DNA-DNA Cross-links Research Articles

Impact of high (1950 MHz) and extremely low (50 Hz) frequency electromagnetic fields on DNA damage caused by occupationally relevant exposures in human derived cell lines

Epidemiological studies indicate that electromagnetic fields (EMF) are associated with cancer in humans. Exposure to mobile phone specific high frequency fields (HF-EMF) may lead to increased glioma risks, while low frequency radiation (LF-EMF) is associated with childhood leukemia. We studied the impact of HF-EMF (1950 MHz, UMTS signal) on DNA stability in an astrocytoma cell line (1321N1), and the effect of LF-EMF (50 Hz) in human derived lymphoma (Jurkat) cells. To find out if these fields affect chemically induced DNA damage, co-exposure experiments were performed. The cells were exposed to HF-EMF or LF-EMF and treated simultaneously and sequentially with mutagens. The compounds cause DNA damage via different molecular mechanisms, i.e. pyrimidine dimers which are characteristic for UV light (4-nitroquinoline 1-oxide, 4NQO), bulky base adducts (benzo[a]pyrene diolepoxide, BPDE), DNA-DNA and DNA-protein cross links and oxidative damage (NiCl2, CrO3). DNA damage was measured in single cell gel electrophoresis (comet) assays. We found a moderate reduction of basal and 4NQO-induced DNA damage in the astrocytoma line, but no significant alterations of chemically induced DNA migration by the HF and LF fields under all other experimental series. The biological consequences of the moderate reduction remain unclear, but our findings indicate that acute mobile phone and power line specific EMF exposures do not enhance genotoxic effects caused by occupationally relevant chemical exposures.

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.

Development of Comprehensive Ultraperformance Liquid Chromatography-High-Resolution Mass Spectrometry Assays to Quantitate Cisplatin-Induced DNA-DNA Cross-Links.

Cisplatin (CP) is a common antitumor drug that is used to treat many solid tumors. The activity of CP is attributed to the formation of DNA-DNA cross-links, which consist of 1,2-intra-, 1,3-intra-, and interstrand cross-links. To better understand how each intrastrand cross-link contributes to the activity of CP, we have developed comprehensive ultraperformance liquid chromatography-selective ion monitoring (UPLC-SIM) assays to quantify 1,2-GG-, 1,2-AG-, 1,3-GCG-, and 1,3-GTG-intrastrand cross-links. The limit of quantitation for the developed assays ranged from 5 to 50 fmol or as low as 6 cross-links per 108 nucleotides. To demonstrate the utility of the UPLC-SIM assays, we first performed in vitro cross-link formation kinetics experiments. We confirmed that the 1,2-GG-intrastrand cross-links were the most abundant intrastrand cross-link and formed at a faster rate compared to 1,2-AG- and 1,3-intrastrand cross-links. Furthermore, we investigated the repair kinetics of intrastrand cross-links in CP-treated wild-type and nucleotide excision repair (NER)-deficient U2OS cells. We observed a slow decrease of both 1,2- and 1,3-intrastrand cross-links in wild-type cells and no evidence of direct repair in the NER-deficient cells. Taken together, we have demonstrated that our assays are capable of accurately quantifying intrastrand cross-links in CP-treated samples and can be utilized to better understand the activity of CP.

Effects of antimony exposure on DNA damage and genome-wide variation in zebrafish (Danio rerio) liver

Antimony (Sb) is a potentially toxic and carcinogenic cumulative contaminant that poses a serious threat to aquatic ecosystems. To better clarify the genotoxicity of Sb and its mechanism of action. In this study, we investigated DNA damage and genome-wide variation in the liver of a model organism, zebrafish (Danio rerio), under subacute Sb exposure and explored its potential toxicological mechanisms. The results showed that medium and high concentrations of Sb significantly reduced the total antioxidant capacity and increased the content of reactive oxygen species in zebrafish liver, and further studies revealed that it increased oxidative DNA damage and DNA-DNA cross-link (DDC), but had little effect on DNA-protein cross-link (DPC). The result of resequencing showed that the mutation sites of the genes with high concentrations of Sb were higher than those with medium concentrations, and the mutation was mainly a single nucleotide. The pathways significantly enriched for nonsynonymous single nucleotide polymorphisms (SNPs) and insertion/deletion mutations (InDels) variant genes in the coding regions of both the medium and high Sb-treated groups were ECM-receptor interactions, and the high Sb-treated group also included lysine degradation, hematopoietic cell lineage, and cytokine-cytokine receptor interactions. This suggests that ECM-receptor interactions play an important role in the mechanism of antimony toxicity to the liver of zebrafish.

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.

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.

Quantitative NanoLC/NSI+-HRMS Method for 1,3-Butadiene Induced bis-N7-guanine DNA-DNA Cross-Links in Urine.

1,3-Butadiene (BD) is a common environmental and industrial chemical widely used in plastic and rubber manufacturing and also present in cigarette smoke and automobile exhaust. BD is classified as a known human carcinogen based on evidence of carcinogenicity in laboratory animals treated with BD by inhalation and epidemiological studies revealing an increased risk of leukemia and lymphohematopoietic cancers in workers occupationally exposed to BD. Upon exposure via inhalation, BD is bioactivated to several toxic epoxides including 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB); these are conjugated with glutathione and excreted as 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA), and 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA). Exposure to DEB generates monoalkylated DNA adducts, DNA-DNA crosslinks, and DNA-protein crosslinks, which can cause base substitutions, genomic rearrangements, and large genomic deletions. In this study, we developed a quantitative nanoLC/NSI+-HRMS methodology for 1,4-bis-(gua-7-yl)-2,3-butanediol (bis-N7G-BD) adducts in urine (LOD: 0.1 fmol/mL urine, LOQ: 1.0 fmol/mL urine). This novel method was used to quantify bis-N7G-BD in urine of mice treated with 590 ± 150 ppm BD for 2 weeks (6 h/day, 5 days/week). Bis-N7G-BD was detected in urine of male and female BD-exposed mice (574.6 ± 206.0 and 571.1 ± 163.4 pg/mg of creatinine, respectively). In addition, major urinary metabolites of BD, bis-BDMA, MHBMA and DHBMA, were measured in the same samples. Urinary bis-N7G-BD adduct levels correlated with DEB-derived metabolite bis-BDMA (r = 0.80, Pearson correlation), but not with the EB-derived DNA adducts (EB-GII) or EB-derived metabolites MHBMA and DHBMA (r = 0.24, r = 0.14, r = 0.18, respectively, Pearson correlations). Urinary bis-N7G-BD could be employed as a novel non-invasive biomarker of exposure to BD and bioactivation to its most mutagenic metabolite, DEB. This method will be useful for future studies of 1,3-butadiene exposure and metabolism.

Effects of GSTT1 Genotype on the Detoxification of 1,3-Butadiene Derived Diepoxide and Formation of Promutagenic DNA-DNA Cross-Links in Human Hapmap Cell Lines.

Smoking is a leading cause of lung cancer, accounting for 81% of lung cancer cases. Tobacco smoke contains over 5000 compounds, of which more than 70 have been classified as human carcinogens. Of the many tobacco smoke constituents, 1,3-butadiene (BD) has a high cancer risk index due to its tumorigenic potency and its abundance in cigarette smoke. The carcinogenicity of BD has been attributed to the formation of several epoxide metabolites, of which 1,2,3,4-diepoxybutane (DEB) is the most toxic and mutagenic. DEB is formed by two oxidation reactions carried out by cytochrome P450 monooxygenases, mainly CYP2E1. Glutathione-S-transferase theta 1 (GSTT1) facilitates the conjugation of DEB to glutathione as the first step of its detoxification and subsequent elimination via the mercapturic acid pathway. Human biomonitoring studies have revealed a strong association between GSTT1 copy number and urinary concentrations of BD-mercapturic acids, suggesting that it plays an important role in the metabolism of BD. To determine the extent that GSTT1 genotype affects the susceptibility of individuals to the toxic and genotoxic properties of DEB, GSTT1 negative and GSTT1 positive HapMap lymphoblastoid cell lines were treated with DEB, and the extent of apoptosis and micronuclei (MN) formation was assessed. These toxicological end points were compared to the formation of DEB-GSH conjugates and 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) DNA-DNA cross-links. GSTT1 negative cell lines were more sensitive to DEB-induced apoptosis as compared to GSTT1 positive cell lines. Consistent with the protective effect of GSH conjugation against DEB-derived apoptosis, GSTT1 positive cell lines formed significantly more DEB-GSH conjugate than GSTT1 negative cell lines. However, GSTT1 genotype did not affect formation of MN or bis-N7G-BD cross-links. These results indicate that GSTT1 genotype significantly influences BD metabolism and acute toxicity.

Cross-linking of the DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of cisplatin

1,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980–995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI+-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI+-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH3)2{d(GpG)}] cross-links which were detected by HPLC-ESI+-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.

Unhooking of an interstrand cross-link at DNA fork structures by the DNA glycosylase NEIL3

Interstrand DNA-DNA cross-links (ICLs) are generated by endogenous processes, drugs, and environmental toxins. Understanding the cellular pathways by which various ICLs are repaired is critical to understanding their biological effects. Recent studies showed that replication-dependent repair of an ICL derived from the reaction of an abasic (AP) site with an adenine residue (dA) on the opposing strand of duplex DNA proceeds via a novel mechanism in which the DNA glycosylase NEIL3 unhooks the ICL. Here we examined the ability of the glycosylase domain of murine NEIL3 (MmuNEIL3-GD) to unhook dA-AP ICLs. The enzyme selectively unhooks the dA-AP ICL located at the duplex/single-strand junction of splayed duplexes that model the strand-separated DNA at the leading edge of a replication fork. We show that the ability to unhook the dA-AP ICL is a specialized function of NEIL3 as this activity is not observed in other BER enzymes. Importantly, NEIL3 only unhooks the dA-AP ICL when the AP residue is located on what would be the leading template strand of a model replication fork. The same specificity for the leading template strand was observed with a 5,6-dihydrothymine monoadduct, demonstrating that this preference is a general feature of the glycosylase and independent of the type of DNA damage. Overall, the results show that the glycosylase domain of NEIL3, lacking the C-terminal NPL4 and GRF zinc finger motifs, is competent to unhook the dA-AP ICL in splayed substrates and independently enforces important substrate preferences on the repair process.

Treosulfan Metabolite (S, S-EBDM) Pharmacokinetics Influences Regimen Related Toxicity in Patients with Beta Thalassaemia Major Undergoing HSCT

A toxicity reduced conditioning regimen containing Treosulfan (Treo), Fludarabine (Flu), Thiotepa in patients with class III high risk thalassemia major (TM) has significantly improved transplant outcomes compared to the historical Busulfan/cyclophosphamide based myeloablative regimen (Mathews et al, 2013). However, complications related to mixed chimerism, rejection and regimen related toxicity remain. Analysis of Treo pharmacokinetics (PK) in TM showed significant inter individual variation but no association with HSCT outcome (Mohanan et al, 2018). This may be attributed to the fact that only the pro-drug Treo was measured and not its active metabolites. Treo, administered as a prodrug, is converted non-enzymatically to its active metabolite monoepoxide 2S, 3S-1,2-epoxybutane-3,4-diol-4-methanesulfonate (S, S-EBDM) and (2S,3S)- 1,2:3,4-diepoxybutane (S, S-DEB) that causes cytotoxicity. We have now assessed the PK of both Treo and S, S-EBDM and the factors which may influence the inter-individual variation in PK, and their impact on HSCT outcome in patients with TM. Fifty-six patients with TM undergoing HSCT between January 2016 and June 2019 with Flu/Treo based conditioning regimen (Flu 40mg/m2/day x 4 days as a 1hr infusion and Treo as 14g/m2/day x 3 days infused at 5g/hr) were included in this analysis. Selected functional polymorphisms in NAD(P)H Quinone Dehydrogenase-1 (NQO1) and Glutathione S-transferase (GST) genes that showed association with Treo PK based on our previous report (Pai et al, Abstract. Proceedings of BMT Tandem Meetings 2018) were screened. Plasma Treo and S, S-EBDM were measured using an LC-MS/MS method. Treo and S, S-EBDM PK were estimated using nonlinear mixed effects modeling via Monolix 5.0.1. The covariates tested were age, sex, body weight, BSA, serum ferritin, NQO1 3'UTR (rs10517) and GSTA1*B polymorphisms. The influence of Treo & S, S-EBDM PK and polymorphisms on graft rejection, regimen related toxicities (RRT), chimerism status, overall and event-free survival were estimated using logistic regression analysis. A two-compartment model best described the PK of Treo and S, S-EBDM. Two and 11-fold inter-individual variation was observed in Treo and S, S-EBDM PK respectively. Among the covariates tested, Treo Cl decreased with age (p=0.03) while, S, S-EBDM apparent Cl increased in patients with NQO1 3'UTR variant (p=0.02) and decreased in patients with GSTA1*B variant genotype (p=0.02). Neither Treo AUC nor clearance showed association with HSCT outcome. However, high S, S-EBDM AUC (213. Vs 130 mg*hr/mL, p=0.016) and low S, S-EBDM apparent Cl (68 Vs 122 L/hr/m2, p=0.013) were significantly associated with increased incidence of sinusoidal obstruction syndrome (SOS). Also, on quartile analysis, patients with S, S-EBDM AUC >210 mg*hr/mL and Treo/S, S-EBDM AUC ratio >18 mg*hr/mL showed increased incidence of SOS (p=0.006 & p=0.026 respectively). Multivariate analysis adjusting for known clinical risk factors also showed in addition to age, only high S, S-EBDM AUC to be significantly associated with increased incidence of SOS (p=0.013). No association between rejection or mixed chimerism and Treo/S, S-EBDM PK was observed, probably due to very low incidence of graft rejection in this cohort (4/56; 7%). Our study suggests that active metabolite S, S-EBDM influences SOS following HSCT in TM patients. It is well acknowledged that Treo to S, S-EBDM conversion is a non-enzymatic nucleophilic substitution reaction and there is a difference in the disposition of Treo and S, S-EBDM in different organs including the liver (Romanski et al, 2017). Treo metabolites exert their cytotoxic activity by both DNA-DNA and DNA-protein crosslinks in these organs. The role of Treo metabolites in exhausting essential cellular proteins like GSH and NQO1 as well as the effect of GST/NQO1 variants in influencing Treo metabolism in liver which may then impact the incidence and severity of SOS need further evaluation. Disclosures No relevant conflicts of interest to declare.

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.

Kinetics of in Vitro Guanine- N7-Alkylation in Calf Thymus DNA by (2 S,3 S)-1,2-Epoxybutane-3,4-diol 4-methanesulfonate and (2 S,3 S)-1,2:3,4-Diepoxybutane: Revision of the Mechanism of DNA Cross-Linking by the Prodrug Treosulfan.

Prodrug treosulfan, originally registered for treatment of ovarian cancer, has gained a use in conditioning prior to hematopoietic stem cell transplantation. Treosulfan converts nonenzymatically to the monoepoxide intermediate (EBDM), and then to (2 S,3 S)-1,2:3,4-diepoxybutane (DEB). The latter alkylates DNA forming mainly (2' S,3' S)- N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (THBG) and (2 S,3 S)-1,4-bis(guan-7'-yl)butane-2,3-diol cross-link (bis-N7G-BD) via the intermediate epoxide adduct (EHBG). It is believed that DNA cross-linking by DEB is a primary mechanism for the anticancer and myeloablative properties of treosulfan, but clear evidence is lacking. Recently, we have proved that EBDM alkylates DNA producing (2' S,3' S)- N-7-(2',3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)-guanine (HMSBG) and that free HMSBG converts to EHBG. In this paper, we investigated the kinetics of HMSBG, bis-N7G-BD, and THBG in DNA in vitro to elucidate the contribution of EBDM and DEB to treosulfan-dependent DNA-DNA cross-linking. Calf thymus DNA was exposed to ( A) 100 μM treosulfan, ( B) 200 μM treosulfan, and ( C) DEB at a concentration 100 μM, exceeding that produced by 200 μM treosulfan. Following mild acid thermal hydrolysis of DNA, ultrafiltration, and off-line HPLC purification, the guanine adducts were quantified by LC-MS/MS. Both bis-N7G-BD and THBG reached highest concentrations in the DNA in experiment B. Ratios of the maximal concentration of bis-N7G-BD and THBG to DEB (adduct Cmax/DEB Cmax) in experiments A and B were 1.7-3.0-times greater than in experiment C. EHBG converted to the bis-N7G-BD cross-link at a much higher rate constant (0.20 h-1) than EBDM and DEB initially alkylated the DNA (1.8-3.4 × 10-5 h-1), giving rise to HMSBG and EHBG, respectively. HMSBG decayed unexpectedly slowly (0.022 h-1) compared with the previously reported behavior of the free adduct (0.14 h-1), which revealed the inhibitory effect of the DNA environment on the adduct epoxidation to EHBG. A kinetic simulation based on the obtained results and the literature pharmacokinetic parameters of treosulfan, EBDM, and DEB suggested that in patients treated with the prodrug, EBDM could produce the vast majority of EHBG and bis-N7G-BD via HMSBG. In conclusion, EBDM can produce DNA-DNA lesions independently of DEB, and likely plays a greater role in DNA cross-linking after in vivo administration of treosulfan than DEB. These findings compel revision of the previously proposed mechanism of the pharmacological action of treosulfan and contribute to better understanding of the importance of EBDM for biological effects.

Intercalation-enhanced "Click" Crosslinking of DNA.

DNA-DNA cross-linking agents constitute an important family of chemotherapeutics that non-specifically react with endogenous nucleophiles and therefore exhibit undesirable side effects. Here we report a cationic Sondheimer diyne derivative "DiMOC" that exhibits weak, reversible intercalation into duplex DNA (Kd =15 μm) where it undergoes tandem strain-promoted cross-linking of azide-containing DNA to give DNA-DNA interstrand crosslinks (ICLs) with an exceptionally high apparent rate constant kapp =2.1×105 m-1 s-1 . This represents a 21 000-fold rate enhancement as compared the reaction between DIMOC and 5-(azidomethyl)-2'-deoxyuridine (AmdU) nucleoside. As single agents, 5'-bispivaloyloxymethyl (POM)-AmdU and DiMOC exhibited low cytotoxicity, but highly toxic DNA-DNA ICLs were generated by metabolic incorporation of AmdU groups into cellular DNA, followed by treatment of the cells with DiMOC. These results provide the first examples of intercalation-enhanced bioorthogonal chemical reactions on DNA, and furthermore, the first strain-promoted double click (SPDC) reactions inside of living cells.

Understanding the interactions of diruthenium anticancer agents with amino acids.

The dinuclear anticancer agents 1,n-bis{chlorido[3-(oxo-κO)-2-methyl-4-(1H)-pyridinonato-κO4](η6-p-cymene)-ruthenium(II)}alkane (PyRu 2n ) exhibit high antiproliferative activity in human cancer cell. Reactivity studies with DNA and protein revealed uncommon protein-DNA and DNA-DNA crosslinking ability. We report here studies on the reactions of the diruthenium organometallics PyRu 26 and PyRu 28 in comparison with a mononuclear analogue PyRu3 with amino acids using mass spectrometry and NMR spectroscopy. The compounds behave very similarly, independent of the spacer length between the metal center and of the nuclearity of the complexes. Incubation with L-cysteine (Cys) results in fast release of the pyridone ligand, with the Ru complexes able to form Cys adducts. In contrast, L-methionine forms, initially, adducts with the metal centers, but over time, the adducts decompose. Similar behavior was observed for the reaction with L-histidine with [Ru(η6-p-cymene)(L-histidine)] species detected.

Sequence‐Dependent Diastereospecific and Diastereodivergent Crosslinking of DNA by Decarbamoylmitomycin C

Mitomycin C (MC), a potent antitumor drug, and decarbamoylmitomycin C (DMC), a derivative lacking the carbamoyl group, form highly cytotoxic DNA interstrand crosslinks. The major interstrand crosslink formed by DMC is the C1'' epimer of the major crosslink formed by MC. The molecular basis for the stereochemical configuration exhibited by DMC was investigated using biomimetic synthesis. The formation of DNA-DNA crosslinks by DMC is diastereospecific and diastereodivergent: Only the 1''S-diastereomer of the initially formed monoadduct can form crosslinks at GpC sequences, and only the 1''R-diastereomer of the monoadduct can form crosslinks at CpG sequences. We also show that CpG and GpC sequences react with divergent diastereoselectivity in the first alkylation step: 1"S stereochemistry is favored at GpC sequences and 1''R stereochemistry is favored at CpG sequences. Therefore, the first alkylation step results, at each sequence, in the selective formation of the diastereomer able to generate an interstrand DNA-DNA crosslink after the "second arm" alkylation. Examination of the known DNA adduct pattern obtained after treatment of cancer cell cultures with DMC indicates that the GpC sequence is the major target for the formation of DNA-DNA crosslinks in vivo by this drug.

Interaction of DNA with water soluble complex of Nickle and formation of DNA cross-links

Interaction of double stranded DNA with bulky and hydrophobic Salen type Schiff base complex: [N, N′ Bis [3- tert-butyl-5-[triphenyl-phosphonium – methyl] - salicylidene] 1,2 ethylene-diamine nickel(III) acetate (refer to Ni Salen complex) was extensively investigated using the spectroscopic techniques and gel electrophoresis. Absorption titration experiment showed the hypochromic effect and the significant red shift of the complex absorption. In competition experiments with ethidium bromide (EB), Ni Salen complex exhibited non-competitive binding at high concentrations. UV-vis absorption and fluorescence emission data agreed on a binding constant of (1.64 ± 0.01) μM−1, thereby showing the strong interaction of the complex with DNA; also, a binding site size of 2.33 ± 0.01 base pairs per complex was achieved.Thermal denaturation experiment showed that Tm of calf thymus-DNA was increased by approximately 10 °C at a molar ratio of the dye/base of 0.2. The CD spectra of DNA exhibited an increase in both positive and negative peaks without any shift in the position of bands upon addition of the complex. The amplitude of the LD spectra of DNA was decreased in the presence of the complex. Reduced linear dichroism (LDred) revealed that the transition moment of complex was parallel to the DNA helix axis. Gel electrophoresis experiments confirmed that Ni Salen complex had no nuclease/DNA cleaving activity; also, DNA-DNA cross links were formed at high concentrations of complex, leading to the aggregation of DNA.

The evaluation of Cr-curcumin-DNA complexation by experimental and theoretical approaches

ABSTRACTChromium(III) chloride mediates DNA-DNA cross-linking. Some chromium complexes promote programmed cell death in specific ligand environment through binding to DNA. One strategy that can be supposed for reduction of Cr3+ binding affinity to DNA is using curcumin as a chelator. In the current study, the [Cr(Curcumin)(EtOH)2](NO3)2 (CCC) was synthesized and characterized by UV/Vis, FT-IR, CHN and spectrophotometric titration techniques. The mole ratio plot revealed a 1:1 complex between Cr3+ and curcumin in solution. Binding interaction of this complex with calf thymus-DNA (CT-DNA) was investigated using UV/Vis, circular dichroism (CD), FT-IR and cyclic voltammetry. The intrinsic binding constants of CCC with DNA, measured by UV/Vis and cyclic voltammetry, were 1.60 × 105 and 1.13 × 105, respectively. The thermodynamic studies showed that the reaction is enthalpy and entropy favoured. CD analysis revealed that only Λ-CCC interacts with DNA and Δ-CCC form has no tendency towards DNA. Based on FT-IR studies, it was understood that CCC interacts with DNA via minor groove binding. The docking simulation was carried out for finding the binding mode of CCC to DNA, too. All of data demonstrated that the curcumin significantly reduced the affinity of Cr3+ to the DNA and the form of Δ-CCC has no interaction with DNA.