1,2,3,4-Diepoxybutane (DEB) is a bifunctional alkylating agent that exhibits both cytotoxic and promutagenic properties. DEB is the ultimate carcinogenic species of the major industrial chemical 1,3-butadiene (BD), as well as the active form of the antitumor prodrug treosulfan. DEB is tumorigenic in laboratory animals and is capable of inducing a variety of genotoxic outcomes, including point mutations, large deletions, and chromosomal aberrations. These potent biological effects are thought to result from the ability of DEB to form DNA-DNA cross-links by consecutive alkylation of two nucleobases within a DNA duplex. Earlier studies have provided evidence for the formation of interstrand DNA-DEB lesions involving guanine nucleobases, but the covalent structure of DEB-induced DNA cross-link has not been previously elucidated. In the present work, the major DNA-DNA cross-link of DEB has been identified as 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD). The DNA-derived N7-N7 guanine DEB cross-link was characterized by comparing its mass spectra, UV spectra, and chromatographic properties to an authentic standard prepared by an independent synthesis. Calf thymus DNA treated with relatively low concentrations of DEB (5-50 microM) contained similar numbers of bis-N7G-BD and the corresponding monoadducts (N7-trihydroxybutyl-guanine), while higher DEB exposures produced predominantly monoalkylated lesions. Although both lesions spontaneously depurinate at physiological conditions giving rise to abasic sites in DNA, bis-N7G-BD lesions have a longer half-life in double-stranded DNA than the N7-guanine monoadducts. These studies provide the first rigorous characterization of the covalent structure and hydrolytic stability of the major DEB-induced DNA-DNA cross-link.
1,2,3,4-Diepoxybutane (DEB) is a bifunctional electrophile capable of forming DNA-DNA and DNA-protein cross-links. DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can then form 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) lesions. All three optical isomers of DEB are produced metabolically from 1,3-butadiene, but S,S-DEB is the most cytotoxic and genotoxic. In the present work, interstrand and intrastrand DNA-DNA cross-linking by individual DEB stereoisomers was investigated by PAGE, mass spectrometry, and stable isotope labeling. S,S-, R,R-, and meso-diepoxides were synthesized from l-dimethyl-2,3-O-isopropylidene-tartrate, d-dimethyl-2,3-O-isopropylidene-tartrate, and meso-erythritol, respectively. Total numbers of bis-N7G-BD lesions (intrastrand and interstrand) in calf thymus DNA treated separately with S,S-, R,R-, or meso-DEB (0.01-0.5 mM) were similar as determined by capillary HPLC-ESI(+)-MS/MS of DNA hydrolysates. However, denaturing PAGE has revealed that S,S-DEB produced the highest number of interchain cross-links in 5'-GGC-3'/3'-CCG-5' sequences. Intrastrand adduct formation by DEB was investigated by a novel methodology based on stable isotope labeling HPLC-ESI(+)-MS/MS. Meso DEB treatment of DNA duplexes containing 5'-[1,7, NH(2)-(15)N(3),2-(13)C-G]GC-3'/3'-CCG-5' and 5'-GGC-3'/3'-CC[(15)N(3),2-(13)C-G]-5' trinucleotides gave rise to comparable numbers of 1,2-intrastrand and 1,3-interstrand bis-N7G-BD cross-links, while S,S DEB produced few intrastrand lesions. R,R-DEB treated DNA contained mostly 1,3-interstrand bis-N7G-BD, along with smaller amounts of 1,2-interstrand and 1,2-intrastrand adducts. The effects of DEB stereochemistry on its ability to form DNA-DNA cross-links may be rationalized by the spatial relationships between the epoxy alcohol side chains in stereoisomeric N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine adducts and their DNA environment. Different cross-linking specificities of DEB stereoisomers provide a likely structural basis for their distinct biological activities.
1,3-butadiene (BD) is a major industrial chemical used in rubber and plastics production and is recognized as an animal and human carcinogen. Although the exact mechanism of BD-induced carcinogenesis is unknown, chemical reactions of epoxide metabolites of BD with DNA to form nucleobase adducts are likely to contribute to multistage carcinogenesis. Among BD-derived epoxy metabolites, 1,2:3,4-diepoxybutane (DEB) appears to be the most genotoxic and carcinogenic, probably because of its bifunctional nature. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)guanine monoadducts, which can then be hydrolyzed to N7-(2',3',4'-trihydroxy-1'-yl)guanine or can react with another site in double-stranded DNA to form 1,4-bis(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links. While (2',3',4'-trihydroxy-1'-yl)guanine lesions have been previously quantified in vivo, they cannot be used as a biomarker of DEB because the same lesions are also formed by another, more prevalent BD metabolite, 1,2-epoxy-3,4-butanediol. In contrast, bis-N7G-BD can only be formed from DEB, potentially providing a specific biomarker of DEB formation. We have developed a quantitative HPLC-ESI+-MS/MS method for measuring racemic and meso forms of bis-N7G-BD in DNA extracted from tissues of BD-exposed laboratory animals. In our approach, bis-N7G-BD adducts are released from DNA as free bases by neutral thermal hydrolysis, purified by solid-phase extraction, and subjected to HPLC-ESI+-MS/MS analysis. Selected reaction monitoring is performed by following the loss of a guanine moiety from protonated molecules of bis-N7G-BD and the formation of protonated guanine under collision-induced dissociation. Quantitative analysis of racemic and meso forms of bis-N7G-BD is based on isotope dilution with the corresponding 15N-labeled internal standards. The lower limit of quantification of our current method is 10-20 fmol/0.1 mg of DNA. The accuracy and precision of the new method were determined by spiking control mouse liver DNA with racemic and meso forms of bis-N7G-BD (10 fmol each), followed by sample processing and HPLC-ESI+-MS/MS analysis. Calculated amounts of racemic and meso forms of bis-N7G-BD were within 20% of the theoretical value (9.7 +/- 2 and 9.2 +/- 1.9 fmol, respectively, N = 4). DNA extracted from liver and lung tissues of mice exposed to 625 ppm butadiene for 5 days contained 3.2 +/- 0.4 and 1.8 +/- 0.5 racemic adducts per 10(6) guanines, respectively, while the amounts of meso-bis-N7G-BD were below the detection limits of our method (1 per 10(7) guanines). Control animals did not contain either bis-N7G-BD lesion. Sensitive and specific quantitative methods for bis-N7G-BD analysis developed in this work provide a unique biomarker of DEB-induced DNA alkylation following exposure to BD.
Nitrogen mustards (NMs) are useful chemotherapeutic agents in the treatment of lymphoma, leukemia, multiple myeloma, and ovarian carcinoma. The antitumor activity of NMs has been attributed to their ability to cross-link the twin strands of DNA. The resulting bifunctional lesions, if not repaired, can inhibit DNA replication and transcription, eventually leading to cell cycle arrest, apoptosis, and the inhibition of tumor growth. The predominant bifunctional DNA lesions of NM have been reported to involve the distal guanine bases in the opposite strands of 5'-GNC sequences. In the present work, the formation of guanine-adenine and adenine-adenine adducts of N,N-bis(2-chloroethyl)methylamine (mechlorethamine) in double-stranded DNA is demonstrated. Guanine-adenine cross-links of mechlorethamine were identified as N-(2-[N3-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine (N3A-N7G-EMA), N-(2-[N1-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine, and N-(2-[N(6)-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine. All three adducts were produced interstrand, while N3A-N7G-EMA was the dominant intrastrand G-A cross-link. The prevalent adenine-adenine mechlorethamine lesions have the structure of N,N-bis(2-[N3-adenyl]ethyl)methylamine (bis-N3A-EMA). DNA-derived lesions have the same HPLC retention times, UV spectra, and MS/MS fragmentation patterns as the authentic standards prepared independently. bis-N3A-EMA lesions were produced in a concentration-dependent manner in calf thymus DNA treated with increasing amounts of mechlorethamine. Furthermore, HPLC-ESI-MS/MS analysis was used to demonstrate the formation of analogous N3-N3 adenine lesions in DNA treated with aromatic nitrogen mustards, N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid and L-phenylalanine mustard. The presence of cross-linked adenine-adenine lesions may explain the enhanced cytotoxicity and mutagenicity of NMs in cells deficient in N3-alkyladenine glycosylase.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.