and Mitochondrial Damage of the Cooked Meat Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Human Neuroblastoma Cells.

Animal fat and iron-rich diets are risk factors for Parkinson's disease (PD). The heterocyclic aromatic amines (HAAs) harman and norharman are neurotoxicants formed in many foods and beverages, including cooked meats, suggesting a role for red meat in PD. The structurally related carcinogenic HAAs 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) also form in cooked meats. We investigated the cytotoxicity, DNA-damaging potential, and mitochondrial damage of HAAs and their genotoxic HONH-HAA metabolites in galactose-dependent SH-SY5Y cells, a human neuroblastoma cell line relevant for PD-related neurotoxicity. All HAAs and HONH-HAAs induced weak toxicity except HONH-PhIP, which was 1000-fold more potent than the other chemicals. HONH-PhIP DNA adduct formation occurred at 300-fold higher levels than adducts formed with HONH-MeIQx and HONH-AαC, assuming similar cellular uptake rates. PhIP-DNA adduct levels occurred at concentrations as low as 1 nM and were threefold or higher and more persistent in mitochondrial DNA than nuclear DNA. N-Acetyltransferases (NATs), sulfotransferases, and kinases catalyzed PhIP-DNA binding and converted HONH-PhIP to highly reactive ester intermediates. DNA binding assays with cytosolic, mitochondrial, and nuclear fractions of SH-SY5Y fortified with cofactors revealed that cytosolic AcCoA-dependent enzymes, including NAT1, mainly carried out HONH-PhIP bioactivation to form N-acetoxy-PhIP, which binds to DNA. Furthermore, HONH-PHIP and N-acetoxy-PhIP inhibited mitochondrial complex-I, -II, and -III activities in isolated SH-SY5Y mitochondria. Mitochondrial respiratory chain complex dysfunction and DNA damage are major mechanisms in PD pathogenesis. Our data support the possible role of PhIP in PD etiology.

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.

Anthracyclines React with Apurinic/Apyrimidinic Sites in DNA.

The combination of doxorubicin (Adriamycin) and cyclophosphamide, referred to as AC chemotherapy, is commonly used for the clinical treatment of breast and other cancers. Both agents target DNA with cyclophosphamide causing alkylation damage and doxorubicin stabilizing the topoisomerase II-DNA complex. We hypothesize a new mechanism of action whereby both agents work in concert. DNA alkylating agents, such as nitrogen mustards, increase the number of apurinic/apyrimidinic (AP) sites through deglycosylation of labile alkylated bases. Herein, we demonstrate that anthracyclines with aldehyde-reactive primary and secondary amines form covalent Schiff base adducts with AP sites in a 12-mer DNA duplex, calf thymus DNA, and MDA-MB-231 human breast cancer cells treated with nor-nitrogen mustard and the anthracycline mitoxantrone. The anthracycline-AP site conjugates are characterized and quantified by mass spectrometry after NaB(CN)H3 or NaBH4 reduction of the Schiff base. If stable, the anthracycline-AP site conjugates represent bulky adducts that may block DNA replication and contribute to the cytotoxic mechanism of therapies involving combinations of anthracyclines and DNA alkylating agents.

Synthesis and Characterization of 15N5-Labeled Aflatoxin B1-Formamidopyrimidines and Aflatoxin B1-N7-Guanine from a Partial Double-Stranded Oligodeoxynucleotide as Internal Standards for Mass Spectrometric Measurements.

Aflatoxin B1 (AFB1) exposure through contaminated food is a primary contributor to hepatocellular carcinogenesis worldwide. Hepatitis B viral infections in livers dramatically increase the carcinogenic potency of AFB1 exposures. Liver cytochrome P450 oxidizes AFB1 to the epoxide, which in turn reacts with N7-guanine in DNA, producing the cationic trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct (AFB1-N7-Gua). The opening of the imidazole ring of AFB1-N7-Gua under physiological conditions causes the formation of the cis- and trans-diastereomers of 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua). These adducts primarily lead to G → T mutations, with AFB1-FapyGua being significantly more mutagenic than AFB1-N7-Gua. The unequivocal identification and accurate quantification of these AFB1-Gua adducts as biomarkers are essential for a fundamental understanding and prevention of AFB1-induced hepatocellular carcinogenesis. Among a variety of analytical techniques used for this purpose, liquid chromatography-tandem mass spectrometry, with the use of the stable isotope-labeled analogues of AFB1-FapyGua and AFB1-N7-Gua as internal standards, provides the greatest accuracy and sensitivity. cis-AFB1-FapyGua-15N5, trans-AFB1-FapyGua-15N5, and AFB1-N7-Gua-15N5 have been synthesized and used successfully as internal standards. However, the availability of these standards from either academic institutions or commercial sources ceased to exist. Thus, quantitative genomic data regarding AFB1-induced DNA damage in animal models and humans remain challenging to obtain. Previously, AFB1-N7-Gua-15N5 was prepared by reacting AFB1-exo-8,9-epoxide with the uniformly 15N5-labeled DNA isolated from algae grown in a pure 15N-environment, followed by alkali treatment, resulting in the conversion of AFB1-N7-Gua-15N5 to AFB1-FapyGua-15N5. In the present work, we used a different and simpler approach to synthesize cis-AFB1-FapyGua-15N5, trans-AFB1-FapyGua-15N5, and AFB1-N7-Gua-15N5 from a partial double-stranded 11-mer Gua-15N5-labeled oligodeoxynucleotide, followed by isolation and purification. We also show the validation of these 15N5-labeled standards for the measurement of cis-AFB1-FapyGua, trans-AFB1-FapyGua, and AFB1-N7-Gua in DNA of livers of AFB1-treated mice.

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.

The Cooked Meat Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine Hair Dosimeter, DNA Adductomics Discovery, and Associations with Prostate Cancer Pathology Biomarkers.

Well-done cooked red meat consumption is linked to aggressive prostate cancer (PC) risk. Identifying mutation-inducing DNA adducts in the prostate genome can advance our understanding of chemicals in meat that may contribute to PC. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic aromatic amine (HAA) formed in cooked meat, is a potential human prostate carcinogen. PhIP was measured in the hair of PC patients undergoing prostatectomy, bladder cancer patients under treatment for cystoprostatectomy, and patients treated for benign prostatic hyperplasia (BPH). PhIP hair levels were above the quantification limit in 123 of 205 subjects. When dichotomizing prostate pathology biomarkers, the geometric mean PhIP hair levels were higher in patients with intermediate and elevated-risk prostate-specific antigen values than lower-risk values <4 ng/mL (p = 0.03). PhIP hair levels were also higher in patients with intermediate and high-risk Gleason scores ≥7 compared to lower-risk Gleason score 6 and BPH patients (p = 0.02). PC patients undergoing prostatectomy had higher PhIP hair levels than cystoprostatectomy or BPH patients (p = 0.02). PhIP-DNA adducts were detected in 9.4% of the patients assayed; however, DNA adducts of other carcinogenic HAAs, and benzo[a]pyrene formed in cooked meat, were not detected. Prostate specimens were also screened for 10 oxidative stress-associated lipid peroxidation (LPO) DNA adducts. Acrolein 1,N2-propano-2'-deoxyguanosine adducts were detected in 54.5% of the patients; other LPO adducts were infrequently detected. Acrolein adducts were not associated with prostate pathology biomarkers, although DNA adductomic profiles differed between PC patients with low and high-grade Gleason scores. Many DNA adducts are of unknown origin; however, dG adducts of formaldehyde and a series of purported 4-hydroxy-2-alkenals were detected at higher abundance in a subset of patients with elevated Gleason scores. The PhIP hair biomarker and DNA adductomics data support the paradigm of well-done cooked meat and oxidative stress in aggressive PC risk.

Metabolism and biomarkers of heterocyclic aromatic amines in humans.

Heterocyclic aromatic amines (HAAs) form during the high-temperature cooking of meats, poultry, and fish. Some HAAs also arise during the combustion of tobacco. HAAs are multisite carcinogens in rodents, inducing cancer of the liver, gastrointestinal tract, pancreas, mammary, and prostate glands. HAAs undergo metabolic activation by N-hydroxylation of the exocyclic amine groups to produce the proposed reactive intermediate, the heteroaryl nitrenium ion, which is the critical metabolite implicated in DNA damage and genotoxicity. Humans efficiently convert HAAs to these reactive intermediates, resulting in HAA protein and DNA adduct formation. Some epidemiologic studies have reported an association between frequent consumption of well-done cooked meats and elevated cancer risk of the colorectum, pancreas, and prostate. However, other studies have reported no associations between cooked meat and these cancer sites. A significant limitation in epidemiology studies assessing the role of HAAs and cooked meat in cancer risk is their reliance on food frequency questionnaires (FFQ) to gauge HAA exposure. FFQs are problematic because of limitations in self-reported dietary history accuracy, and estimating HAA intake formed in cooked meats at the parts-per-billion level is challenging. There is a critical need to establish long-lived biomarkers of HAAs for implementation in molecular epidemiology studies designed to assess the role of HAAs in health risk. This review article highlights the mechanisms of HAA formation, mutagenesis and carcinogenesis, the metabolism of several prominent HAAs, and the impact of critical xenobiotic-metabolizing enzymes on biological effects. The analytical approaches that have successfully biomonitored HAAs and their biomarkers for molecular epidemiology studies are presented.

Cytotoxicity and genotoxicity of the carcinogen aristolochic acid I (AA-I) in human bladder RT4 cells.

Aristolochic acid (AA-I) induces upper urothelial tract cancer (UUTC) and bladder cancer (BC) in humans. AA-I forms the 7-(2'-deoxyadenosin-N6-yl)aristolactam I (dA-AL-I) adduct, which induces multiple A:T-to-T:A transversion mutations in TP53 of AA-I exposed UTUC patients. This mutation is rarely reported in TP53 of other transitional cell carcinomas and thus recognized as an AA-I mutational signature. A:T-to-T:A transversion mutations were recently detected in bladder tumors of patients in Asia with known AA-I-exposure, implying that AA-I contributes to BC. Mechanistic studies on AA-I genotoxicity have not been reported in human bladder. In this study, we examined AA-I DNA adduct formation and mechanisms of toxicity in the human RT4 bladder cell line. The biological potencies of AA-I were compared to 4-aminobiphenyl, a recognized human bladder carcinogen, and several structurally related carcinogenic heterocyclic aromatic amines (HAA), which are present in urine of smokers and omnivores. AA-I (0.05-10 µM) induced a concentration- and time-dependent cytotoxicity. AA-I (100 nM) DNA adduct formation occurred at over a thousand higher levels than the principal DNA adducts formed with 4-ABP or HAAs (1 µM). dA-AL-I adduct formation was detected down to a 1 nM concentration. Studies with selective chemical inhibitors provided evidence that NQO1 is the major enzyme involved in AA-I bio-activation in RT4 cells, whereas CYP1A1, another enzyme implicated in AA-I toxicity, had a lesser role in bio-activation or detoxification of AA-I. AA-I DNA damage also induced genotoxic stress leading to p53-dependent apoptosis. These biochemical data support the human mutation data and a role for AA-I in BC.

Comprehensive Analysis of DNA Adducts Using Data-Independent wSIM/MS2 Acquisition and wSIM-City.

A novel software has been created to comprehensively characterize covalent modifications of DNA through mass spectral analysis of enzymatically hydrolyzed DNA using the neutral loss of 2'-deoxyribose, a nearly universal MS2 fragmentation process of protonated 2'-deoxyribonucleosides. These covalent modifications termed DNA adducts form through xenobiotic exposures or by reaction with endogenous electrophiles and can induce mutations during cell division and initiate carcinogenesis. DNA adducts are typically present at trace levels in the human genome, requiring a very sensitive and comprehensive data acquisition and analysis method. Our software, wSIM-City, was created to process mass spectral data acquired by a wide selected ion monitoring (wSIM) with gas-phase fractionation and coupled to wide MS2 fragmentation. This untargeted approach can detect DNA adducts at trace levels as low as 1.5 adducts per 109 nucleotides. This level of sensitivity is sufficient for comprehensive analysis and characterization of DNA modifications in human specimens.

Risk of two common glandular cell-type cancers (breast and colorectal cancers) in Chinese occupational chefs: a nationwide ecological study in Taiwan.

OBJECTIVES: Cooking oil fumes (COFs) contain many carcinogens. We investigated the association between COFs and incidence risk of colorectal cancer and female breast in chefs. METHODS: We identified Chinese food chefs and non-Chinese food chefs from Taiwan's national database of certified chefs in 1984-2007. In total, 379,275 overall and 259,450 females had not been diagnosed as having any cancer before chef certification. We followed these chefs in Taiwan's Cancer Registry Database (1979-2010) and Taiwan's National Death Statistics Database (1985-2011) for newly diagnosed colorectal cancer and female breast cancer. RESULTS: A total of 4,218,135 and 2,873,515 person-years were included in our analysis of colorectal cancer and female breast cancer incidence, respectively. Compared to non-Chinese food chefs, the Chinese food chefs had an adjusted IRR for colorectal cancer of 1.65 (95% CI 1.17-2.33). The risk of colorectal cancer was even higher among female Chinese food chefs certified for more than 5 years (adjusted incident rate ratio (IRR) = 2.39, 95% CI 1.38-4.12). For female breast cancer, the risk was also significant (adjusted IRR = 1.40, 95% CI 1.10-1.78) and the risks were even higher in female Chinese food chefs certified for more than 5 years (adjusted IRR = 1.74, 95% CI 1.37-2.22). CONCLUSIONS: This study found that Chinese food chefs had an increased risk of colorectal cancer and female breast cancer, particularly female chefs who had worked for more than 5 years. Future human and animal studies are necessary to re-confirm these findings.

Dietary Heterocyclic Amine Intake and Risk of Esophageal Squamous Cell Carcinoma in Rural Uganda.

Dietary exposure to 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) in cooked meats maybe responsible for the high burden of Esophageal squamous cell carcinoma (ESCC) in southwestern Uganda. We conducted a pilot case-control study among 31 histologically confirmed ESCC cases and 54 age, gender, and residence matched healthy community controls sampled from the general population at the time of accrual of each case in southwestern Uganda. We collected data including smoking, alcohol consumption, diet, and scalp hair samples analyzed for normalized PhlP (adjusted per gram of melanin). We used logistic regression to determine the association of PhlP and ESCC. Overall, the mean normalized PhIP (ng/g melanin) was 44.79 (SD 148.08), higher among women compared to men (130.68 vs. 9.00, p = 0.03), lowest among healthy men [8.31 (SD 8.52) ng/g melanin] and highest among healthy women 158.39 (SD 288.75) ng/g melanin. In fully adjusted models, covariates associated with greater odds of ESCC included ever smoking 2 to 3 pack years of cigarettes (aOR 7.75 (95% CI 1.90, 31.50) and those 3 or more pack years (aOR5.82, 95%CI 1.25, 27.11), drinking 3 to 4 alcoholic drinks daily (aOR8.00, 95%CI 2.31, 27.74), and normalized PhIP above 75th percentile (8.65 ng/g of melanin) (aOR4.27, 95%CI 1.12, 16.24). In conclusion, high PhIP levels maybe associated with ESCC in a rural Uganda, a high ESCC burden setting. Further study with larger sample with a wider geographical representation is needed to validate scalp hair PhIP for assessment of ESCC risk.

Additive Effects of Arsenic and Aristolochic Acid in Chemical Carcinogenesis of Upper Urinary Tract Urothelium.

BACKGROUND: Aristolochic acids (AA) and arsenic are chemical carcinogens associated with urothelial carcinogenesis. Here we investigate the combined effects of AA and arsenic toward the risk of developing upper tract urothelial carcinoma (UTUC). METHODS: Hospital-based (n = 89) and population-based (2,921 cases and 11,684 controls) Taiwanese UTUC cohorts were used to investigate the association between exposure to AA and/or arsenic and the risk of developing UTUC. In the hospital cohort, AA exposure was evaluated by measuring aristolactam-DNA adducts in the renal cortex and by identifying A>T TP53 mutations in tumors. In the population cohort, AA exposure was determined from prescription health insurance records. Arsenic levels were graded from 0 to 3 based on concentrations in well water and the presence of arseniasis-related diseases. RESULTS: In the hospital cohort, 43, 26, and 20 patients resided in grade 0, 1+2, and 3 arseniasis-endemic areas, respectively. Aristolactam-DNA adducts were present in >90% of these patients, indicating widespread AA exposure. A>T mutations in TP53 were detected in 28%, 44%, and 22% of patients residing in grade 0, 1+2, and 3 arseniasis-endemic areas, respectively. Population studies revealed that individuals who consumed more AA-containing herbs had a higher risk of developing UTUC in both arseniasis-endemic and nonendemic areas. Logistic regression showed an additive effect of AA and arsenic exposure on the risk of developing UTUC. CONCLUSIONS: Exposure to both AA and arsenic acts additively to increase the UTUC risk in Taiwan. IMPACT: This is the first study to investigate the combined effect of AA and arsenic exposure on UTUC.

Mutagenicity of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in human TP53 knock-in (Hupki) mouse embryo fibroblasts.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a possible human carcinogen formed in cooked fish and meat. PhIP is bioactivated by cytochrome P450 enzymes to form 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a genotoxic metabolite that reacts with DNA leading to the mutation-prone DNA adduct N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP). Here, we studied N-OH-PhIP-induced whole genome mutagenesis in human TP53 knock-in (Hupki) mouse embryo fibroblasts (HUFs) immortalised and subjected to whole genome sequencing (WGS). In addition, mutagenicity of N-OH-PhIP in TP53 and the lacZ reporter gene were assessed. TP53 mutant frequency in HUF cultures treated with N-OH-PhIP (2.5 µM for 24 h, n = 90) was 10% while no TP53 mutations were found in untreated controls (DMSO for 24 h, n = 6). All N-OH-PhIP-induced TP53 mutations occurred at G:C base pairs with G > T/C > A transversions accounting for 58% of them. TP53 mutations characteristic of those induced by N-OH-PhIP have been found in human tumours including breast and colorectal, which are cancer types that have been associated with PhIP exposure. LacZ mutant frequency increased 25-fold at 5 µM N-OH-PHIP and up to ~350 dG-C8-PhIP adducts/108 nucleosides were detected by ultra-performance liquid chromatography-electrospray ionisation multistage scan mass spectrometry (UPLC-ESI-MS3) at this concentration. In addition, a WGS mutational signature defined by G > T/C > A transversions was present in N-OH-PhIP-treated immortalised clones, which showed similarity to COSMIC SBS4, 18 and 29 signatures found in human tumours.

Mass Spectrometric Quantitation of Apurinic/Apyrimidinic Sites in Tissue DNA of Rats Exposed to Tobacco-Specific Nitrosamines and in Lung and Leukocyte DNA of Cigarette Smokers and Nonsmokers.

Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N7POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 108 nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and nonsmokers. Rats treated with 5 or 21 mg/kg bw NNK for 4 days by s.c. injection had 2-6 and 2-17 times more AP sites than controls in liver and lung DNA (p < 0.05). Increases in AP sites were also found in liver DNA of rats exposed for 10 and 30 weeks (p < 0.05) but not for 50 and 70 weeks to 5 ppm of NNK in their drinking water. Levels of N7POBG were significantly correlated with AP sites in rats treated with NNK. In rats treated with 14 ppm (S)-NNN in their drinking water for 10 weeks, increased AP site formation compared to controls was observed in oral and nasal respiratory mucosa DNA (p < 0.05). No significant increase in AP sites was found in human lung and leukocyte DNA of cigarette smokers compared to nonsmokers, although AP sites in leukocyte DNA were significantly correlated with urinary levels of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). This is the first study to use mass spectrometry based methods to examine AP site formation by carcinogenic tobacco-specific nitrosamines in laboratory animals and to evaluate AP sites in DNA of smokers and nonsmokers.