DNA modifications: Biomarkers for the exposome?

The concept of the exposome is the encompassing of all the environmental exposures, both exogenous and endogenous, across the life course. Many, if not all, of these exposures can result in the generation of reactive species, and/or the modulation of cellular processes, that can lead to a breadth of modifications of DNA, the nature of which may be used to infer their origin. Because of their role in cell function, such modifications have been associated with various major human diseases, including cancer, and so their assessment is crucial. Historically, most methods have been able to only measure one or a few DNA modifications at a time, limiting the information available. With the development of DNA adductomics, which aims to determine the totality of DNA modifications, a far more comprehensive picture of the DNA adduct burden can be gained. Importantly, DNA adductomics can facilitate a "top-down" investigative approach whereby patterns of adducts may be used to trace and identify the originating exposure source. This, together with other 'omic approaches, represents a major tool for unraveling the complexities of the exposome and hence allow a better a understanding of the environmental origins of disease.

A Novel Adductomics Workflow Incorporating FeatureHunter Software: Rapid Detection of Nucleic Acid Modifications for Studying the Exposome.

Exposure to the physicochemical agents that interact with nucleic acids (NA) may lead to modification of DNA and RNA (i.e., NA modifications), which have been associated with various diseases, including cancer. The emerging field of NA adductomics aims to identify both known and unknown NA modifications, some of which may also be associated with proteins. One of the main challenges for adductomics is the processing of massive and complex data generated by high-resolution tandem mass spectrometry (HR-MS/MS). To address this, we have developed a software called "FeatureHunter", which provides the automated extraction, annotation, and classification of different types of key NA modifications based on the MS and MS/MS spectra acquired by HR-MS/MS, using a user-defined feature list. The capability and effectiveness of FeatureHunter was demonstrated by analyzing various NA modifications induced by formaldehyde or chlorambucil in mixtures of calf thymus DNA, yeast RNA and proteins, and by analyzing the NA modifications present in the pooled urines of smokers and nonsmokers. The incorporation of FeatureHunter into the NA adductomics workflow offers a powerful tool for the identification and classification of various types of NA modifications induced by reactive chemicals in complex biological samples, providing a valuable resource for studying the exposome.

Genetic and epigenetic instability induced by betel quid associated chemicals.

Over the years, betel quid chewing and tobacco use have attracted considerable interest as they are implicated as the most likely causative risk factors of oral and esophageal cancers. Although areca nut use and betel quid chewing may lead to apoptosis, chronic exposure to areca nut and slaked lime may promote pre-malignant and malignant transformation of oral cells. The putative mutagenic and carcinogenic mechanisms may involve endogenous nitrosation of areca and tobacco alkaloids as well as the presence of direct alkylating agents in betel quid and smokeless tobacco. Metabolic activation of carcinogenic N-nitrosamines by phase-I enzymes is required not only to elicit the genotoxicity via the reactive intermediates but also to potentiate the mutagenicity with the sporadic alkylations of nucleotide bases, resulting in the formation of diverse DNA adducts. Persistent DNA adducts provides the impetus for genetic and epigenetic lesions. The genetic and epigenetic factors cumulatively influence the development and progression of disorders such as cancer. Accumulation of numerous genetic and epigenetic aberrations due to long-term betel quid (with or without tobacco) chewing and tobacco use culminates into the development of head and neck cancers. We review recent evidence that supports putative mechanisms for mutagenicity and carcinogenicity of betel quid chewing along with tobacco (smoking and smokeless) use. The detailed molecular mechanisms of the extent of accumulation and patterns of genetic alterations, indicative of the prior exposure to carcinogens and alkylating agents because of BQ chewing and tobacco use, have not yet been elucidated.

Mixture analysis on associations between semen quality and sperm DNA integrity and occupational exposure to polycyclic aromatic hydrocarbons.

The objective of this study was to assess relationships between exposure to PAHs at occupational levels and outcomes of human semen quality and sperm DNA integrity. Personal breathing zone air samples were collected to quantify exposure of 16 targeted PAHs to coke-oven workers at a steel company in southern Taiwan. Semen quality, including concentration, motility, morphology, and viability, were assessed. Sperm DNA fragmentation, 8-oxodGuo, bulky PAH adducts, and benzo[a]pyrene diol epoxide-DNA adducts served as biomarkers for assessment of sperm DNA integrity. The Bayesian Kernel Machine Regression modeling was employed to estimate mixture effects of the PAH mixture on the outcomes of semen quality and sperm DNA integrity and to identify individual compounds of PAH mixtures associated with the mixture effects. Exposure to the PAH mixture was inversely associated with sperm viability, while benzo(b)fluoranthene (B[b]F) was identified as the main predictor for sperm viability. Exposure to the PAH mixture also exhibited a positive trend with sperm DNA fragmentation. B[b]F and benzo(a)anthracene (B[a]A) were identified as individual PAH compounds associated with increased sperm DNA fragmentation.

Connecting chemical exposome to human health using high-resolution mass spectrometry-based biomonitoring: Recent advances and future perspectives.

Compared with the rapid advances in genomics leading to broad understanding of human disease, the linkage between chemical exposome and diseases is still under investigation. High-resolution mass spectrometry (HRMS) is expected to accelerate the process via relatively accurate and precise biomonitoring of human exposome. This review covers recent advancements in biomonitoring of exposed environmental chemicals (chemical exposome) using HRMS described in the 124 articles that resulted from a systematic literature search on Medline and Web of Science databases. The analytical strategic aspects, including the selection of specimens, sample preparation, instrumentation, untargeted versus targeted analysis, and workflows for MS-based biomonitoring to explore the environmental chemical space of human exposome, are deliberated. Applications of HRMS in human exposome investigation are presented by biomonitoring (1) exposed chemical compounds and their biotransformation products; (2) DNA/protein adducts; and (3) endogenous compound perturbations. Challenges and future perspectives are also discussed.

Nucleic acid adductomics - The next generation of adductomics towards assessing environmental health risks.

This Discussion article aims to explore the potential for a new generation of assay to emerge from cellular and urinary DNA adductomics which brings together DNA-RNA- and, to some extent, protein adductomics, to better understand the role of the exposome in environmental health. Components of the exposome have been linked to an increased risk of various, major diseases, and to identify the precise nature, and size, of risk, in this complex mixture of exposures, powerful tools are needed. Modification of nucleic acids (NA) is a key consequence of environmental exposures, and a goal of cellular DNA adductomics is to evaluate the totality of DNA modifications in the genome, on the basis that this will be most informative. Consequently, an approach which encompasses modifications of all nucleic acids (NA) would be potentially yet more informative. This article focuses on NA adductomics, which brings together the assessment of both DNA and RNA modifications, including modified (2'-deoxy)ribonucleosides (2'-dN/rN), modified nucleobases (nB), plus: DNA-DNA, RNA-RNA, DNA-RNA, DNA-protein, and RNA-protein crosslinks (DDCL, RRCL, DRCL, DPCL, and RPCL, respectively). We discuss the need for NA adductomics, plus the pros and cons of cellular vs. urinary NA adductomics, and present some evidence for the feasibility of this approach. We propose that NA adductomics provides a more comprehensive approach to the study of nucleic acid modifications, which will facilitate a range of advances, including the identification of novel, unexpected modifications e.g., RNA-RNA, and DNA-RNA crosslinks; key modifications associated with mutagenesis; agent-specific mechanisms; and adductome signatures of key environmental agents, leading to the dissection of the exposome, and its role in human health/disease, across the life course.

Elevated Nitrite/Nitrate Ratio as a Potential Biomarker for the Differential Diagnosis of Pleural Effusions.

Pleural effusions (PEs) are common in clinical practice and can be due to many different underlying diseases such as cancer, congestive heart failure, or pneumonia. An accurate differential diagnostic categorization is essential, as the treatment and prognosis of PEs largely depend on its cause. In this study, we tested the hypothesis that nitrite and nitrate concentrations in PEs are associated with the inflammation and infection conditions. We therefore measured the nitrite and nitrate levels in 143 PE samples using a sensitive liquid chromatography-tandem mass spectrometry method and investigated their diagnostic potential in differentiating PEs. The results showed that nitrite concentrations and nitrite/nitrate ratios were higher in exudates than in transudates (NO2-: 2.12 vs. 1.49 µM; NO2-/NO3-: 23.3 vs. 14.0). Both the nitrite concentrations and the nitrite/nitrate ratios were positively correlated with the three Light's criteria. Moreover, the receiver operating characteristic curve analysis revealed that the nitrite/nitrate ratio with an area under the curve of 0.71 could be a potential diagnostic biomarker in separating infectious PEs (IPEs) from other types of PEs. Taken together, the nitrite/nitrate ratio not only reflected the statuses of inflammation, but also the nitrate reduction by pathogenic bacteria infection in the pleural cavity. The nitrite/nitrate ratio could be a better biomarker in the differential diagnosis of PEs than the nitrite concentration alone.

Validation of a high-throughput method for simultaneous determination of areca nut and tobacco biomarkers in hair using microwave-assisted extraction and isotope dilution liquid chromatography tandem mass spectrometry.

For people with habits of chewing betel nuts and smoking, the probability of suffering from oral cancer is ten to a hundred times higher than others. Due to the serious health consequences of areca nut and tobacco, a reliable cessation program is needed. Hair is the best option to document long-term exposure. Unfortunately, the research on betel nut in hair did not attract much attention. In this study, a high-throughput method based on microwave-assisted extraction (MAE) and isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed to measure the four biomarkers of betel nuts and cigarettes, including areca alkaloids (arecoline), tobacco alkaloids (nicotine), and their metabolites (arecaidine and cotinine). The hair sample was washed, cut, weighed, and incubated for 3 min MAE with methanol/trifluoroacetic acid, then evaporated and reconstituted for LC-MS/MS analysis. The total experiment time was 50 min. The lower limits of quantification (LOQ) were 5-10 pg/mg. The intra-day and inter-day precision were 2.2-7.6%. Intra-day and inter-day accuracy were - 6.1-8.2%. The method showed good linearity (r2 > 0.995) over LOQ - 1000 pg/mg concentration ranges. It was successfully applied to analyze 11 subjects of regular areca nut chewers, also smokers. Eight samples were black hair; three samples were naturally black hair with partially gray hair. Measured concentrations in black hair were in the range 56.9 pg/mg to 3.2 ng/mg for arecoline, 12.8 pg/mg to 222.2 pg/mg for arecaidine, 3.8 ng/mg to 33.4 ng/mg for nicotine and 1.1 ng/mg to 6.1 ng/mg for cotinine. The results showed lower levels in gray hair. This method was utilized successfully to analyze pg/mg levels of arecoline, arecaidine, nicotine, and cotinine, and good recoveries were obtained. The mean concentration of arecaidine and cotinine in hair was 15% and 20% of arecoline and nicotine, respectively. A good positive correlation was found between the concentrations of these compounds and self-report. This method improved extraction speed, concentration, and analysis of samples and is useful for monitoring betel nut and smoking cessation programs.

Association between oxidized nucleobases and mitochondrial DNA damage with long-term mortality in patients with sepsis.

BACKGROUND: Sepsis not only leads to short-term mortality during hospitalization, but is also associated with increased mortality during long-term follow-up after hospital discharge. Metabolic stress during sepsis may cause oxidative damage to both nuclear and mitochondrial DNA (mtDNA) and RNA, which could affect long-term health and life span. Therefore, the aim of this study was to assess the association of sepsis with oxidized nucleobases and (mt)DNA damage and long-term all-cause mortality in septic patients. METHODS: 91 patients with sepsis who visited the emergency department (ED) of the University Medical Center Groningen between August 2012 and June 2013 were included. Urine and plasma were collected during the ED visit. Septic patients were matched with 91 healthy controls. Death rate was obtained until June 2020.The degree of oxidation of DNA, RNA and free nucleobases were assessed in urine by mass-spectrometry. Lipid peroxidation was assessed in plasma using a TBAR assay. Additionally, plasma levels of mtDNA and damage to mtDNA were determined by qPCR. RESULTS: Sepsis patients denoted higher levels of oxidated DNA, RNA, free nucleobases and lipid peroxidation than controls (all p < 0.01). Further, sepsis patients displayed an increase in plasma mtDNA with an increase in mtDNA damage compared to matched controls (p < 0.01). Kaplan meier survival analyses revealed that high degrees of RNA- and nucleobase oxidation were associated with higher long-term all-cause mortality after sepsis (p < 0.01 and p = 0.01 respectively). Of these two, high RNA oxidation was associated with long-term all-cause mortality, independent of adjustment for age, medical history and sepsis severity (HR 1.29 [(1.17-1.41, 95% CI] p < 0.01). CONCLUSIONS: Sepsis is accompanied with oxidation of nuclear and mitochondrial DNA and RNA, where RNA oxidation is an independent predictor of long-term all-cause mortality. In addition, sepsis causes mtDNA damage and an increase in cell free mtDNA in plasma.

Simultaneous determination of areca nut- and tobacco-specific alkaloids in saliva by LC-MS/MS: Distribution and transformation of alkaloids in oral cavity.

Areca nut and tobacco are frequently used in combination. Cigarette smoking and betel quid (BQ) chewing habits impose greater oral cancer risk than either habit alone. Saliva is a better noninvasive diagnostic material as it is in direct contact with oral mucosa and cancerous lesions. This study describes the application of isotope-dilution LC-MS/MS for simultaneous quantitation of five areca nut-specific alkaloids (ASAs) and three tobacco-specific alkaloids (TSAs) in human saliva. With this method, we demonstrate that the distribution of ASAs vary significantly in smokers who chew BQ habitually, due to the hydrolysis of ASAs and metabolic activity in the oral cavity. The alkaline condition formed due to slaked lime in BQ, plays an important role in the distribution of ASAs and TSAs, by catalyzing the hydrolysis of ester forms of ASAs to their respective carboxylic acid forms besides facilitating the TSA (i.e., nicotine) absorption in the oral cavity. Moreover, our results reveal that oral mucosa rather than saliva contributes to the metabolism of ASAs at oral cavity. Less than 2.1% of ASAs were metabolized by saliva, as determined by in vitro test. Our findings may provide a better insight into the pathobiology of oral carcinogenesis due to BQ chewing.

Is high resolution a strict requirement for mass spectrometry-based cellular DNA adductomics?

Exposure to endogenous and exogenous factors can result in the formation of a wide variety of DNA adducts, and these may lead to gene mutations, thereby contributing to the development of cancer. DNA adductomics, a novel tool for exposomics, aims to detect the totality of DNA adducts. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) is the state-of-the-art method for DNA adductomic analysis, although its high cost has limited widespread use. In this study, we compared the analytical performance between HRMS and the more popular/accessible triple-quadrupole MS (QqQ-MS). We initially developed and optimized a hybrid quadrupole-linear ion trap-orbitrap MS (Q-LIT-OT-MS) method, considering the detection of both purine and pyrimidine adducts. LC-Q-LIT-OT-MS and LC-QqQ-MS methods were compared by non-targeted screening of formaldehyde-induced DNA adducts. Using the results from Q-LIT-OT-MS as the gold standard, QqQ-MS successfully detected 12 out of 18 formaldehyde-induced DNA adducts/inter-strand crosslinks (ICLs). QqQ-MS however also produced nine false-positive results owing to the inherent instrumental mass resolution limits. To discriminate the false-positive results from the accurate ones, we firstly introduced a statistical analysis, partial least squares-discriminant analysis, which efficiently excluded the false signals. Six DNA adducts/ICLs were not detected by QqQ-MS, due to insufficient sensitivity. This could be overcome by employing a selected reaction monitoring scan mode with multiple injections. Overall, this study demonstrated that high resolution may not be a strict requirement for MS-based DNA adductomics. LC-QqQ-MS with statistical analysis, could also provide a comparable performance as HRMS for pre-screening purposes.

Global DNA methylation levels in white blood cells of patients with chronic heroin use disorder. A prospective study.

BACKGROUND: Increasing scientific evidence shows the significant role of epigenetic mechanisms in drug use disorder, abstinence and relapse. Studies on human subjects are limited compared to those on animals, for various reasons such as poly-substance abuse, high drop-out rate and technical difficulties. OBJECTIVES: Our goal was to evaluate whether a monitored abstinence period of 21 days could induce changes in global DNA methylation in chronic heroin users. METHOD: In the current study, we present data on global DNA methylation on a set of 18 male patients with chronic heroin use disorder, carefully selected based on inclusion and exclusion criteria, who were hospitalized and closely monitored during a 21-day detoxification program, one of the few where no opioid agonist is administered. The participants were sampled twice, once upon enrolment to the program and once upon completion. RESULTS: According to our results, no difference in global DNA methylation was detected between samples collected upon enrolment and samples collected upon completion of the program. CONCLUSION: The findings of this study do not rule out the possibility that the 21-day abstinence period was not long enough to observe changes in global DNA methylation, or that abstinence induced site-specific methylation changes (but not global changes), that certainly merit further evaluation.

Biomarkers of nucleic acid oxidation - A summary state-of-the-art.

Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2'-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2'-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 µL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo, but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease.

Markers of oxidative stress and toxicant exposure among young waterpipe smokers in the USA.

Waterpipe (aka hookah) tobacco smokers are exposed to toxicants that can lead to oxidative DNA and RNA damage, a precursor to chronic disease formation. This study assessed toxicant exposure and biomarkers of DNA [8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG)] and RNA [8-oxo-7,8-dihydroguanosine (8-oxoGuo)] oxidative damage during smoking of flavored and non-flavored waterpipe tobacco. Thirty waterpipe smokers completed two counterbalanced 2-h lab waterpipe smoking sessions (flavored vs. non-flavored waterpipe tobacco). Urinary concentrations of 8-oxodG and 8-oxoGuo and expired carbon monoxide (eCO) were measured before and after the smoking sessions. A significant increase in the urinary concentrations of 8-oxodG (from 2.12 ± 0.83 to 2.35 ± 0.91 ng/mg creatinine, p = 0.024) and 8-oxoGuo (from 2.96 ± 0.84 to 3.45 ± 0.76 ng/mg creatinine, p = 0.003) were observed after smoking the non-flavored and flavored waterpipe tobacco, respectively. Our results also showed that the mean ± SD of eCO increased significantly after smoking the flavored (from 1.3 ± 1.1 to 20.3 ± 23.6 ppm, p < 0.001) and non-flavored waterpipe tobacco (from 1.8 ± 1.2 to 24.5 ± 26.1 ppm, p < 0.001). There were no significant differences in the means of 8-oxodG (p = 0.576), 8-oxoGuo (p = 0.108), and eCO (p = 0.170) between the flavored and non-flavored tobacco sessions. Smoking non-flavored and flavored waterpipe tobacco leads to oxidative stress and toxicant exposure. Our findings add to the existing evidence about the adverse effects of waterpipe tobacco smoking (WTS) and the need for strong policies to inform and protect young people from the risks of WTS.

Alkylating and oxidative stresses in smoking and non-smoking patients with COPD: Implications for lung carcinogenesis.

Chronic obstructive pulmonary disease (COPD) is a disease characterized by chronic inflammation and irreversible airway obstruction. Cigarette smoking is the predominant risk factor for developing COPD. It is well-known that the COPD is also strongly associated with an increased risk of developing lung cancer. Cigarette smoke contains elevated concentrations of oxidants and various carcinogens (e.g., tobacco-derived nitrosamines) that can cause oxidative and alkylating stresses, which can also arise from inflammation. However, it is surprising that, except for oxidative stress, little information is available on the burden of alkylating stress and the detoxification efficiency of tobacco-derived carcinogens in COPD patients. In this study, we used LC-MS/MS to measure the archetypical tobacco-specific carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), three biomarkers of oxidative stress (8-oxo-7,8-dihydroguanine, 8-oxoGua; 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodGuo; 8-oxo-7,8-dihydroguanosine, 8-oxoGuo) and two biomarkers of alkylating stress (N7-methylguanine, N7-MeGua and N3-methyladenine, N3-MeAde), in the urine of smoking and non-smoking COPD patients and healthy controls. Our results showed that not only was oxidative stress significantly elevated in the COPD patients compared to the controls, but also alkylating stress. Significantly, levels of alkylating stress (i.e., N7-MeGua) were highly correlated with the COPD severity and not affected by age and smoking status. Furthermore, COPD smokers had significantly higher ratios of free NNAL to the total NNAL than control smokers, implying a lower detoxification efficiency of NNK in COPD smokers. This ratio was even higher in COPD smokers with stages 3-4 than in COPD smokers with stages 1-2. Taken together, our results demonstrated that the detoxification efficiency of tobacco-derived carcinogens (e.g., NNK) was associated with the pathogenesis and possibly the progression of COPD. In addition to oxidative stress, alkylating stress derived from chronic inflammation appears to be also dominant in COPD patients.

Influence of skin melanisation and ultraviolet radiation on biomarkers of systemic oxidative stress.

Skin melanisation ranges widely across human populations. Melanin has antioxidant properties and also acts as a filter to solar ultraviolet radiation (UVR) incident upon the skin. In this study we firstly examined whether melanin level might influence baseline levels of systemic oxidative stress, in 65 humans in vivo from the same geographical area ranging from the lightest to darkest skin type (phototype I-VI). This was examined in winter-time (latitude 53.5°N). Remarkably, we found that urinary biomarkers of oxidatively-generated DNA damage (8-oxodG) and RNA damage (8-oxoGuo) were significantly correlated with skin lightness (L*), such that 14-15% of the variation in their baseline levels could be explained by skin colour. Next we exposed 15 humans at the extremes of skin melanisation to a simulated summer-time exposure of solar UVR (95% UVA, 5% UVB; dose standardised to sunburn threshold), following which they provided a sample of every urine void over the next five days. We found that UVR induced a small but significant increase in urinary 8-oxodG and 8-oxoGuo, with differing kinetics between skin types. Thus greater melanisation is associated with protection against systemic oxidative stress, which may reflect melanin's antioxidant properties, and solar UVR exposure also influences systemic oxidative stress levels in humans. These novel findings may have profound implications for human physiology and health.

DNA Crosslinkomics: A Tool for the Comprehensive Assessment of Interstrand Crosslinks Using High Resolution Mass Spectrometry.

DNA-DNA crosslinks, especially interstrand crosslinks (ICLs), cause cytotoxicity via blocking replication and transcription. Most measurements of ICLs lack sensitivity and structural information. Here, a high resolution, accurate mass spectrometry (HRMS) method was developed to comprehensively determine the untargeted, totality of DNA crosslinks, a.k.a. DNA crosslinkomics. Two novel features were introduced into this method: the accurate mass neutral losses of both two 2-deoxyribose (dR) and one dR groups will screen for ICLs as modified dinucleosides; the accurate mass neutral losses of both of the two nucleobases and one nucleobase will detect unstable DNA crosslinks, that could undergo depurination. Our crosslinkomics approach was tested by screening for crosslinks in formaldehyde- and chlorambucil-treated calf thymus DNA. The results showed that all expected drug-bridged crosslinks were detected successfully, along with various unexpected crosslinks. Using HRMS, the molecular formula and chemical structures of these unexpected crosslinks were determined. The formation of apurinic/apyrimidinic (AP) site-derived crosslinks, at levels comparable to those for drug-bridged crosslinks, highlighted their novel, potential role in cytotoxicity. Our new crosslinkomics approach can detect expected and unexpected environmental and drug-induced crosslinks in biological samples. This broadens the existing cellular DNA adductome and offers the potential to become a powerful tool in precision medicine.