Liquid Chromatography-Mass Spectrometry Screening of Cyclophosphamide DNA Damage In Vitro and in Patients Undergoing Chemotherapy Treatment.

DNA alkylating drugs have been used as frontline medications to treat cancer for decades. Their chemical reaction with DNA leads to the blockage of DNA replication, which impacts cell replication. While this impacts rapidly dividing cancerous cells, this process is not selective and results in highly variable and often severe side effects in patients undergoing alkylating-drug based therapies. The development of biomarkers to identify patients who effectively respond with tolerable toxicities vs patients who develop serious side effects is needed. Cyclophosphamide (CPA) is a commonly used chemotherapeutic drug and lacks biomarkers to evaluate its therapeutic effect and toxicity. Upon administration, CPA is metabolically activated and converted to phosphoramide mustard and acrolein, which are responsible for its efficacy and toxicity, respectively. Previous studies have explored the detection of the major DNA adduct of CPA, the interstrand DNA-DNA cross-link G-NOR-G, finding differences in the cross-link amount between Fanconi Anemia and non-Fanconi Anemia patients undergoing chemotherapy treatment. In this study, we take advantage of our DNA adductomic approach to comprehensively profile CPA's and its metabolites' reactions with DNA in vitro and in patients undergoing CPA-based chemotherapy. This investigation led to the detection of 40 DNA adducts in vitro and 20 DNA adducts in patients treated with CPA. Moreover, acrolein-derived DNA adducts were quantified in patient samples. The results suggest that CPA-DNA damage is very complex, and an evaluation of DNA adduct profiles is necessary when evaluating the relationship between CPA-DNA damage and patient outcome.

Characterization and quantitation of busulfan DNA adducts in the blood of patients receiving busulfan therapy.

DNA alkylating drugs have been used as cancer chemotherapy with variable outcomes. The establishment of predictive biomarkers to identify patients who will effectively respond to treatment would allow for the development of personalized therapies. As the degree of interaction of alkylating drug with DNA plays a key role in their mechanism of action, our hypothesis is that the measurement of the DNA adducts formed by alkylating drugs could be used to inform patient stratification. Beginning with busulfan, we took advantage of our DNA adductomic approach to characterize DNA adducts formed by reacting busulfan with calf-thymus DNA. Samples collected from six patients undergoing busulfan-based chemotherapy prior to allogeneic hematopoietic cell transplantation were analyzed for the presence of busulfan-derived DNA adducts. Among the 15 adducts detected in vitro, 12 were observed in the patient blood confirming the presence of a large profile of DNA adducts in vivo. Two of the detected adducts were structurally confirmed by comparison with synthetic standards and quantified in patients. These data confirm our ability to comprehensively characterize busulfan-derived DNA damage and set the stage for the development of methods to support personalized chemotherapy.

Identification of New Markers of Alcohol-Derived DNA Damage in Humans.

Alcohol consumption is a risk factor for the development of several cancers, including those of the head and neck and the esophagus. The underlying mechanisms of alcohol-induced carcinogenesis remain unclear; however, at these sites, alcohol-derived acetaldehyde seems to play a major role. By reacting with DNA, acetaldehyde generates covalent modifications (adducts) that can lead to mutations. Previous studies have shown a dose dependence between levels of a major acetaldehyde-derived DNA adduct and alcohol exposure in oral-cell DNA. The goal of this study was to optimize a mass spectrometry (MS)-based DNA adductomic approach to screen for all acetaldehyde-derived DNA adducts to more comprehensively characterize the genotoxic effects of acetaldehyde in humans. A high-resolution/-accurate-mass data-dependent constant-neutral-loss-MS3 methodology was developed to profile acetaldehyde-DNA adducts in purified DNA. This resulted in the identification of 22 DNA adducts. In addition to the expected N2-ethyldeoxyguanosine (after NaBH3CN reduction), two previously unreported adducts showed prominent signals in the mass spectra. MSn fragmentation spectra and accurate mass were used to hypothesize the structure of the two new adducts, which were then identified as N6-ethyldeoxyadenosine and N4-ethyldeoxycytidine by comparison with synthesized standards. These adducts were quantified in DNA isolated from oral cells collected from volunteers exposed to alcohol, revealing a significant increase after the exposure. In addition, 17 of the adducts identified in vitro were detected in these samples confirming our ability to more comprehensively characterize the DNA damage deriving from alcohol exposures.

Quantitative Liquid Chromatography-Nanoelectrospray Ionization-High-Resolution Tandem Mass Spectrometry Analysis of Acrolein-DNA Adducts and Etheno-DNA Adducts in Oral Cells from Cigarette Smokers and Nonsmokers.

Cigarette smoking is an important source of human exposure to toxicants and carcinogens and contributes significantly to cancer morbidity and mortality worldwide. Acrolein, a widespread environmental pollutant, is present in relatively high amounts in cigarette smoke and can react directly with DNA to form DNA adducts, which serve as important biomarkers for the assessment of exposure to acrolein and its potential role in smoking related cancer. Etheno-DNA adducts are promutagenic DNA lesions that can derive from exogenous chemicals as well as endogenous sources, including lipid peroxidation. In this study, we developed a combined method for the quantitation of (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8,-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)-one (α-OH-Acr-dGuo), (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8,-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dGuo), 1,N6-etheno-dAdo (εdAdo), and 3,N4-etheno-dCyd (εdCyd) adducts in oral rinse and cytobrush DNA from smokers and nonsmokers by liquid chromatography-nanoelelctrospray ionization-high-resolution tandem mass spectrometry (LC-NSI-HRMS/MS). For oral rinse samples, there was a statistically significant difference between the levels of α-OH-Acr-dGuo, γ-OH-Acr-dGuo, εdAdo, and εdCyd in smokers (12.1 ± 17.9, 163 ± 227, 182 ± 568, and 194 ± 400 adducts/109 nucleotides, respectively) and nonsmokers (1.85 ± 2.08, 5.95 ± 4.23, 7.69 ± 11.7, and 6.07 ± 10.9 adducts/109 nucleotides, respectively). For cytobrush samples, there was a statistically significant difference between the levels of γ-OH-Acr-dGuo and εdAdo in smokers (259 ± 540 and 82.9 ± 271 adducts/109 nucleotides, respectively) and nonsmokers (7.37 ± 5.09 and 16.2 ± 30.2 adducts/109 nucleotides, respectively) but not for α-OH-Acr-dGuo and εdCyd. Our results demonstrate that oral mucosa cells are an excellent source of material for evaluating DNA adducts to be used as biomarkers of tobacco smoke exposure and molecular changes potentially related to cancer.

Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca.

Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC50 = 29 ± 4 µM) through the inhibition of NpsA (KD = 29 ± 4 nM).

Nanoscale battery cathode materials induce DNA damage in bacteria.

The increasing use of nanoscale lithium nickel manganese cobalt oxide (Li x Ni y Mn z Co1-y-z O2, NMC) as a cathode material in lithium-ion batteries poses risk to the environment. Learning toxicity mechanisms on molecular levels is critical to promote proactive risk assessment of these complex nanomaterials and inform their sustainable development. We focused on DNA damage as a toxicity mechanism and profiled in depth chemical and biological changes linked to DNA damage in two environmentally relevant bacteria upon nano-NMC exposure. DNA damage occurred in both bacteria, characterized by double-strand breakage and increased levels of many putative chemical modifications on bacterial DNA bases related to direct oxidative stress and lipid peroxidation, measured by cutting-edge DNA adductomic techniques. Chemical probes indicated elevated intracellular reactive oxygen species and transition metal ions, in agreement with DNA adductomics and gene expression analysis. By integrating multi-dimensional datasets from chemical and biological measurements, we present rich mechanistic insights on nano-NMC-induced DNA damage in bacteria, providing targets for biomarkers in the risk assessment of reactive materials that may be extrapolated to other nano-bio interactions.

Targeted High Resolution LC/MS3 Adductomics Method for the Characterization of Endogenous DNA Damage.

DNA can be damaged through covalent modifications of the nucleobases by endogenous processes. These modifications, commonly referred to as DNA adducts, can persist and may lead to mutations, and ultimately to the initiation of cancer. A screening methodology for the majority of known endogenous DNA adducts would be a powerful tool for investigating the etiology of cancer and for the identification of individuals at high-risk to the detrimental effects of DNA damage. This idea led to the development of a DNA adductomic approach using high resolution data-dependent scanning, an extensive MS2 fragmentation inclusion list of known endogenous adducts, and neutral loss MS3 triggering to profile all DNA modifications. In this method, the detection of endogenous DNA adducts is performed by observation of their corresponding MS3 neutral loss triggered events and their relative quantitation using the corresponding full scan extracted ion chromatograms. The method's inclusion list consists of the majority of known endogenous DNA adducts, compiled, and reported here, as well as adducts specific to tobacco exposure included to compare the performance of the method with previously developed targeted approaches. The sensitivity of the method was maximized by reduction of extraneous background signal through the purification and minimization of the amount of commercially obtained enzymes used for the DNA hydrolysis. In addition, post-hydrolysis sample purification was performed using off-line HPLC fraction collection to eliminate the highly abundant unmodified bases, and to avoid introduction of plasticizers found in solid-phase extraction cartridges. Also, several instrument parameters were evaluated to optimize the ion signal intensities and fragmentation spectra quality. The method was tested on an animal model of lung carcinogenesis where A/J mice were exposed to the tobacco specific lung carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) with its effects enhanced by co-exposure to the pro-inflammatory agent lipopolysaccharide (LPS). Lung DNA were screened for endogenous DNA adducts known to result from oxidative stress and LPS-induced lipid peroxidation, as well as for adducts due to NNK exposure. The relative quantitation of the detected DNA adducts was performed using parallel reaction monitoring MS2 analysis, demonstrating a general workflow for analysis of endogenous DNA adducts.

Correction: Alessia Stornetta et al. Alcohol-Derived Acetaldehyde Exposure in the Oral Cavity. Cancers 2018, 10, 20.

The authors would like to make a correction to their published paper[...].

Alcohol-Derived Acetaldehyde Exposure in the Oral Cavity.

Alcohol is classified by the International Agency for Research on Cancer (IARC) as a human carcinogen and its consumption has been associated to an increased risk of liver, breast, colorectum, and upper aerodigestive tract (UADT) cancers. Its mechanisms of carcinogenicity remain unclear and various hypotheses have been formulated depending on the target organ considered. In the case of UADT cancers, alcohol's major metabolite acetaldehyde seems to play a crucial role. Acetaldehyde reacts with DNA inducing modifications, which, if not repaired, can result in mutations and lead to cancer development. Despite alcohol being mainly metabolized in the liver, several studies performed in humans found higher levels of acetaldehyde in saliva compared to those found in blood immediately after alcohol consumption. These results suggest that alcohol-derived acetaldehyde exposure may occur in the oral cavity independently from liver metabolism. This hypothesis is supported by our recent results showing the presence of acetaldehyde-related DNA modifications in oral cells of monkeys and humans exposed to alcohol, overall suggesting that the alcohol metabolism in the oral cavity is an independent cancer risk factor. This review article will focus on illustrating the factors modulating alcohol-derived acetaldehyde exposure and effects in the oral cavity.

A High Resolution/Accurate Mass (HRAM) Data-Dependent MS3 Neutral Loss Screening, Classification, and Relative Quantitation Methodology for Carbonyl Compounds in Saliva.

Reactive carbonyl compounds (RCCs) are ubiquitous in the environment and are generated endogenously as a result of various physiological and pathological processes. These compounds can react with biological molecules inducing deleterious processes believed to be at the basis of their toxic effects. Several of these compounds are implicated in neurotoxic processes, aging disorders, and cancer. Therefore, a method characterizing exposures to these chemicals will provide insights into how they may influence overall health and contribute to disease pathogenesis. Here, we have developed a high resolution accurate mass (HRAM) screening strategy allowing simultaneous identification and relative quantitation of DNPH-derivatized carbonyls in human biological fluids. The screening strategy involves the diagnostic neutral loss of hydroxyl radical triggering MS3 fragmentation, which is only observed in positive ionization mode of DNPH-derivatized carbonyls. Unique fragmentation pathways were used to develop a classification scheme for characterizing known and unanticipated/unknown carbonyl compounds present in saliva. Furthermore, a relative quantitation strategy was implemented to assess variations in the levels of carbonyl compounds before and after exposure using deuterated d 3 -DNPH. This relative quantitation method was tested on human samples before and after exposure to specific amounts of alcohol. The nano-electrospray ionization (nano-ESI) in positive mode afforded excellent sensitivity with detection limits on-column in the high-attomole levels. To the best of our knowledge, this is the first report of a method using HRAM neutral loss screening of carbonyl compounds. In addition, the method allows simultaneous characterization and relative quantitation of DNPH-derivatized compounds using nano-ESI in positive mode. Graphical Abstract ᅟ.