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.

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.

Identification of Formaldehyde-Induced DNA-RNA Cross-Links in the A/J Mouse Lung Tumorigenesis Model.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco products, and exposure to it is likely one of the factors contributing to the development of lung cancer in cigarette smokers. To exert its carcinogenic effects, NNK must be metabolically activated into highly reactive species generating a wide spectrum of DNA damage. We have identified a new class of DNA adducts, DNA-RNA cross-links found for the first time in NNK-treated mice lung DNA using our improved high-resolution accurate mass segmented full scan data-dependent neutral loss MS3 screening strategy. The levels of these DNA-RNA cross-links were found to be significantly higher in NNK-treated mice compared to the corresponding controls, which is consistent with higher levels of formaldehyde due to NNK metabolism as compared to endogenous levels. We hypothesize that this DNA-RNA cross-linking occurs through reaction with NNK-generated formaldehyde and speculate that this phenomenon has broad implications for NNK-induced carcinogenesis. The structures of these cross-links were characterized using high-resolution LC-MS2 and LC-MS3 accurate mass spectral analysis and comparison to a newly synthesized standard. Taken together, our data demonstrate a previously unknown link between DNA-RNA cross-link adducts and NNK and provide a unique opportunity to further investigate how these novel NNK-derived DNA-RNA cross-links contribute to carcinogenesis in the future.

Identification of new candidate biomarkers to support doxorubicin treatments in canine cancer patients.

BACKGROUND: Both human and veterinary cancer chemotherapy are undergoing a paradigm shift from a "one size fits all" approach to more personalized, patient-oriented treatment strategies. Personalized chemotherapy is dependent on the identification and validation of biomarkers that can predict treatment outcome and/or risk of toxicity. Many cytotoxic chemotherapy agents, including doxorubicin, base their mechanism of action by interaction with DNA and disruption of normal cellular processes. We developed a high-resolution/accurate-mass liquid chromatography-mass spectrometry DNA screening approach for monitoring doxorubicin-induced DNA modifications (adducts) in vitro and in vivo. We used, for the first time, a new strategy involving the use of isotope-labeled DNA, which greatly facilitates adduct discovery. The overall goal of this work was to identify doxorubicin-DNA adducts to be used as biomarkers to predict drug efficacy for use in veterinary oncology. RESULTS: We used our novel mass spectrometry approach to screen for adducts in purified DNA exposed to doxorubicin. This initial in vitro screening identified nine potential doxorubicin-DNA adduct masses, as well as an intense signal corresponding to DNA-intercalated doxorubicin. Two of the adduct masses, together with doxorubicin and its metabolite doxorubicinol, were subsequently detected in vivo in liver DNA extracted from mice exposed to doxorubicin. Finally, the presence of these adducts and analytes was explored in the DNA isolated from dogs undergoing treatment with doxorubicin. The previously identified nine DOX-DNA adducts were not detected in these preliminary three samples collected seven days post-treatment, however intercalated doxorubicin and doxorubicinol were detected. CONCLUSIONS: This work sets the stage for future evaluation of doxorubicin-DNA adducts and doxorubicin-related molecules as candidate biomarkers to personalize chemotherapy protocols for canine cancer patients. It demonstrates our ability to combine in one method the analysis of DNA adducts and DNA-intercalated doxorubicin and doxorubicinol. The last two analytes interestingly, were persistent in samples from canine patients undergoing doxorubicin chemotherapy seven days after treatment. The presence of doxorubicin in all samples suggests a role for it as a promising biomarker for use in veterinary chemotherapy. Future studies will involve the analysis of more samples from canine cancer patients to elucidate optimal timepoints for monitoring intercalated doxorubicin and doxorubicin-DNA adducts and the correlation of these markers with therapy outcome.

Human cancer genomes harbor the mutational signature of tobacco-specific nitrosamines NNN and NNK.

Tobacco usage is linked to multiple cancer types and accounts for a quarter of all cancer-related deaths. Tobacco smoke contains various carcinogenic compounds, including polycyclic aromatic hydrocarbons (PAH), though the mutagenic potential of many tobacco-related chemicals remains largely unexplored. In particular, the highly carcinogenic tobacco-specific nitrosamines NNN and NNK form pre-mutagenic pyridyloxobutyl (POB) DNA adducts. In the study presented here, we identified genome-scale POB-induced mutational signatures in cell lines and rat tumors, while also investigating their role in human cancer. These signatures are characterized by T>N and C>T mutations forming from specific POB adducts damaging dT and dC residues. Analysis of 2,780 cancer genomes uncovered POB signatures in ∼180 tumors; from cancer types distinct from the ones linked to smoking-related signatures SBS4 and SBS92. This suggests that, unlike PAH compounds, the POB pathway may contribute uniquely to the mutational landscapes of certain hematological malignancies and cancers of the kidney, breast, prostate and pancreas.

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.