In vitro investigation and mass spectrometric characterization of novel DNA adducts of sesquimustard.

Zearalenone-14-glucoside (ZEN-14G), a key modified mycotoxin, has attracted a great deal of attention due to the possible conversion to its free form of zearalenone (ZEN) exerting toxicity. In this study, the toxicokinetics of ZEN-14G were investigated in rats after oral and intravenous administration. The plasma concentrations of ZEN-14G and its major five metabolites were quantified using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The data were analyzed via non-compartmental analysis using software WinNonlin 6.3. The results indicated that ZEN-14G was rapidly hydrolyzed into ZEN in vivo. In addition, the major parameters of ZEN-14G following intravenous administration were: area under the plasma concentration–time curve (AUC), 1.80 h·ng/mL; the apparent volume of distribution (VZ), 7.25 L/kg; and total body clearance (CL), 5.02 mL/h/kg, respectively. After oral administration, the typical parameters were: AUC, 0.16 h·ng/mL; VZ, 6.24 mL/kg; and CL, 4.50 mL/h/kg, respectively. The absolute oral bioavailability of ZEN-14G in rats was about 9%, since low levels of ZEN-14G were detected in plasma, which might be attributed to its extensive metabolism. Therefore, liquid chromatography high-resolution mass spectrometry (LC-HRMS) was adopted to clarify the metabolic profile of ZEN-14G in rats’ plasma. As a result, eight metabolites were identified in which ZEN-14-glucuronic acid (ZEN-14GlcA) had a large yield from the first time-point and continued accumulating after oral administration, indicating that ZEN-14-glucuronic acid could serve a potential biomarker of ZEN-14G. The obtained outcomes would prompt the accurate safety evaluation of ZEN-14G.

Acetaldehyde is an ubiquitous genotoxic compound that has been classified as a possible carcinogen to humans. It can react with DNA to form primarily a Schiff base N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) adduct. An online column-switching valve liquid chromatography tandem mass spectrometry (LC-MS/MS) selected reaction monitoring (SRM) method was developed for the determination of N(2)-ethylidene-dG adducts in DNA following reduction with sodium cyanoborohydride (NaBH(3)CN) to the chemically stable N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) adduct. Accurate quantitation of the adduct was obtained by the addition of the [(15)N(5)]N(2)-ethyl-dG stable isotope-labeled internal standard prior to enzymatic hydrolysis of the DNA samples to 2'-deoxynucleosides with the incorporation of NaBH(3)CN in the DNA hydrolysis buffer. The method required 50 microg of hydrolyzed DNA on column for the analysis, and the limit of detection for N(2)-ethyl-dG was 2.0 fmol. The analysis of calf thymus DNA treated in vitro with acetaldehyde (ranging from 0.5 to 100 mM) or with the smoke generated from 1, 5, and 10 cannabis cigarettes showed linear dose-dependent increases in the level of N(2)-ethyl-dG adducts (r = 0.954 and r = 0.999, respectively). Similar levels (332.8 +/- 21.9 vs 348.4 +/- 19.1 adducts per 10(8) 2'-deoxynucleosides) of N(2)-ethyl-dG adducts were detected following the exposure of calf thymus DNA to 10 tobacco or 10 cannabis cigarettes. No significant difference was found in the levels of N(2)-ethyl-dG adducts in human lung DNA obtained from nonsmokers (n = 4) and smokers (n = 4) with the average level observed as 13.3 +/- 0.7 adducts per 10(8) 2'-deoxynucleosides. No N(2)-ethyl-dG adducts were detected in any of the DNA samples following analysis with the omission of NaBH(3)CN from the DNA hydrolysis buffer. In conclusion, these results provide evidence for the DNA damaging potential of cannabis smoke, implying that the consumption of cannabis cigarettes may be detrimental to human health with the possibility to initiate cancer development.

Elevated aflatoxin B(1)-albumin adducts (AFB(1)-Alb) have been associated with an increased risk for HCC development. However, there are no studies in humans, correlating albumin adducts in blood with liver DNA adducts. Forty frozen tumor tissues and 39 paired plasma samples from HCC patients were collected in Taiwan, to determine the relationship between albumin adducts in blood and DNA adducts in liver tissue as well as mutations in p53 and methylation of p16. AFB(1)- and polycyclic aromatic hydrocarbon (PAH)-DNA adducts in tissue and albumin adducts in plasma were determined by immunohistochemistry and competitive ELISA, respectively. Plasma AFB(1)-Alb adducts in subjects with low, medium and high levels of AFB(1)-DNA adducts in tumor tissues were 51.0 +/- 36.5, 70.5 +/- 48.1 and 84.9 +/- 48.2 fmol/mg, respectively (p(trend) = 0.05). No significant correlation was found for PAH. Fourteen of 40 (36%) tissues were positive for mutant p53 protein by immunohistochemistry; 11 of 40 tissue DNA samples (28%) were positive for p53 mutations, but not their corresponding plasma DNAs. p16 was methylated in 24 of 40 (62%) tissues and 12 of 39 (32%) plasma DNAs. Significant correlations were observed between AFB(1)-Alb adducts and p53 mutations and p16 methylation. These data suggest that genetic, epigenetic and environmental exposure biomarkers in plasma may help in estimating the risk for the development of HCC.

Screening for DNA adducts in ovarian follicles exposed to benzo[a]pyrene and cigarette smoke condensate using liquid chromatography-tandem mass spectrometry

DNA adducts are produced both exogenously and endogenously via exposure to various DNA-damaging agents. Two lipid peroxidation (LPO) products, 4-oxo-2(E)-nonenal (4-ONE) and 4-oxo-2(E)-hexenal (4-OHE), induce substituted etheno-DNA adducts in cells and chemically treated animals, but the adduct levels in humans have never been reported. It is important to investigate the occurrence of 4-ONE- and 4-OHE-derived DNA adducts in humans to further understand their potential impact on human health. In this study, we conducted DNA adductome analysis of several human specimens of pulmonary DNA as well as various LPO-induced DNA adducts in 68 human autopsy tissues, including colon, heart, kidney, liver, lung, pancreas, small intestine, and spleen, by liquid chromatography tandem mass spectrometry. In the adductome analysis, DNA adducts derived from 4-ONE and 4-OHE, namely, heptanone-etheno-2'-deoxycytidine (HεdC), heptanone-etheno-2'-deoxyadenosine (HεdA), and butanone-etheno-2'-deoxycytidine (BεdC), were identified as major adducts in one human pulmonary DNA. Quantitative analysis revealed 4-ONE-derived HεdC, HεdA, and heptanone-etheno-2'-deoxyguanosine (HεdG) to be ubiquitous in various human tissues at median values of 10, 15, and 8.6 adducts per 10(8) bases, respectively. More importantly, an extremely high level (more than 100 per 10(8) bases) of these DNA adducts was observed in several cases. The level of 4-OHE-derived BεdC was highly correlated with that of HεdC (R(2) = 0.94), although BεdC was present at about a 7-fold lower concentration than HεdC. These results suggest that 4-ONE- and 4-OHE-derived DNA adducts are likely to be significant DNA adducts in human tissues, with potential for deleterious effects on human health.

The formation and repair of N2-(trans-isosafrol-3'-yl)-2'-deoxyguanosine (S-3'-N2-dG) DNA adduct derived from the spice and herbal alkenylbenzene constituent safrole were investigated. DNA adduct formation and repair were studied in vitro and using molecular dynamics (MD) simulations. DNA adduct formation was quantified using liquid chromatography-mass spectrometry (LCMS) in wild type and NER (nucleotide excision repair) deficient CHO cells and also in HepaRG cells and primary rat hepatocytes after different periods of repair following exposure to safrole or 1'-hydroxysafrole (1'-OH safrole). The slower repair of the DNA adducts found in NER deficient cells compared to that in CHO wild type cells indicates a role for NER in repair of S-3'-N2-dG DNA adducts. However, DNA repair in liver cell models appeared to be limited, with over 90% of the adducts remaining even after 24 or 48 h recovery. In our further studies, MD simulations indicated that S-3'-N2-dG adduct formation causes only subtle changes in the DNA structure, potentially explaining inefficient activation of NER. Inefficiency of NER mediated repair of S-3'-N2-dG adducts points at persistence and potential bioaccumulation of safrole DNA adducts upon daily dietary exposure.

Advances in sulfur mustard-induced DNA adducts: Characterization and detection

The development of analytical approaches for identification of DNA adducts may provide important tools to help clarify their role as biomarkers of exposure and as potential markers of cancer risk. New sensitive mass spectrometric methods are needed to screen for previously unidentified modified bases to allow for identification of adducts of multiple types, potentially leading to the identification of base modification patterns in DNA from human tissues. Liquid chromatography-mass spectrometry (LC-MS) is now the method of choice for characterization and quantitation of DNA adducts in tissue samples. We have developed a unique LC-High Resolution-MSn-based DNA adductomics approach which has the potential to identify previously uncharacterized DNA adducts in human tissues because it possesses the required combination of sensitivity, specificity, and generation of chemical information that is absent from previous MS techniques. DNA hydrolysis produces deoxynucleosides (dR) which upon ionization can undergo fragmentation resulting in loss of deoxyribose, resulting in an overall [M+H - 116.0474]+ ion. This universally observed fragmentation can be used to identify the analyte as a dR. The methodology described here uses the appearance of this fragment ion at high mass tolerance to trigger a second fragmentation step (MS3) leading to production of fragment ion spectra which provide additional structural information. An additional fragmentation step (MS4) is triggered for those MS3 spectra, which contain ions corresponding to one of the four nucleobases to provide confirmation of base identity. The final output is an LC chromatogram of DNA adducts in which each peak provides an accurate mass sufficient for unambiguous molecular formula determination and extensive fragmentation data potentially leading to its structural identification. We performed preliminary experiments to test the performance of our approach on mixtures of 18 DNA adducts selected to represent modifications of all four nucleobases and modifications at various reactive sites and with differing polarity. All 18 DNA adducts showed clear, sharp and symmetric peaks in the full scan and [M+H - 116.0474]+ ions upon MS2 fragmentation which then triggered the MS3 event on the product ions. The fragmentation spectrum of each adduct contained the ion corresponding to the mass of the base it was derived from and triggered the fragmentation of the base ion in the MS4 event. To test the effect of a real sample matrix on DNA adduct detection, the mixture of 18 DNA adducts was added to a human leukocyte DNA sample which had previously been passed through the purification and enrichment steps used for our LC-MS/MS analysis of N2-ethyl-dGuo. All 18 DNA adduct standards were detected as clear and sharp peaks. These results clearly demonstrate the potential of this method for DNA adduct detection and identification in human tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4791. doi:1538-7445.AM2012-4791

Investigating the discriminatory potential of urinary DNA adductomics in smokeless tobacco-treated rats and head-neck cancer patients

Environmental carcinogens exert their carcinogenic effects by forming DNA adducts. This type of DNA damage can also be formed endogenously as a result of, e.g., oxidative damage. Unrepaired DNA adducts may induce mutations in critical genes, leading to the initiation of chemical carcinogenesis. Therefore, detection, identification, and quantification of DNA adducts is essential for cancer risk assessment. Over the last 50 years, the major DNA adducts formed by different classes of environmental carcinogens were characterized. With the development of techniques such as 32P-postlabeling, their measurement was implemented into molecular epidemiology. Advances in liquid chromatography-tandem mass spectrometry (LC-MS ) made the measurement of adducts more precise and allowed to gain knowledge about their identity and structures. Therefore, opened the way to DNA adductomics, the "omics" approach investigating DNA adducts comprehensively, similarly to proteomics. This review presents the historical perspective of DNA adducts research and the emerging field of adductomics.

The formation of DNA adducts and the subsequent generation of mutations in the genome is believed to be a pivotal event in the pathogenesis of cancer, but several obstacles have hampered a concrete demonstration of the molecular species, quantity, and significance of DNA adducts in human tissues. For the last few years, we have attempted to demonstrate the existence of multiple DNA adducts in human tissues using a single experiment involving liquid chromatography tandem mass spectrometry. This procedure discloses a hundred to a few hundred peaks with distinct mass/charge and retention times in each sample. In previous experiments, we validated the presence of some tobacco-related and other DNA adducts in the lungs of a smoker and a non-smoker. Recently, we demonstrated the presence of lipid peroxidation-induced DNA adducts in several autopsy tissues including the lung, liver, pancreas, and spleen. In this presentation, we would like to show an adductome map for two regions of the human stomach in which the prevalence of gastric cancer differs. The DNA samples were obtained under anti-oxidation conditions from the gastric mucosa from regions adjacent to, but distant from, the cancer lesion in 22 patients with gastric cancer (12 cases in China and 10 cases in Japan) who underwent a gastrectomy. We identified 141 peaks in the Japanese sample and 159 peaks in the Chinese sample. Ninety-two of these peaks were present in both the Japanese and Chinese samples. Seven lipid peroxidation-related DNA adducts (1,N6-etheno-2′-deoxyadenosine [ϵdA], butanone-etheno-2′-deoxycytidine [BϵdC], butanone-etheno-2′-deoxy-5-methylcytidine [BϵmedC], butanone-etheno-2′-deoxyadenosine [BϵdA], heptanone-etheno-2′-deoxycytidine [HϵdC], heptanone-etheno-2′-deoxyadenosine [HϵdA], and heptanone-etheno-2′-deoxyguanosine [HϵdG]) were identified in a total of 22 gastric mucosa samples. The levels of these adducts ranged from 0 to 30,000 per 109 bases, and the levels of BedC, BedA, and HedA were higher in the Japanese gastric mucosa samples. Next, the mutation spectrum for p53 in gastric cancer tissues from the same hospital in China was investigated and compared with that of gastric cancer tissues obtained in Japan. The prevalence of a G-to-A transition in a non-GC area was higher in those derived from China; that is, G-to-A transitions in a GC rich area, which has been assumed to be related to endogenous inflammation, were more prevalent in the Japanese gastric cancer specimens. We interpreted these results as indicating that inflammation can mediate carcinogenesis via lipid peroxidation-induced adducts, and the mutation process that may consequently arise may play a greater role in Japanese gastric cancers than in Chinese gastric cancers. To delve into the mechanism of the cellular reactions to these adducts, oligonucleotides containing ϵdA, ϵdC, BϵdC, BϵdG, HϵdC, and HϵdG were synthesized and used as substrates for a gel retardation assay using the recombinant proteins of 8 different base excision repair genes. In contrast to their considerable capacity to repair smaller modified bases, such as ϵdA and ϵdC, the base excision repair gene products seemed to be less capable of repairing the larger modified bases BϵdC, BϵdG, HϵdC, and HϵdG, except for a few combinations. While a comprehensive understanding of the modifications of DNA bases, both endogenous and exogenous, and the corresponding host machineries might seem like a distant goal and an adductome approach is admittedly at a burgeoning stage, combing every un-annotated spot in an adductome map could help to realize a comprehensive understanding of the effects of exposures of various kinds. Citation Format: Haruhiko Sugimura, Hong Tao, Nobuya Kurabe, Masanori Goto, Yoshitaka Matsushima, Hidetaka Yamada, Kazuya Shinmura, Yohei Miyagi, Akira Tsuburaya, Takaki Yoshikawa, Yukari Totsuka, Hitoshi Nakagama, Yaping Wang, Tomonari Matsuda. DNA adductome: An ultimate exposome of human tissues. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 47.

Mass spectrometric analysis of sulfur mustard-induced biomolecular adducts: Are DNA adducts suitable biomarkers of exposure?

Quinones are potent genotoxic agents through generating reactive oxygen species or undergo covalent bonding to modify the nucleobases of DNA. The formation of DNA adducts is an early indication of cancer, as it may lead to gene mutation, thereby initiating tumor development. The present review aims to summarize different types of quinone-induced DNA damages and the corresponding DNA adducts. In addition to the considerably stable naphthoquinone and halobenzoquinones in the environment, quinones are also metabolically activated from estrogen, bisphenol A, ochratoxin A, polychlorinated biphenyls, benzo[a]pyrene and polybrominated diphenyl ethers, in which the inducement from these pollutants to produce DNA adducts were systematically discussed. Liquid chromatography-mass spectrometry (LC-MS)-based method with high accuracy and sensitivity for the detection of DNA adducts in mononucleotides, DNA duplexes, and biological samples was methodically summarized. It is believed that the presence of dione moiety in quinones is an imperative element that induce the formation of DNA adducts. The investigation on DNA adducts is helpful for understanding the generation, transformation, and repair of DNA damages, and for elucidating the underlying mechanism of carcinogenesis. This review provides a new perspective to assess the toxicological potential of quinones, and intends to put forward strategies of analyzing DNA adducts, which act as biomarkers, for indicating the potential health risks of multiDNA damages.

ABSTRACTOccupational exposure of workers to 1-bromopropane (1-BP) has raised concerns in industry for many years. Despite the known toxicity of this chemical, molecular events attributed to exposure to 1-BP have not been extensively studied. The aim of the present study was to examine the effects of 1-BP exposure on adduct formation with DNA and glutathione (GSH) in male Sprague–Dawley rats in an attempt to determine the early stages of toxicity. Following 6 h after either single or daily exposure to 1-BP for 3 days, N7-propyl guanine and S-propyl GSH were quantified in several organs by using liquid chromatography-mass spectrometry (LC-MS/MS). The results showed that N7-propyl guanine was maximally formed in liver followed by spleen, testes, and lung in both dose- and time-dependent manners. However, DNA adduct was not detected in cardiac tissue. In the case of S-propyl GSH, this compound was formed in the following order in various organs: liver > testes > spleen > kidney > lung > heart. In a subsequent in vitro study, formation of N7-propyl guanine initiated by 1-BP in calf thymus DNA was not markedly affected by addition of liver homogenates, which indicated that this chemical may be acting as a direct alkylating agent. In contrast, an in vitro study with free GSH demonstrated that 1-BP reduced GSH and elevated production of S-propyl GSH, and that the production of this adduct was significantly higher in the presence of active liver homogenates. Data indicated that formation of GSH adducts initiated by 1-BP might be associated with an enzyme-driven process. Although further characterization is necessary, it would appear that N7-propyl guanine and S-propyl GSH might serve as useful markers in cases of exposure assessment of 1-BP.

The measurement of DNA adducts provides important information about human exposure to genotoxic chemicals and can be employed to elucidate mechanisms of DNA damage and repair. DNA adducts can serve as biomarkers for interspecies comparisons of the biologically effective dose of procarcinogens and permit extrapolation of genotoxicity data from animal studies for human risk assessment. One major challenge in DNA adduct biomarker research is the paucity of fresh frozen biopsy samples available for study. However, archived formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis of disease are often available. We have established robust methods to recover DNA free of crosslinks from FFPE tissues under mild conditions which permit quantitative measurements of DNA adducts by liquid chromatography-mass spectrometry. The technology is versatile and can be employed to screen for DNA adducts formed with a wide range of environmental and dietary carcinogens, some of which were retrieved from section-cuts of FFPE blocks stored at ambient temperature for up to nine years. The ability to retrospectively analyze FFPE tissues for DNA adducts for which there is clinical diagnosis of disease opens a previously untapped source of biospecimens for molecular epidemiology studies that seek to assess the causal role of environmental chemicals in cancer etiology.