HPLC-UV, MALDI-TOF-MS and ESI-MS/MS Analysis of the Mechlorethamine DNA Crosslink at a Cytosine-Cytosine Mismatch Pair

Rojsitthisak, Pornchai; Jongaroonngamsang, Nutthapon; Romero, Rebecca M.; Haworth, Ian S.

doi:10.1371/journal.pone.0020745

Cited by 16 publications

(19 citation statements)

References 50 publications

(67 reference statements)

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“…The major adducts are from reaction at N7-dG and N3-dA with minor reaction at N3-dC, N1-dA and O 6 -dG. 8, 14-21 In addition, cross-links have been characterized between N7-positions of dG, N3-positions of dA, and between the N7-position dG and N3-position of dA. 9, 10 Cationic N7-dG adducts can also undergo a secondary reaction involving the addition of hydroxide to C8 followed by imidazole ring-opening to form an N 5 -substituted formamidopyrimidine adduct (Fapy-dG) in which the N 5 -substituent is derived from the alkylating agent.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of Bis(2-chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

Gruppi

Hejazi

Christov

et al. 2015

Chem. Res. Toxicol.

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A robust, quantitative ultraperformance liquid chromatography ion trap multistage scanning mass spectrometric (UPLC/MS3) method was established to characterize and measure five deoxyguanosine (dG) adducts formed by reaction of the chemotherapeutic nitrogen mustard (NM) bis-(2-chloroethyl)ethylamine with calf thymus (CT) DNA. In addition to the known N7-guanine (NM-G) adduct and its crosslink (G-NM-G), the ring-opened formamidopyrimidine (FapyG) mono-adduct (NM-FapyG) and cross-links in which one (FapyG-NM-G) or both (FapyG-NM-FapyG) guanines underwent ring-opening to FapyG units were identified. Authentic standards of all adducts were synthesized and characterized by NMR and mass spectrometry. These adducts were quantified in CT DNA treated with NM (1 μM) as their deglycosylated bases. A two-stage neutral thermal hydrolysis was developed to mitigate the artifactual formation of ring-opened FapyG adducts involving hydrolysis of the cationic adduct at 37 °C, followed by hydrolysis of the FapyG adducts at 95 °C. The limit of quantification values ranged between 0.3 and 1.6 adducts per 107 DNA bases, when the equivalent of 5 μg DNA hydrolysate was assayed on column. The principal adduct formed was the G-NM-G cross-link, followed by the NM-G mono-adduct; the FapyG-NM-FapyG adduct was at the limit of detection. The NM-FapyG adducts formed in CT DNA at a level of ~20% that of the NM-G adduct. NM-FapyG has not been previously quanitified and the FapyG-NM-G and FapyG-NM-FapyG adducts have not be previously characterized. Our validated analytical method was then applied to measure DNA adduct formation in the MDA-MB-231 mammary tumor cell line exposed to NM (100 μM) for 24 h. The major adduct formed was NM-G (970 adducts per 107 bases), followed by G-NM-G (240 adducts per 107 bases) and NM-FapyG (180 adducts per 107 bases), and lastly the FapyG-NM-G cross-link adduct (6.0 adducts per 107 bases). These lesions are expected to contribute to the NM-mediated toxicity and genotoxicity in vivo.

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Section: Introductionmentioning

confidence: 99%

Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of Bis(2-chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

Gruppi

Hejazi

Christov

et al. 2015

Chem. Res. Toxicol.

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“…Herein, we tested this hypothesis by chronically challenging both wild-type (Wt) and DNA repair-deficient Ercc1 −/Δ mice with a subtoxic dose of the chemotherapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage. MEC, a nitrogen mustard alkylating agent, reacts primarily with the N7 position of guanine residues to form monoadducts and 1,3 G-G interstrand crosslinks [17; 18]. A prototype of alkylating agents, MEC has been used extensively as an anticancer chemotherapeutic drug [19; 20].…”

Section: Introductionmentioning

confidence: 99%

Genotoxic stress accelerates age-associated degenerative changes in intervertebral discs

Nasto

Wang

Robinson

et al. 2013

Mechanisms of Ageing and Development

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Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1−/Δ mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5x in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments were significantly increased. Additionally, new PG synthesis was reduced 2-3x in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1−/Δ mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.

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“…For example, the detection of an intact mechlorethamine-crosslinked duplex reported by Rojsitthisak (Rojsitthisak et al, 2011). Mechlorethamine is a type of nitrogen mustard which has been used clinically for chemotherapeutic applications as it prevents DNA replication, thus affording its significant toxicity.…”

Section: Mass Spectrometry For Analysis Of Nucleic Acidsmentioning

confidence: 99%

“…Mechlorethamine crosslinks DNA preferentially at a mismatch C-C base pairs (Romero et al, 1999). Rojsitthisak et al used negative mode MALDI to detect the molecular ion of a short synthetic duplex with a C-C mismatch base pair, as described in a later section (Rojsitthisak et al, 2011). …”

Section: Mass Spectrometry For Analysis Of Nucleic Acidsmentioning

confidence: 99%

“…A recent study by Rojsitthisak et al illustrated the use of MS/MS methods to identify the connectivities of mismatched cytosine bases crosslinked by the nitrogen mustard mechlorethamine (Rojsitthisak et al, 2011). Mismatched base pairs may arise from DNA damage or mutation, and self-regulating enzymes may not recognize the mismatch, thus preventing repair of the mismatches.…”

Section: Characterization Of Dna/drug Adducts By Tandem Mass Specmentioning

confidence: 99%

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Tandem mass spectrometry for characterization of covalent adducts of DNA with anticancer therapeutics

Silvestri

Brodbelt

2012

Mass Spectrometry Reviews

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The chemotherapeutic activities of many anticancer and antibacterial drugs arise from their interactions with nucleic acid substrates. Some of these ligands interact with DNA in a way that causes conformational changes or damage to the nucleic acid targets, ultimately altering recognition by key DNA-specific enzymes, interfering with DNA transcription or prohibiting replication, and terminating cell growth and proliferation. The design and synthesis of ligands that bind to nucleic acids remains a dynamic field in medicinal chemistry and pharmaceutical research. The quest for more selective and efficacious DNA-interactive anti-cancer chemotherapeutics has likewise catalyzed the need for sensitive analytical methods that can provide structural information about the nature of the resulting DNA adducts and provide insight into the mechanistic pathways of the DNA/drug interactions and the impact on the cellular processes in biological systems. This review focuses on the array of tandem mass spectrometric strategies developed and applied for characterization of covalent adducts formed between DNA and anti-cancer ligands.

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HPLC-UV, MALDI-TOF-MS and ESI-MS/MS Analysis of the Mechlorethamine DNA Crosslink at a Cytosine-Cytosine Mismatch Pair

Cited by 16 publications

References 50 publications

Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of Bis(2-chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of Bis(2-chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

Genotoxic stress accelerates age-associated degenerative changes in intervertebral discs

Tandem mass spectrometry for characterization of covalent adducts of DNA with anticancer therapeutics

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