Covalent DNA–Protein Cross-Linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells

<i>N</i>,<i>N</i>-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and <i>N</i>,<i>N</i>-bis-(2-chloroethyl)-amine (nornitrogen mustard, NOR) are the two biologically active metabolites of cyclophosphamide, a DNA alkylating drug commonly used to treat lymphomas, breast cancer, certain brain cancers, and autoimmune diseases. PM and NOR are reactive bis-electrophiles capable of cross-linking cellular biomolecules to form covalent DNA–DNA and DNA–protein cross-links (DPCs). In the present work, a mass spectrometry-based proteomics approach was employed to characterize PM- and NOR-mediated DNA–protein cross-linking in human cells. Following treatment of human fibrosarcoma cells (HT1080) with cytotoxic concentrations of PM, over 130 proteins were found to be covalently trapped to DNA, including those involved in transcriptional regulation, RNA splicing/processing, chromatin organization, and protein transport. HPLC-ESI⁺-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of <i>N</i>-[2-[cysteinyl]­ethyl]-<i>N</i>-[2-(guan-7-yl)­ethyl]­amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N7 position of guanines in DNA. Cys-NOR-N7G adduct numbers were higher in NER-deficient xeroderma pigmentosum cells (XPA) as compared with repair proficient cells. Furthermore, both XPA and FANCD2 deficient cells were sensitized to PM treatment as compared to that of wild type cells, suggesting that Fanconi anemia and nucleotide excision repair pathways are involved in the removal of cyclophosphamide-induced DNA damage

NanoHPLC-nanoESI⁺-MS/MS Quantitation of <i>Bis</i>-N7-Guanine DNA–DNA Cross-Links in Tissues of B6C3F1 Mice Exposed to subppm Levels of 1,3-Butadiene

1,3-Butadiene (BD) is an important industrial chemical and a common environmental pollutant present in urban air. BD is classified as a human carcinogen based on epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its potent carcinogenicity in laboratory mice. A diepoxide metabolite of BD, 1,2,3,4-diepoxybutane (DEB), is considered the ultimate carcinogenic species of BD due to its ability to form genotoxic DNA–DNA cross-links. We have previously employed capillary HPLC-ESI⁺-MS/MS (liquid chromatography-electrospray ionization tandem mass spectrometry) methods to quantify DEB-induced DNA–DNA conjugates, e.g. 1,4-<i>bis</i>-(guan-7-yl)-2,3-butanediol (<i>bis</i>-N7G-BD), 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), and <i>1,N</i>^<i>6</i>-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (<i>1,N</i>^<i>6</i>-HMHP-dA), in tissues of laboratory mice exposed to 6.25–625 ppm BD (Goggin et al. <i>Cancer Res.</i> <b>2009</b>, <i>69</i>(6), 2479–2486). However, typical BD human exposure levels are 0.01 to 3.2 ppb in urban air and 1–2.0 ppm in an occupational setting, requiring greater detection sensitivity for these critical lesions. In the present study, a nanoHPLC-nanoESI⁺-MS/MS method was developed for ultrasensitive, accurate, and precise quantitation of <i>bis</i>-N7G-BD in tissues of laboratory mice treated with low ppm and subppm concentrations of BD. The LOD value of the new method is 0.5 fmol/100 μg DNA, and the LOQ is 1.0 fmol/100 μg DNA, making it possible to quantify <i>bis</i>-N7G-BD adducts present at concentrations of 3 per 10⁹ nucleotides. <i>Bis</i>-N7G-BD adduct amounts in liver tissues of mice exposed to 0.5, 1.0, and 1.5 ppm BD for 2 weeks were 5.7 ± 3.3, 9.2 ± 1.5, and 18.6 ± 6.9 adducts per 10⁹ nucleotides, respectively, suggesting that <i>bis</i>-N7G-BD adduct formation is more efficient under low exposure conditions. To our knowledge, this is the first quantitative analysis of DEB specific DNA adducts following low ppm and subppm exposure to BD

1,3-Butadiene-Induced Adenine DNA Adducts Are Genotoxic but Only Weakly Mutagenic When Replicated in <i>Escherichia coli</i> of Various Repair and Replication Backgrounds

The adverse effects of the human carcinogen 1,3-butadiene (BD) are believed to be mediated by its DNA-reactive metabolites such as 3,4-epoxybut-1-ene (EB) and 1,2,3,4-diepoxybutane (DEB). The specific DNA adducts responsible for toxic and mutagenic effects of BD, however, have yet to be identified. Recent <i>in vitro</i> polymerase bypass studies of BD-induced adenine (BD-dA) adducts show that DEB-induced <i>N</i>⁶,<i>N</i>⁶-DHB-dA (DHB = 2,3-dihydroxybutan-1,4-diyl) and 1,<i>N</i>⁶-γ-HMHP-dA (HMHP = 2-hydroxy-3-hydroxymethylpropan-1,3-diyl) adducts block replicative DNA polymerases but are bypassed by human polymerases η and κ, leading to point mutations and deletions. In contrast, EB-induced <i>N</i>⁶-HB-dA (HB = 2-hydroxy-3-buten-1-yl) does not block DNA synthesis and is nonmutagenic. In the present study, we employed a newly established <i>in vivo</i> lesion-induced mutagenesis/genotoxicity assay via next-generation sequencing to evaluate the <i>in vivo</i> biological consequences of <i>S</i>-<i>N</i>⁶-HB-dA, <i>R,R</i>-<i>N</i>⁶,<i>N</i>⁶-DHB-dA, <i>S</i>,<i>S</i>-<i>N</i>⁶,<i>N</i>⁶-DHB-dA, and <i>R</i>,<i>S</i>-1,<i>N</i>⁶-γ-HMHP-dA. In addition, the effects of AlkB-mediated direct reversal repair, MutM and MutY catalyzed base excision repair, and DinB translesion synthesis on the BD-dA adducts in bacterial cells were investigated. BD-dA adducts showed the expected inhibition of DNA replication <i>in vivo</i> but were not substantively mutagenic in any of the genetic environments investigated. This result is in contrast with previous <i>in vitro</i> observations and opens the possibility that <i>E. coli</i> repair and bypass systems other than the ones studied here are able to minimize the mutagenic properties of BD-dA adducts

Capillary HPLC-Accurate Mass MS/MS Quantitation of N7-(2,3,4-Trihydroxybut-1-yl)-guanine Adducts of 1,3-Butadiene in Human Leukocyte DNA

1,3-Butadiene (BD) is a high volume industrial chemical commonly used in polymer and rubber production. It is also present in cigarette smoke, automobile exhaust, and urban air, leading to widespread exposure of human populations. Upon entering the body, BD is metabolized to electrophilic epoxides, 3,4-epoxy-1-butene (EB), diepoxybutane (DEB), and 3,4-epoxy-1,2-diol (EBD), which can alkylate DNA nucleobases. The most abundant BD epoxide, EBD, modifies the N7-guanine positions in DNA to form N7-(2, 3, 4-trihydroxybut-1-yl) guanine (N7-THBG) adducts, which can be useful as biomarkers of BD exposure and metabolic activation to DNA-reactive epoxides. In the present work, a capillary HPLC-high resolution ESI⁺-MS/MS (HPLC-ESI⁺-HRMS/MS) methodology was developed for accurate, sensitive, and reproducible quantification of N7-THBG in cell culture and in human white blood cells. In our approach, DNA is subjected to neutral thermal hydrolysis to release N7-guanine adducts from the DNA backbone, followed by ultrafiltration, solid-phase extraction, and isotope dilution HPLC-ESI⁺-HRMS/MS analysis on an Orbitrap Velos mass spectrometer. Following method validation, N7-THBG was quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of DEB and in DNA isolated from blood of smokers, nonsmokers, individuals participating in a smoking cessation program, and occupationally exposed workers. N7-THBG concentrations increased linearly from 31.4 ± 4.84 to 966.55 ± 128.05 adducts per 10⁹ nucleotides in HT1080 cells treated with 1–100 μM DEB. N7-THBG amounts in leukocyte DNA of nonsmokers, smokers, and occupationally exposed workers were 7.08 ± 5.29, 8.20 ± 5.12, and 9.72 ± 3.80 adducts per 10⁹ nucleotides, respectively, suggesting the presence of an endogenous or environmental source for this adduct. The availability of sensitive HPLC-ESI⁺-HRMS/MS methodology for BD-induced DNA adducts in humans will enable future population studies of interindividual and ethnic differences in BD bioactivation to DNA-reactive epoxides

Major Groove Orientation of the (2<i>S</i>)‑<i>N</i>⁶‑(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine DNA Adduct Induced by 1,2-Epoxy-3-butene

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. It is oxidized by cytochrome P450 monooxygenases to 1,2-epoxy-3-butene (EB), which alkylates DNA. BD exposures lead to large numbers of mutations at A:T base pairs even though alkylation of guanines is more prevalent, suggesting that one or more adenine adducts of BD play a role in BD-mediated genotoxicity. However, the etiology of BD-mediated genotoxicity at adenine remains poorly understood. EB alkylates the <i>N</i>⁶ exocyclic nitrogen of adenine to form <i>N</i>⁶-(hydroxy-3-buten-1-yl)-2′-dA ((2<i>S</i>)-<i>N</i>⁶-HB-dA) adducts (Tretyakova, N., Lin, Y., Sangaiah, R., Upton, P. B., and Swenberg, J. A. (1997) Carcinogenesis 18, 137−147). The structure of the (2<i>S</i>)-<i>N</i>⁶-HB-dA adduct has been determined in the 5′-d­(C¹G²G³A⁴<u>C</u>^<u>5</u><u>Y</u>^<u>6</u><u>A</u>^<u>7</u>G⁸A⁹A¹⁰G¹¹)-3′:5′-d­(C¹²T¹³T¹⁴C¹⁵T¹⁶T¹⁷G¹⁸T¹⁹ C²⁰C²¹G²²)-3′ duplex [Y = (2<i>S</i>)-<i>N</i>⁶-HB-dA] containing codon 61 (underlined) of the human N-<i>ras</i> protooncogene, from NMR spectroscopy. The (2<i>S</i>)-<i>N</i>⁶-HB-dA adduct was positioned in the major groove, such that the butadiene moiety was oriented in the 3′ direction. At the C_α carbon, the methylene protons of the modified nucleobase Y⁶ faced the 5′ direction, which placed the C_β carbon in the 3′ direction. The C_β hydroxyl group faced toward the solvent, as did carbons C_γ and C_δ. The C_β hydroxyl group did not form hydrogen bonds with either T¹⁶ <i>O</i>⁴ or T¹⁷ <i>O</i>⁴. The (2<i>S</i>)-<i>N</i>⁶-HB-dA nucleoside maintained the <i>anti</i> conformation about the glycosyl bond, and the modified base retained Watson–Crick base pairing with the complementary base (T¹⁷). The adduct perturbed stacking interactions at base pairs C⁵:G¹⁸, Y⁶:T¹⁷, and A⁷:T¹⁶ such that the Y⁶ base did not stack with its 5′ neighbor C⁵, but it did with its 3′ neighbor A⁷. The complementary thymine T¹⁷ stacked well with both 5′ and 3′ neighbors T¹⁶ and G¹⁸. The presence of the (2<i>S</i>)-<i>N</i>⁶-HB-dA resulted in a 5 °C reduction in the <i>T</i>_m of the duplex, which is attributed to less favorable stacking interactions and adduct accommodation in the major groove

Mapping Structurally Defined Guanine Oxidation Products along DNA Duplexes: Influence of Local Sequence Context and Endogenous Cytosine Methylation

DNA oxidation by reactive oxygen species is nonrandom, potentially leading to accumulation of nucleobase damage and mutations at specific sites within the genome. We now present the first quantitative data for sequence-dependent formation of structurally defined oxidative nucleobase adducts along <i>p53</i> gene-derived DNA duplexes using a novel isotope labeling-based approach. Our results reveal that local nucleobase sequence context differentially alters the yields of 2,2,4-triamino-2<i>H</i>-oxal-5-one (Z) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) in double stranded DNA. While both lesions are overproduced within endogenously methylated ^MeCG dinucleotides and at 5′ Gs in runs of several guanines, the formation of Z (but not OG) is strongly preferred at solvent-exposed guanine nucleobases at duplex ends. Targeted oxidation of ^MeCG sequences may be caused by a lowered ionization potential of guanine bases paired with ^MeC and the preferential intercalation of riboflavin photosensitizer adjacent to ^MeC:G base pairs. Importantly, some of the most frequently oxidized positions coincide with the known <i>p53</i> lung cancer mutational “hotspots” at codons 245 (GGC), 248 (CGG), and 158 (CGC) respectively, supporting a possible role of oxidative degradation of DNA in the initiation of lung cancer

Isotope Dilution nanoLC/ESI⁺‑HRMS³ Quantitation of Urinary N7-(1-Hydroxy-3-buten-2-yl) Guanine Adducts in Humans and Their Use as Biomarkers of Exposure to 1,3-Butadiene

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a known human carcinogen. Occupational exposure to BD in the polymer and monomer industries is associated with an increased incidence of lymphoma. BD is present in automobile exhaust, cigarette smoke, and forest fires, raising concern about potential exposure of the general population to this carcinogen. Following inhalation exposure, BD is bioactivated to 3,4-epoxy-1-butene (EB). If not detoxified, EB is capable of modifying guanine and adenine bases of DNA to form nucleobase adducts, which interfere with accurate DNA replication and cause cancer-initiating mutations. We have developed a nanoLC/ESI⁺-HRMS³ methodology for N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human urine (limit of detection: 0.25 fmol/mL urine; limit of quantitation: 1.0 fmol/mL urine). This new method was successfully used to quantify EB-GII in urine of F344 rats treated with 0–200 ppm of BD, occupationally exposed workers, and smokers belonging to two different ethnic groups. EB-GII amounts increased in a dose-dependent manner in urine of laboratory rats exposed to 0, 62.5, or 200 ppm of BD. Urinary EB-GII levels were significantly increased in workers occupationally exposed to 0.1–2.2 ppm of BD (1.25 ± 0.51 pg/mg of creatinine) as compared to administrative controls exposed to <0.01 ppm of BD (0.22 ± 0.08 and pg/mg of creatinine) (<i>p</i> = 0.0024), validating the use of EB-GII as a biomarker of human exposure to BD. EB-GII was also detected in smokers’ urine with European American smokers excreting significantly higher amounts of EB-GII than African American smokers (0.48 ± 0.09 vs 0.12 ± 0.02 pg/mg of creatinine, <i>p</i> = 3.1 × 10^–7). Interestingly, small amounts of EB-GII were observed in animals and humans with no known exposure to BD, providing preliminary evidence for its endogenous formation. Urinary EB-GII adduct levels and urinary mercapturic acids of BD (MHBMA, DHBMA) were compared in a genotyped multiethnic smoker cohort

Kinetics of <i>O</i>⁶‑Pyridyloxobutyl-2′-deoxyguanosine Repair by Human <i>O</i>⁶‑alkylguanine DNA Alkyltransferase

Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN) are potent carcinogens believed to contribute to the development of lung tumors in smokers. NNK and NNN are metabolized to DNA-reactive species that form a range of nucleobase adducts, including bulky <i>O</i>⁶-[4-oxo-4-(3-pyridyl)­but-1-yl]­deoxyguanosine (<i>O</i>⁶-POB-dG) lesions. If not repaired, <i>O</i>⁶-POB-dG adducts induce large numbers of G → A and G → T mutations. Previous studies have shown that <i>O</i>⁶-POB-dG can be directly repaired by <i>O</i>⁶-alkylguanine-DNA alkyltransferase (AGT), which transfers the pyridyloxobutyl group from <i>O</i>⁶-alkylguanines in DNA to an active site cysteine residue within the protein. In the present study, we investigated the influence of DNA sequence context and endogenous cytosine methylation on the kinetics of AGT-dependent repair of <i>O</i>⁶-POB-dG in duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific <i>O</i>⁶-POB-dG adducts within <i>K-ras</i> and <i>p53</i> gene-derived DNA sequences were incubated with recombinant human AGT protein, and the kinetics of POB group transfer was monitored by isotope dilution HPLC-ESI⁺-MS/MS analysis of <i>O</i>⁶-POB-dG remaining in DNA over time. We found that the second-order rates of AGT-mediated repair were influenced by DNA sequence context (10-fold differences) but were only weakly affected by the methylation status of neighboring cytosines. Overall, AGT-mediated repair of <i>O</i>⁶-POB-dG was 2–7 times slower than that of <i>O</i>⁶-Me-dG adducts. To evaluate the contribution of AGT to <i>O</i>⁶-POB-dG repair in human lung, normal human bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating agent, and <i>O</i>⁶-POB-dG adduct repair over time was monitored by HPLC-ESI⁺-MS/MS. We found that HBEC cells were capable of removing <i>O</i>⁶-POB-dG lesions, and the repair rates were significantly reduced in the presence of an AGT inhibitor (<i>O</i>⁶-benzylguanine). Taken together, our results suggest that AGT plays an important role in protecting human lung against tobacco nitrosamine-mediated DNA damage and that inefficient AGT repair of <i>O</i>⁶-POB-dG at a specific sequences contributes to mutational spectra observed in smoking-induced lung cancer

Base Excision Repair of <i>N</i>⁶<i>-</i>Deoxyadenosine Adducts of 1,3-Butadiene

The important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including <i>N</i>⁶-(2-hydroxy-3-buten-1-yl)-2′-deoxyadenosine (<i>N</i>⁶-HB-dA), 1,<i>N</i>⁶-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,<i>N</i>⁶-HMHP-dA), and <i>N</i>⁶,<i>N</i>⁶-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (<i>N</i>⁶,<i>N</i>⁶-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In this study, base excision repair of BD-induced 2′-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site-specific and stereospecific (<i>S</i>)-<i>N</i>⁶-HB-dA, (<i>R</i>,<i>S</i>)-1,<i>N</i>⁶-HMHP-dA, and (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA adducts were prepared by a postoligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with the relative excision efficiency decreasing in the following order: (<i>S</i>)-<i>N</i>⁶-HB-dA > (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA > (<i>R</i>,<i>S</i>)-1,<i>N</i>⁶-HMHP-dA. The extent of strand cleavage at the adduct site was decreased in the presence of BER inhibitor methoxyamine and by competitor duplexes containing known BER substrates. Similar strand cleavage assays conducted using several eukaryotic DNA glycosylases/lyases (AAG, Mutyh, hNEIL1, and hOGG1) have failed to observe correct incision products at the BD-dA lesion sites, suggesting that a different BER enzyme may be involved in the removal of BD-dA adducts in human cells