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.

Mass Spectral Library for DNA Adductomics.

Endogenous electrophiles, ionizing and non-ionizing radiation, and hazardous chemicals present in the environment and diet can damage DNA by forming covalent adducts. DNA adducts can form in critical cancer driver genes and, if not repaired, may induce mutations during cell division, potentially leading to the onset of cancer. The detection and quantification of specific DNA adducts are some of the first steps in studying their role in carcinogenesis, the physiological conditions that lead to their production, and the risk assessment of exposure to specific genotoxic chemicals. Hundreds of different DNA adducts have been reported in the literature, and there is a critical need to establish a DNA adduct mass spectral database to facilitate the detection of previously observed DNA adducts and characterize newly discovered DNA adducts. We have collected synthetic DNA adduct standards from the research community, acquired MSn (n = 2, 3) fragmentation spectra using Orbitrap and Quadrupole-Time-of-Flight (Q-TOF) MS instrumentation, processed the spectral data and incorporated it into the MassBank of North America (MoNA) database, and created a DNA adduct portal Web site (https://sites.google.com/umn.edu/dnaadductportal) to serve as a central location for the DNA adduct mass spectra and metadata, including the spectral database downloadable in different formats. This spectral library should prove to be a valuable resource for the DNA adductomics community, accelerating research and improving our understanding of the role of DNA adducts in disease.

Redox modulation of oxidatively-induced DNA damage by ascorbate enhances both in vitro and ex-vivo DNA damage formation and cell death in melanoma cells.

Elevated genomic instability in cancer cells suggests a possible model-scenario for their selective killing via the therapeutic delivery of well-defined levels of further DNA damage. To examine this scenario, this study investigated the potential for redox modulation of oxidatively-induced DNA damage by ascorbate in malignant melanoma (MM) cancer cells, to selectively enhance both DNA damage and MM cell killing. DNA damage was assessed by Comet and ɣH2AX assays, intracellular oxidising species by dichlorofluorescein fluorescence, a key antioxidant enzymatic defence by assessment of catalase activity and cell survival was determined by clonogenic assay. Comet revealed that MM cells had higher endogenous DNA damage levels than normal keratinocytes (HaCaT cells); this correlated MM cells having higher intracellular oxidising species and lower catalase activity, and ranked with MM cell melanin pigmentation. Comet also showed MM cells more sensitive towards the DNA damaging effects of exogenous H2O2, and that ascorbate further enhanced this H2O2-induced damage in MM cells; again, with MM cell sensitivity to induced damage ranking with degree of cell pigmentation. Furthermore, cell survival data indicated that ascorbate enhanced H2O2-induced clonogenic cell death selectively in MM cells whilst protecting HaCaT cells. Finally, we show that ascorbate serves to enhance the oxidising effects of the MM therapeutic drug Elesclomol in both established MM cells in vitro and primary cell cultures ex vivo. Together, these results suggest that ascorbate selectively enhances DNA damage and cell-killing in MM cells. This raises the option of incorporating ascorbate into clinical oxidative therapies to treat MM.

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.

Accumulation of 8-oxodG within the human mitochondrial genome positively associates with transcription.

Mitochondrial DNA (mtDNA) can be subject to internal and environmental stressors that lead to oxidatively generated damage and the formation of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG). The accumulation of 8-oxodG has been linked to degenerative diseases and aging, as well as cancer. Despite the well-described implications of 8-oxodG in mtDNA for mitochondrial function, there have been no reports of mapping of 8-oxodG across the mitochondrial genome. To address this, we used OxiDIP-Seq and mapped 8-oxodG levels in the mitochondrial genome of human MCF10A cells. Our findings indicated that, under steady-state conditions, 8-oxodG is non-uniformly distributed along the mitochondrial genome, and that the longer non-coding region appeared to be more protected from 8-oxodG accumulation compared with the coding region. However, when the cells have been exposed to oxidative stress, 8-oxodG preferentially accumulated in the coding region which is highly transcribed as H1 transcript. Our data suggest that 8-oxodG accumulation in the mitochondrial genome is positively associated with mitochondrial transcription.

Development of a Novel, Automated High-Throughput Device for Performing the Comet Assay.

A comet assay is a trusted and widely used method for assessing DNA damage in individual eukaryotic cells. However, it is time-consuming and requires extensive monitoring and sample manipulation by the user. This limits the throughput of the assay, increases the risk of errors, and contributes to intra- and inter-laboratory variability. Here, we describe the development of a device which automates high throughput sample processing for a comet assay. This device is based upon our patented, high throughput, vertical comet assay electrophoresis tank, and incorporates our novel, patented combination of assay fluidics, temperature control, and a sliding electrophoresis tank to facilitate sample loading and removal. Additionally, we demonstrated that the automated device performs at least as well as our "manual" high throughput system, but with all the advantages of a fully "walkaway" device, such as a decreased need for human involvement and a decreased assay run time. Our automated device represents a valuable, high throughput approach for reliably assessing DNA damage with the minimal operator involvement, particularly if combined with the automated analysis of comets.

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.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

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.

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage.

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

Perspectives on Cyclobutane Pyrimidine Dimers-Rise of the Dark Dimers.

Some early reports demonstrate that levels of cyclobutane pyrimidine dimers (CPD) may increase after UVR exposure had ended, although these observations were treated as artifacts. More recently, it has been shown unequivocally that CPD formation does occur post-irradiation, with maximal levels occurring after about 2-3 h. These lesions have been termed "dark CPD" (dCPD). Subsequent studies have confirmed their presence in vitro, in mouse models and in human skin in vivo. Melanin carbonyls have a role in the formation of dCPD, but they have also been observed in amelanotic systems, indicating other, unknown process(es) exist. In both cases, the formation of dCPD can be prevented by the presence of certain antioxidants. We lack data on the spectral dependence of dCPD, but it is unlikely to be the same as for incident CPD (iCPD), which are formed only during irradiation. There is evidence that iCPD and dCPD may have different repair kinetics, although this remains to be elucidated. It is also unknown whether iCPD and dCPD have different biological properties. The formation of dCPD in human skin in vivo has implications for post solar exposure photoprotection, and skin carcinogenesis, with a need for this to be investigated further.

Genome-wide mapping of genomic DNA damage: methods and implications.

Exposures from the external and internal environments lead to the modification of genomic DNA, which is implicated in the cause of numerous diseases, including cancer, cardiovascular, pulmonary and neurodegenerative diseases, together with ageing. However, the precise mechanism(s) linking the presence of damage, to impact upon cellular function and pathogenesis, is far from clear. Genomic location of specific forms of damage is likely to be highly informative in understanding this process, as the impact of downstream events (e.g. mutation, microsatellite instability, altered methylation and gene expression) on cellular function will be positional-events at key locations will have the greatest impact. However, until recently, methods for assessing DNA damage determined the totality of damage in the genomic location, with no positional information. The technique of "mapping DNA adductomics" describes the molecular approaches that map a variety of forms of DNA damage, to specific locations across the nuclear and mitochondrial genomes. We propose that integrated comparison of this information with other genome-wide data, such as mutational hotspots for specific genotoxins, tumour-specific mutation patterns and chromatin organisation and transcriptional activity in non-cancerous lesions (such as nevi), pre-cancerous conditions (such as polyps) and tumours, will improve our understanding of how environmental toxins lead to cancer. Adopting an analogous approach for non-cancer diseases, including the development of genome-wide assays for other cellular outcomes of DNA damage, will improve our understanding of the role of DNA damage in pathogenesis more generally.

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.

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.

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.

Towards a comprehensive view of 8-oxo-7,8-dihydro-2'-deoxyguanosine: Highlighting the intertwined roles of DNA damage and epigenetics in genomic instability.

8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a major product of DNA oxidation, is a pre-mutagenic lesion which is prone to mispair, if left unrepaired, with 2'-deoxyadenosine during DNA replication. While unrepaired or incompletely repaired 8-oxodG has classically been associated with genome instability and cancer, it has recently been reported to have a role in the epigenetic regulation of gene expression. Despite the growing collection of genome-wide 8-oxodG mapping studies that have been used to provide new insight on the functional nature of 8-oxodG within the genome, a comprehensive view that brings together the epigenetic and the mutagenic nature of the 8-oxodG is still lacking. To help address this gap, this review aims to provide (i) a description of the state-of-the-art knowledge on both the mutagenic and epigenetic roles of 8-oxodG; (ii) putative molecular models through which the 8-oxodG can cause genome instability; (iii) a possible molecular model on how 8-oxodG, acting as an epigenetic signal, could cause the translocations and deletions which are associated with cancer.

How Robust is the Evidence for a Role of Oxidative Stress in Autism Spectrum Disorders and Intellectual Disabilities?

Growing interest in the pathogenesis of autism spectrum disorders (ASDs) and other intellectual and developmental disabilities (IDD) has led to emerging evidence implicating a role for oxidative stress. However, understanding the strength of this association is made challenging by the use of a variety of purported biomarkers of oxidative stress, many of which have either uncertain specificity or flawed methods of analysis. This review aims to address this issue, which is widespread in the ASD and IDD literature, by providing readers with information concerning the strengths and limitations of the choice and analysis of biomarkers of oxidative stress. We highlight that biomarkers and assays should be specific, sensitive, reproducible, precise, robust, and chosen with careful consideration. Future studies should be sufficiently powered and address sample collection, processing, and storage which are, additionally, poorly considered, sources of bad practice, and potential errors. Only with these issues considered, will the data lead to conclusions as to the precise role of oxidative stress in ASDs and IDD.

Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results.

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.