Interactions with DNA Models of the Oxaliplatin Analog (cis-1,3-DACH)PtCl2.

It is generally accepted that adjacent guanine residues in DNA are the primary target for platinum antitumor drugs and that differences in the conformations of the Pt-DNA adducts can play a role in their antitumor activity. In this study, we investigated the effect of the carrier ligand cis-1,3-diaminocyclohexane (cis-1,3-DACH) upon formation, stability, and stereochemistry of the (cis-1,3-DACH)PtG2 and (cis-1,3-DACH)Pt(d(GpG)) adducts (G = 9-EthlyGuanine, guanosine, 5'- and 3'-guanosine monophosphate; d(GpG) = deoxyguanosil(3'-5')deoxyguanosine). A peculiar feature of the cis-1,3-DACH carrier ligand is the steric bulk of the diamine, which is asymmetric with respect to the Pt-coordination plane. The (cis-1,3-DACH)Pt(5'GMP)2 and (cis-1,3-DACH)Pt(3'GMP)2 adducts show preference for the ΛHT and ∆HT conformations, respectively (HT stands for Head-to-Tail). Moreover, the increased intensity of the circular dichroism signals in the cis-1,3-DACH derivatives with respect to the analogous cis-(NH3)2 species could be a consequence of the greater bite angle of the cis-1,3-DACH carrier ligand with respect to cis-(NH3)2. Finally, the (cis-1,3-DACH)Pt(d(GpG)) adduct is present in two isomeric forms, each one giving a pair of H8 resonances linked by a NOE cross peak. The two isomers were formed in comparable amounts and had a dominance of the HH conformer but with some contribution of the ΔHT conformer which is related to the HH conformer by having the 3'-G base flipped with respect to the 5'-G residue.

An integrated LC-MS/MS platform for noninvasive urinary nucleus acid adductomics: A pilot study for tobacco exposure.

Tobacco smoke exposure significantly increases the level of global nucleoside damage. To evaluate all aspects of nucleic acid (NA) modifications, NA adductomics analyzes DNA, RNA and nucleobase adducts and provides comprehensive data. Liquid chromatography-tandem triple quadrupole mass spectrometry (LC-QQQ-MS/MS) and LC-Zeno-TOF-MS/MS were employed to screen for DNA, RNA and nucleobase adducts, as part of the analytical platform that was designed to combine high sensitivity and high resolution detection. We identified and distinguished urine nucleoside adducts via precursor ion and neutral loss scanning. A total of 245 potential adducts were detected, of which 28 were known adducts. The smoking group had significantly higher concentrations of nucleoside adducts in rat urine than the control group, based on MRM scanning, which was then used to perform relative quantitative analysis of these adducts. Urine nucleoside adducts were further confirmed using LC-Zeno-TOF-MS/MS. This highlights the potential of untargeted detection methods to provide comprehensive data on both known and unknown adducts. These approaches can be used to investigate the interactions among oxidative and alkylation stresses, and epigenetic modifications caused by exposure to tobacco smoke.

Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

Ultrafast Reaction of the Drug Hydralazine with Apurinic/Apyrimidinic Sites in DNA Gives Rise to a Stable Triazolo[3,4-a]phthalazine Adduct.

The clinically used antihypertensive agent hydralazine rapidly generates hydrazone-derived adducts by reaction with apurinic/apyrimidinic (also known as abasic or AP) sites in many different sequences of duplex DNA. The reaction rates are comparable to those of some AP-trapping reagents previously described as "ultrafast." Initially, reversible formation of a hydrazone adduct is followed by an oxidative cyclization reaction that generates a chemically stable triazolo[3,4-a]phthalazine adduct. The net result is that the reaction of hydralazine with AP sites in duplex DNA yields a rapid and irreversible adduct formation. Although the hydrazone and triazolo[3,4-a]phthalazine adducts differ by only two mass units, it was possible to use MALDI-TOF-MS and ESI-QTOF-nanospray-MS to quantitatively characterize mixtures of these adducts by deconvolution of overlapping isotope envelopes. Reactions of hydralazine with the endogenous ketone pyruvate do not prevent the formation of the hydralazine-AP adducts, providing further evidence that these adducts have the potential to form in cellular DNA. AP sites are ubiquitous in cellular DNA, and rapid, irreversible adduct formation by hydralazine could be relevant to the pathogenesis of systemic drug-induced lupus erythematosus experienced by some patients. Finally, hydralazine might be developed as a probe for the detection of AP sites, the study of cellular BER, and marking the location of AP sites in DNA-sequencing analyses.

Endogenous Cellular Metabolite Methylglyoxal Induces DNA-Protein Cross-Links in Living Cells.

Methylglyoxal (MGO) is an electrophilic α-oxoaldehyde generated endogenously through metabolism of carbohydrates and exogenously due to autoxidation of sugars, degradation of lipids, and fermentation during food and drink processing. MGO can react with nucleophilic sites within proteins and DNA to form covalent adducts. MGO-induced advanced glycation end-products such as protein and DNA adducts are thought to be involved in oxidative stress, inflammation, diabetes, cancer, renal failure, and neurodegenerative diseases. Additionally, MGO has been hypothesized to form toxic DNA-protein cross-links (DPC), but the identities of proteins participating in such cross-linking in cells have not been determined. In the present work, we quantified DPC formation in human cells exposed to MGO and identified proteins trapped on DNA upon MGO exposure using mass spectrometry-based proteomics. A total of 265 proteins were found to participate in MGO-derived DPC formation including gene products engaged in telomere organization, nucleosome assembly, and gene expression. In vitro experiments confirmed DPC formation between DNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as well as histone proteins H3.1 and H4. Collectively, our study provides the first evidence for MGO-mediated DNA-protein cross-linking in living cells, prompting future studies regarding the relevance of these toxic lesions in cancer, diabetes, and other diseases linked to elevated MGO levels.

Synthesis of N2-trans-isosafrole-dG-adduct Bearing DNAs and the Bypass Studies with Human TLS Polymerases κ and η.

Safrole is a natural product present in many plants and plant products, including spices and essential oils. During cellular metabolism, it converts to a highly reactive trans-isosafrole (SF) intermediate that reacts with genomic DNA and forms N2-SF-dG and N6-SF-dA DNA adducts, which are detected in the oral tissue of cancer patients with betel quid chewing history. To study the SF-induced carcinogenesis and to probe the role of low fidelity translesion synthesis (TLS) polymerases in bypassing SF adducts, herein, we report the synthesis of N2-SF-dG modified DNAs using phosphoramidite chemistry. The N2-SF-dG modification in the duplex DNA does not affect the thermal stability and retains the B-form of helical conformation, indicating that this adduct may escape the radar of common DNA repair mechanisms. Primer extension studies showed that the N2-SF-dG adduct is bypassed by human TLS polymerases hpolκ and hpolη, which perform error-free replication across this adduct. Furthermore, molecular modeling and dynamics studies revealed that the adduct reorients to pair with the incoming nucleotide, thus allowing the effective bypass. Overall, the results indicate that hpolκ and hpolη do not distinguish the N2-SF-dG adduct, suggesting that they may not be involved in the safrole-induced carcinogenicity.

Aristolochic Acid Exposure via Dermal Contact or Inhalation of Herbal Powders: Evidence of Occupational Exposure in Herbalists with Urothelial Cancer.

Emerging evidence showing urothelial cancer in herbalists is linked to aristolochic acid (AA) exposure; however, the exposure pathway remains unclear. Here, we show that dermal contact and inhalation of fine powders of AA-containing herbs are significant occupational AA exposure pathways for herbalists. We initiated the study by quantifying the amount of AA in the AA-containing powder deposited on gloves and face masks worn by the operators of an AA-containing herb grinding machine. Then, we measured the kinetics of dermal absorption and dissolution of AA from fine powders of AA-containing herbs into artificial sweat and surrogate lung fluid. Lastly, we quantified the mutagenic AA-DNA adduct levels formed in the kidneys of mice exposed to AA-containing fine powders through dermal contact. Our findings highlight an urgent occupational risk that should demand implementation of safety standards for herbalists exposed to AA-containing fine powders.

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.

Formation of DNA Adducts by 1-Methoxy-3-indolylmethylalcohol, a Breakdown Product of a Glucosinolate, in the Mouse: Impact of the SULT1A1 Status-Wild-Type, Knockout or Humanised.

We previously found that feeding rats with broccoli or cauliflower leads to the formation of characteristic DNA adducts in the liver, intestine and various other tissues. We identified the critical substances in the plants as 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate and its degradation product 1-MIM-OH. DNA adduct formation and the mutagenicity of 1-MIM-OH in cell models were drastically enhanced when human sulfotransferase (SULT) 1A1 was expressed. The aim of this study was to clarify the role of SULT1A1 in DNA adduct formation by 1-MIM-OH in mouse tissues in vivo. Furthermore, we compared the endogenous mouse Sult1a1 and transgenic human SULT1A1 in the activation of 1-MIM-OH using genetically modified mouse strains. We orally treated male wild-type (wt) and Sult1a1-knockout (ko) mice, as well as corresponding lines carrying the human SULT1A1-SULT1A2 gene cluster (tg and ko-tg), with 1-MIM-OH. N2-(1-MIM)-dG and N6-(1-MIM)-dA adducts in DNA were analysed using isotope-dilution UPLC-MS/MS. In the liver, caecum and colon adducts were abundant in mice expressing mouse and/or human SULT1A1, but were drastically reduced in ko mice (1.2-10.6% of wt). In the kidney and small intestine, adduct levels were high in mice carrying human SULT1A1-SULT1A2 genes, but low in wt and ko mice (1.8-6.3% of tg-ko). In bone marrow, adduct levels were very low, independently of the SULT1A1 status. In the stomach, they were high in all four lines. Thus, adduct formation was primarily controlled by SULT1A1 in five out of seven tissues studied, with a strong impact of differences in the tissue distribution of mouse and human SULT1A1. The behaviour of 1-MIM-OH in these models (levels and tissue distribution of DNA adducts; impact of SULTs) was similar to that of methyleugenol, classified as "probably carcinogenic to humans". Thus, there is a need to test 1-MIM-OH for carcinogenicity in animal models and to study its adduct formation in humans consuming brassicaceous foodstuff.

Transcription-coupled repair of DNA-protein cross-links depends on CSA and CSB.

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

Biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach using LC-HRMS/MS and chemometrics.

Since its first employment in World War I, chlorine gas has often been used as chemical warfare agent. Unfortunately, after suspected release, it is difficult to prove the use of chlorine as a chemical weapon and unambiguous verification is still challenging. Furthermore, similar evidence can be found for exposure to chlorine gas and other, less harmful chlorinating agents. Therefore, the current study aims to use untargeted high resolution mass spectrometric analysis of chlorinated biomarkers together with machine learning techniques to be able to differentiate between exposure of plants to various chlorinating agents. Green spire (Euonymus japonicus), stinging nettle (Urtica dioica), and feathergrass (Stipa tenuifolia) were exposed to 1000 and 7500 ppm chlorine gas and household bleach, pool bleach, and concentrated sodium hypochlorite. After sample preparation and digestion, the samples were analyzed by liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). More than 150 chlorinated compounds including plant fatty acids, proteins, and DNA adducts were tentatively identified. Principal component analysis (PCA) and linear discriminant analysis (LDA) showed clear discrimination between chlorine gas and bleach exposure and grouping of the samples according to chlorine concentration and type of bleach. The identity of a set of novel biomarkers was confirmed using commercially available or synthetic reference standards. Chlorodopamine, dichlorodopamine, and trichlorodopamine were identified as specific markers for chlorine gas exposure. Fenclonine (Cl-Phe), 3-chlorotyrosine (Cl-Tyr), 3,5-dichlorotyrosine (di-Cl-Tyr), and 5-chlorocytosine (Cl-Cyt) were more abundantly present in plants after chlorine contact. In contrast, the DNA adduct 2-amino-6-chloropurine (Cl-Ade) was identified in both types of samples at a similar level. None of these chlorinated biomarkers were observed in untreated samples. The DNA adducts Cl-Cyt and Cl-Ade could clearly be identified even three months after the actual exposure. This study demonstrates the feasibility of forensic biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach.

Attenuation of Polycyclic Aromatic Hydrocarbon (PAH)-Induced Carcinogenesis and Tumorigenesis by Omega-3 Fatty Acids in Mice In Vivo.

Lung cancer is the leading cause of cancer death worldwide. Polycyclic aromatic hydrocarbons (PAHs) are metabolized by the cytochrome P450 (CYP)1A and 1B1 to DNA-reactive metabolites, which could lead to mutations in critical genes, eventually resulting in cancer. Omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are beneficial against cancers. In this investigation, we elucidated the mechanisms by which omega-3 fatty acids EPA and DHA will attenuate PAH-DNA adducts and lung carcinogenesis and tumorigenesis mediated by the PAHs BP and MC. Adult wild-type (WT) (A/J) mice, Cyp1a1-null, Cyp1a2-null, or Cyp1b1-null mice were exposed to PAHs benzo[a]pyrene (BP) or 3-methylcholanthrene (MC), and the effects of omega-3 fatty acid on PAH-mediated lung carcinogenesis and tumorigenesis were studied. The major findings were as follows: (i) omega-3 fatty acids significantly decreased PAH-DNA adducts in the lungs of each of the genotypes studied; (ii) decreases in PAH-DNA adduct levels by EPA/DHA was in part due to inhibition of CYP1B1; (iii) inhibition of soluble epoxide hydrolase (sEH) enhanced the EPA/DHA-mediated prevention of pulmonary carcinogenesis; and (iv) EPA/DHA attenuated PAH-mediated carcinogenesis in part by epigenetic mechanisms. Taken together, our results suggest that omega-3 fatty acids have the potential to be developed as cancer chemo-preventive agents in people.

Stable Interstrand Cross-Links Generated from the Repair of 1,N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

DNA adduct formation in Saccharomyces cerevisiae following exposure to environmental pollutants, as in vivo model for molecular toxicity studies.

DNA adduction in the model yeast Saccharomyces cerevisiae was investigated after exposure to the fungicide penconazole and the reference genotoxic compound benzo(a)pyrene, for validating yeasts as a tool for molecular toxicity studies, particularly of environmental pollution. The effect of the toxicants on the yeast's growth kinetics was determined as an indicator of cytotoxicity. Fermentative cultures of S. cerevisiae were exposed to 2 ppm of Penconazole during different phases of growth; while 0.2 and 2 ppm of benzo(a)pyrene were applied to the culture medium before inoculation and on exponential cultures. Exponential respiratory cultures were also exposed to 0.2 ppm of B(a)P for comparison of both metabolisms. Penconazole induced DNA adducts formation in the exponential phase test; DNA adducts showed a peak of 54.93 adducts/109 nucleotides. Benzo(a)pyrene induced the formation of DNA adducts in all the tests carried out; the highest amount of 46.7 adducts/109 nucleotides was obtained in the fermentative cultures after the exponential phase exposure to 0.2 ppm; whereas in the respiratory cultures, 14.6 adducts/109 nucleotides were detected. No cytotoxicity was obtained in any experiment. Our study showed that yeast could be used to analyse DNA adducts as biomarkers of exposure to environmental toxicants.

Commentary of the SKLM to the EFSA opinion on risk assessment of N-nitrosamines in food.

Dietary exposure to N-nitrosamines has recently been assessed by the European Food Safety Authority (EFSA) to result in margins of exposure that are conceived to indicate concern with respect to human health risk. However, evidence from more than half a century of international research shows that N-nitroso compounds (NOC) can also be formed endogenously. In this commentary of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG), the complex metabolic and physiological biokinetics network of nitrate, nitrite and reactive nitrogen species is discussed with emphasis on its influence on endogenous NOC formation. Pioneering approaches to monitor endogenous NOC have been based on steady-state levels of N-nitrosodimethylamine (NDMA) in human blood and on DNA adduct levels in blood cells. Further NOC have not been considered yet to a comparable extent, although their generation from endogenous or exogenous precursors is to be expected. The evidence available to date indicates that endogenous NDMA exposure could exceed dietary exposure by about 2-3 orders of magnitude. These findings require consolidation by refined toxicokinetics and DNA adduct monitoring data to achieve a credible and comprehensive human health risk assessment.

Exocyclic DNA adducts and oxidative stress parameters: useful tools for biomonitoring exposure to aldehydes in smokers.

CONTEXT: Exocyclic DNA adducts have been shown to be potential biomarkers of cancer risk related to oxidative stress and exposure to aldehydes in smokers. In fact, aldehydes potentially arise from tobacco combustion directly and endogenously through lipid peroxidation. OBJECTIVE: This study aims to investigate the relationship between a profile of nine aldehydes-induced DNA adducts and antioxidant activities, in order to evaluate new biomarkers of systemic exposure to aldehydes. METHODS: Using our previously published UPLC-MS/MS method, adducts levels were quantified in the blood DNA of 34 active smokers. The levels of antioxidant vitamins (A, C and E), coenzyme Q10, ß-carotene, superoxide dismutase (SOD) and autoantibodies against oxidized low-density lipoprotein were measured. RESULTS: Adducts induced by tobacco smoking-related aldehydes were quantified at levels reflecting an oxidative production from lipid peroxidation. A significant correlation between SOD and crotonaldehyde-induced adducts (p = 0.0251) was also observed. ß-Carotene was negatively correlated with the adducts of formaldehyde (p = 0.0351) and acetaldehyde (p = 0.0413). Vitamin C tended to inversely correlate with acetaldehyde-induced adducts (p = 0.0584). CONCLUSION: These results are promising, and the study is now being conducted on a larger cohort with the aim of evaluating the impact of smoking cessation programs on the evolution of adducts profile and antioxidants activities.

DNA Adducts in Cancer Chemotherapy.

DNA adducting drugs, including alkylating agents and platinum-containing drugs, are prominent in cancer chemotherapy. Their mechanisms of action involve direct interaction with DNA, resulting in the formation of DNA addition products known as DNA adducts. While these adducts are well-accepted to induce cancer cell death, understanding of their specific chemotypes and their role in drug therapy response remain limited. This perspective aims to address this gap by investigating the metabolic activation and chemical characterization of DNA adducts formed by the U.S. FDA-approved drugs. Moreover, clinical studies on DNA adducts as potential biomarkers for predicting patient responses to drug efficacy are examined. The overarching goal is to engage the interest of medicinal chemists and stimulate further research into the use of DNA adducts as biomarkers for guiding personalized cancer treatment.

Mass Spectrometry-Based Method to Measure Aflatoxin B1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 µg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.

Effects of Heavy Metal Co-Exposure on the formation of DNA Adducts from Aristolochic Acid I: Implications for Balkan Endemic Nephropathy Development.

Accumulated evidence has shown that Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with exposure to aristolochic acids (AA), and the associated DNA adduct formation, as a key causative factor of BEN development. Here, we show that coexposure to arsenic, cadmium, and iron increases the DNA adduct formation of AA in cultured kidney cells, while exhibiting both an exposure concentration and duration dependence. In contrast, coexposure to calcium and copper showed a decreasing DNA adduct formation. Because DNA damage is responsible for both the nephrotoxicity and carcinogenicity of AA, these results shed greater light on the endemic nature of BEN.

DNA adductomics aided rapid screening of genotoxic impurities using nucleosides and 3D bioprinted human liver organoids.

Current genotoxicity assessment methods are mainly employed to verify the genotoxic safety of drugs, but do not allow for rapid screening of specific genotoxic impurities (GTIs). In this study, a new approach for the recognition of GTIs has been proposed. It is to expose the complex samples to an in vitro nucleoside incubation model, and then draw complete DNA adduct profiles to infer the structures of potential genotoxic impurities (PGIs). Subsequently, the genotoxicity is confirmed in human by 3D bioprinted human liver organoids. To verify the feasibility of the approach, lansoprazole chloride compound (Lanchlor), a PGI during the synthesis of lansoprazole, was selected as the model drug. After confirming genotoxicity by Comet assay, it was exposed to different models to map and compare the DNA adduct profiles by LC-MS/MS. The results showed Lanchlor could generate diverse DNA adducts, revealing firstly its genotoxicity at molecular mechanism of action. Furthermore, the largest variety and content of DNA adducts were observed in the nucleoside incubation model, while the human liver organoids exhibited similar results with rats. The results showed that the combination of DNA adductomics and 3D bioprinted organoids were useful for the rapid screening of GTIs.