Oxidative Generation of Guanine Radicals by Carbonate Radicals and Their Reactions with Nitrogen Dioxide to Form Site Specific 5-Guanidino-4-nitroimidazole Lesions in Oligodeoxynucleotides

A simple photochemical approach is described for synthesizing site specific, stable 5-guanidino-4-nitroimidazole (NIm) adducts in single- and double-stranded oligodeoxynucleotides containing single and multiple guanine residues. The DNA sequences employed, 5‘-d(ACC CG1C G*2TC CG*3C G4CC) and 5‘-d(ACC CG1C G*2TC C), were a portion of exon 5 of the p53 tumor suppressor gene, including the codons 157 (G*2) and 158 (G*3) mutation hot spots in the former sequence with four Gs and the codon 157 (G*2) mutation hot spot in the latter sequence with two Gs. The nitration of oligodeoxynucleotides was initiated by the selective photodissociation of persulfate anions to sulfate radicals induced by UV laser pulses (308 nm). In aqueous solutions, of bicarbonate and nitrite anions, the sulfate radicals generate carbonate anion radicals and nitrogen dioxide radicals by one electron oxidation of the respective anions. The guanine residue in the oligodeoxynucleotide is oxidized by the carbonate anion radical to form the neutral guanine radical. While the nitrogen dioxide radicals do not react with any of the intact DNA bases, they readily combine with the guanine radicals at either the C8 or the C5 positions. The C8 addition generates the well-known 8-nitroguanine (8-nitro-G) lesions, whereas the C5 attack produces unstable adducts, which rapidly decompose to NIm lesions. The maximum yields of the nitro products (NIm + 8-nitro-G) were typically in the range of 20−40%, depending on the number of guanine residues in the sequence. The ratio of the NIm to 8-nitro-G lesions gradually decreases from 3.4 in the model compound, 2‘,3‘,5‘-tri-O-acetylguanosine, to 2.1−2.6 in the single-stranded oligodeoxynucleotides and to 0.8−1.1 in the duplexes. The adduct of the 5‘-d(ACC CG1C G*2TC C) oligodeoxynucleotide containing the NIm lesion in codon 157 (G2) was isolated in HPLC-pure form. The integrity of this adduct was established by a detailed analysis of exonuclease digestion ladders by matrix-assisted laser desorption ionization with time-of-flight detection MS techniques

Biomonitoring DNA Adducts of Cooked Meat Carcinogens in Human Prostate by Nano Liquid Chromatography–High Resolution Tandem Mass Spectrometry: Identification of 2‑Amino-1-methyl-6-phenylimidazo[4,5‑<i>b</i>]pyridine DNA Adduct

Epidemiologic studies have reported an association between frequent consumption of well-done cooked meats and prostate cancer risk. However, unambiguous physiochemical markers of DNA damage from carcinogens derived from cooked meats, such as DNA adducts, have not been identified in human samples to support this paradigm. We have developed a highly sensitive nano-LC-Orbitrap MSn method to measure DNA adducts of several carcinogens originating from well-done cooked meats, tobacco smoke, and environmental pollution, including 2-amino-1-methyl-6-phenylimidazo­[4,5-b]­pyridine (PhIP), 2-amino-9H-pyrido­[2,3-b]­indole (AαC), 2-amino-3,8-dimethylimidazo­[4,5-f]­quinoxaline (MeIQx), benzo­[a]­pyrene (B­[a]­P), and 4-aminobiphenyl (4-ABP). The limit of quantification (LOQ) of the major deoxyguanosine (dG) adducts of these carcinogens ranged between 1.3 and 2.2 adducts per 109 nucleotides per 2.5 μg of DNA assayed. The DNA adduct of PhIP, N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP) was identified in 11 out of 35 patients, at levels ranging from 2 to 120 adducts per 109 nucleotides. The dG-C8 adducts of AαC and MeIQx, and the B­[a]P adduct, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo­[a]­pyrene (dG-N2-B­[a]­PDE) were not detected in any specimen, whereas N-(deoxyguanosin-8-yl)-4-ABP (dG-C8-4-ABP) was identified in one subject (30 adducts per 109 nucleotides). PhIP–DNA adducts also were recovered quantitatively from formalin fixed paraffin embedded (FFPE) tissues, signifying FFPE tissues can serve as biospecimens for carcinogen DNA adduct biomarker research. Our biomarker data provide support to the epidemiological observations implicating PhIP, one of the most mass-abundant heterocyclic aromatic amines formed in well-done cooked meats, as a DNA-damaging agent that may contribute to the etiology of prostate cancer

Photosensitized Oxidative DNA Damage: From Hole Injection to Chemical Product Formation and Strand Cleavage

Oxidatively generated damage to DNA induced by a pyrenyl photosensitizer residue (Py) covalently attached to a guanine base in the DNA sequence context 5‘-d(CAT[G1Py]CG2TCCTAC) in aerated solutions was monitored from the initial one-electron transfer, or hole injection step, to the formation of chemical end-products monitored by HPLC, mass spectrometry, and high-resolution gel electrophoresis. Hole injection into the DNA was initiated by two-photon excitation of the Py residue with 355 nm laser pulses, thus producing the radical cation Py•+ and hydrated electrons; the latter are trapped by O2, thus forming the superoxide anion O2•-. The decay of the Py•+ radical is correlated with the appearance of the G•+/G(−H)• radical on microsecond time scales, and O2•- combines with guanine radicals at G1 to form alkali-labile 2,5-diamino-4H-imidazolone lesions (Iz1Py). Product formation in the modified strand is smaller by a factor of 2.4 in double-stranded than in single-stranded DNA. In double-stranded DNA, hot piperidine-mediated cleavage at G2 occurs only after G1Py, an efficient hole trap, is oxidized thus generating tandem lesions. An upper limit of hole hopping rates, khh 3 s-1 from G1•+−Py to G2 can be estimated from the known rates of the combination reaction of the G(−H)• and O2•- radicals. The formation of Iz products in the unmodified complementary strand compared to the modified strand in the duplex is ∼10 times smaller. The formation of tandem lesions is observed even at low levels of irradiation corresponding to “single-hit” conditions when less than ∼10% of the oligonucleotide strands are damaged. A plausible mechanism for this observation is discussed

Multiclass Carcinogenic DNA Adduct Quantification in Formalin-Fixed Paraffin-Embedded Tissues by Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry

DNA adducts are a measure of internal exposure to genotoxicants and an important biomarker for human risk assessment. However, the employment of DNA adducts as biomarkers in human studies is often restricted because fresh-frozen tissues are not available. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis are readily accessible. Recently, our laboratory reported that DNA adducts of aristolochic acid, a carcinogenic component of Aristolochia herbs used in traditional Chinese medicines worldwide, can be recovered quantitatively from FFPE tissues. In this study, we have evaluated the efficacy of our method for retrieval of DNA adducts from archived tissue by measuring DNA adducts derived from four other classes of human carcinogens: polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic aromatic amines (HAAs), and N-nitroso compounds (NOCs). Deoxyguanosine (dG) adducts of the PAH benzo­[<i>a</i>]­pyrene (B­[<i>a</i>]­P), 10-(deoxyguanosin-<i>N</i>²-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo­[<i>a</i>]­pyrene (dG-<i>N</i>²–B­[<i>a</i>]­PDE); the aromatic amine 4-aminobiphenyl (4-ABP), <i>N</i>-(deoxyguanosin-8-yl)–4-aminobiphenyl (dG-C8–4-ABP); the HAA 2-amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (PhIP), <i>N</i>-(deoxyguanosin-8-yl)-PhIP (dG-C8–PhIP); and the dG adducts of the NOC 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), <i>O</i>⁶-methyl-dG (<i>O</i>⁶-Me–dG) and <i>O</i>⁶-pyridyloxobutyl-dG (<i>O</i>⁶-POB–dG), formed in liver, lung, bladder, pancreas, or colon were recovered in comparable yields from fresh-frozen and FFPE preserved tissues of rodents treated with the procarcinogens. Quantification was achieved by ultraperformance liquid chromatography coupled with electrospray ionization ion-trap multistage mass spectrometry (UPLC/ESI-IT-MS³). These advancements in the technology of DNA adduct retrieval from FFPE tissue clear the way for use of archived pathology samples in molecular epidemiology studies designed to assess the causal role of exposure to hazardous chemicals with cancer risk

ccRCC cohort characteristics from Aristolochic Acid in the Etiology of Renal Cell Carcinoma

Description of Taiwanese ccRCC cohort</p

Supplementary Table 2 from Aristolochic Acid in the Etiology of Renal Cell Carcinoma

Somatic mutations identified from exome sequencing of ten Taiwanese ccRCC patients</p

Copy number alterations in ten AA-exposed Taiwanese ccRCCs from Aristolochic Acid in the Etiology of Renal Cell Carcinoma

Circus plots of Taiwanese ccRCCs.</p

Supplementary Table 4 from Aristolochic Acid in the Etiology of Renal Cell Carcinoma

Summary of exome sequence analysis of ten Taiwanese ccRCC patients</p

Supplementary Methods and Supplementary Figure Legend from Aristolochic Acid in the Etiology of Renal Cell Carcinoma

Online methods and figures</p

Supplementary Table 3 from Aristolochic Acid in the Etiology of Renal Cell Carcinoma

Primers used for Sanger sequencing</p