Synthesis, Characterization, and in Vitro Quantitation of N -7-Guanine Adducts of Diepoxybutane

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1,3-Butadiene (BD) is a high-volume industrial chemical and a common environmental pollutant. Although BD is classified as a "probable human carcinogen", only limited evidence is available for its tumorigenic effects in occupationally exposed populations. Animal studies show a surprisingly high sensitivity of mice to the carcinogenic effects of BD compared to rats (approximately 10(3)-fold), making interspecies extrapolations difficult. Identification and quantitation of specific BD-induced DNA adducts are important for improving our understanding of the mechanisms of BD biological effects and for explaining the observed species differences. Covalent binding of BD to DNA is probably due to its two epoxy metabolites: 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Both EB and DEB are direct mutagens producing frameshift and point mutations at both A:T and G:C base pairs. DEB is 100 times more mutagenic than EB and is found in quantity only in tissues of the most sensitive species (mouse). This has led to the suggestion that the higher sensitivity of mice to BD could be due to greater exposure to DEB. The present work was initiated in order to isolate and structurally characterize DEB-induced adenine adducts. The adducts were formed by reacting DEB with free adenine (Ade), 2'-deoxyadenosine (2'-dAdo), and calf thymus DNA followed by HPLC separation and analysis of the products by UV spectrophotometry, electrospray ionization mass spectrometry, and nuclear magnetic resonance. The adenine reaction resulted in three products which were identified as N-3-, N-7-, and N-9-(2'-hydroxy-3',4'-epoxybut-1'-yl)adenine. These adducts underwent acid-catalyzed hydrolysis to their corresponding (2',3',4'-trihydroxybut-1'-yl)adenines upon heating or storage. The 2'-dAdo reaction with DEB followed by acid hydrolysis yielded a single adduct, N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-DEB-Ade). N-3-DEB-Ade and N6-DEB-Ade were also found in hydrolysates of calf thymus DNA exposed to DEB. The amounts of N-3-DEB-Ade (13/10(3) normal Ade) and N6-DEB-Ade (5/10(3) normal Ade) were slightly lower than those of the corresponding EB-induced adducts in similar experiments, suggesting comparable reactivity of the two epoxy metabolites of BD toward adenine in DNA. The findings of this study provide a basis for future analyses of BD-induced adenyl DNA adducts in vitro and in vivo.

Section: Resultssupporting

confidence: 68%

Section: Discussionsupporting

confidence: 66%

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Adenine Adducts with Diepoxybutane: Isolation and Analysis in Exposed Calf Thymus DNA

Tretyakova

Sangaiah²,

Yen³

et al. 1997

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“…Thus, this product was characterized as H3. Similarly, the decomposition product eluting at 8.8 min was characterized as 2-amino-7-(2,3,4-trihydroxybutyl)-1,7-dihydro-6H-purin-6-one (H4′), also a known compound (19,20).…”

Section: Characterization Of Decomposition Products Of P5 and P5′mentioning

confidence: 99%

Reaction of 1,2,3,4-Diepoxybutane with 2‘-Deoxyguanosine: Initial Products and Their Stabilities and Decomposition Patterns under Physiological Conditions

Zhang

Elfarra

2005

Abstract1,2,3,4-Diepoxybutane (DEB), an in vivo metabolite of 1,3-butadiene (BD), is a carcinogen and a potent mutagen. Previously, DEB was shown to react with 2′-deoxyguanosine (dG) under physiological conditions to produce seven major nucleoside adducts resulting from alkylation at the N1-(P8 and P9), N7-(P5 and P5′) and both the N1-and N 2 -positions of dG to form six-membered (P4-1 and P4-2) and seven-membered fused ring systems (P6), respectively (Zhang and Elfarra, Chem. Res. Toxicol. 2003, 16, 1606 ibid, 2004, 17, 521). In the present study the stabilities and decomposition products of the seven adducts under in vitro physiological conditions (phosphate buffer containing KCl, pH 7.4, 37 °C) were investigated. The results showed that P4-1, P4-2 and P6 were stable, whereas P5, P5′, P8 and P9 were labile with half-lives of 2.6, 2.7, 16 and 16 h, respectively. P5 and P5′ decomposed initially by the loss of the deoxyribose moiety to yield the corresponding guanine adduct P5D, which exhibited a half-life of 33 h and decomposed through opening of the remaining oxirane ring by dihydrogen phosphate ion, water or chloride ion. Decomposition of P8 yielded P4-1, P6 and nucleoside products resulting from opening of the oxirane ring by dihydrogen phosphate ion, water or chloride ion. Similarly, decomposition of P9 led to formation of P4-2, P6, and nucleoside products resulting from opening of the oxirane ring by dihydrogen phosphate ion, water or chloride ion. These results indicate that the initial products of the reaction of DEB with dG are P5, P5′, P8 and P9, whereas P4-1, P4-2 and P6 are secondary products. The results may also facilitate development of useful biomarkers of exposure to DEB.

“…Numerous DNA adducts of BD, mainly with guanine and adenine, have been reported in the literature. The N-7 guanine adducts of EB (24)(25)(26)(27)(28)(29)(30)(31) and DEB (32), N-1, N-3, N 6 , and N-7 adenine adducts of EB (30,31,(33)(34)(35), and N-3, N 6 , N-7, and N-9 adenine adducts of DEB (36)(37)(38) and N-3-thymidine adducts of EB (39) have been characterized. LC/ESI + /MS/MS (40,41) and 32 P-postlabeling (27,28,34,35,37,(42)(43)(44)(45) combined with chromatographic separation have been used to characterize and/or quantitate the adducts formed in vitro by EB and DEB.…”

Section: Introductionmentioning

confidence: 99%

Molecular Dosimetry of N-7 Guanine Adduct Formation in Mice and Rats Exposed to 1,3-Butadiene

Koç

Tretyakova

Walker

et al. 1999

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126

1,3-Butadiene (BD) is a high-volume chemical used in the production of rubber and plastic. BD is a potent carcinogen in mice and a much weaker carcinogen in rats, and has been classified as a probable human carcinogen. Upon metabolic activation in vivo, it forms DNA-reactive metabolites, 1,2-epoxy-3-butene (EB), 1,2:3, 4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBD). The molecular dosimetry of N-7 guanine adduct formation by these metabolites of BD in liver, lung, and kidney of B6C3F1 mice and F344 rats exposed to 0, 20, 62.5, or 625 ppm BD was studied. The adducts, racemic and meso forms of N-7-(2,3,4-trihydroxybut-1-yl)guanine (THB-Gua), N-7-(2-hydroxy-3-buten-1-yl)guanine (EB-Gua I), and N-7-(1-hydroxy-3-buten-2-yl)guanine (EB-Gua II), were isolated from DNA by neutral thermal hydrolysis, desalted on solid-phase extraction cartridges, and quantitated by LC/ESI(+)/MS/MS. The number of adducts per 10(6) normal guanine bases for a given adduct was higher in mice than rats exposed to 625 ppm BD, but generally similar at lower levels of exposure. The THB-Gua adducts were the most abundant (6-27 times higher than EB-Gua) and exhibited a nonlinear exposure-response relationship. In rats, the exposure-response curves for the formation of THB-Gua adducts reached a plateau after 62.5 ppm, suggesting saturation of metabolic activation. The number of THB-Gua adducts continued to increase in mice between 62.5 and 625 ppm BD. In contrast, the less common EB-Gua adducts had a linear exposure-response relationship in both species. Combining the information from this study with previous data on BD metabolism, we were able to estimate the number of THB-Gua that resulted from DEB and EBD, and conclude that most of the THB-Gua is formed from EBD. We hypothesize that most of the EBD arises from the immediate conversion of DEB to EBD within the endoplasmic reticulum. This study highlights the need for measurements of the levels of EBD in tissues of rats and mice and for the development of a unique biomarker for DEB that is available for binding to DNA.