Vinyl chloride-induced DNA adducts I: Quantitative determination of N²,3-ethenoguanine based on electrophore labeling

Abstract

A sensitive assay for quantitative determination of the vinyl chloride (VC)-induced cyclic DNA adduct N2,3-ethenoguanine (EG) was developed. The method is based on the detection of EG as its di-pentafluorobenzyl derivative (3,5-PFB2-EG). This compound exhibited good gas chromatographic properties and was detected with high sensitivity by gas chromatography with electron capture detection (limit of detection 300 amol/microliters injected solution) or with negative ion chemical ionization mass spectrometry monitoring the [M-181]-fragment ion at m/z 354 (GC-NICI-MS, limit of detection 190 amol/microliters injected solution). EG, its 13C-labeled analog [13C4]-EG and 3,5-PFB2-EG were synthesized and characterized by UV and fluorescence spectrophotometry, 1H- and 13C-NMR spectroscopy and mass spectrometry. The standards were used to optimize the isolation of EG and its derivatization with pentafluorobenzyl bromide (electrophore labeling) at fmol quantities. DNA solutions were spiked with EG, the DNA was depurinated by mild acid hydrolysis, and EG was isolated from the hydrolysates by low-pressure strong cation exchange chromatography with subsequent C18 solid-phase extraction. The extracted EG was electrophore labeled and 3,5-PFB2-EG was detected using GC-NICI-MS. [13C4]EG served as internal standard. 3,5-PFB2-EG was quantitated relative to its 13C-labeled analog by measuring the ion ratio m/z 354/358. The limit of detection for the complete method was 60 fmol EG/mumols guanine. The method was applied to liver DNA from young Sprague-Dawley rats exposed to 600 p.p.m. VC from day 10 through day 14 after birth. The EG concentration in these samples was 1.8 +/- 0.3 pmol/mumols guanine.

Reassigning the Structures of Natural Products Using NMR Chemical Shifts Computed with Quantum Mechanics: A Laboratory Exercise

“…2b,25 In this approach, isotropic shielding constants for several model compounds ( Figure 3) with known shifts in DMSO are computed (after structural optimization). 26 These compounds are chosen to reflect structural features present in the compounds of interest (e.g., 6−8). Guanine derivative 9 (Figure 3) 27 was used to correct 1 H and 13 C chemical shifts for the heterocycles in 6−8, while isopropylbenzene (cumene, 10, Figure 3) 28 was used to correct 1 H and 13 C chemical shifts for their isopropyl groups.…”

“…The data shown here were derived from HF/3-21G//HF/3-21G calculations, which are generated rapidly and are accurate enough to solve the structural problem posed herein. 26 …”

“…26 The learning outcomes of this exercise were as follows: (1) students understood that natural product structures are sometimes misassigned 29 and gained an appreciation for why misassignments occur; (2) students learned how to build and optimize structures of complex organic molecules, and predicted their NMR chemical shifts, using quantum chemistry; and (3) students learned how NMR chemical shifts predicted using quantum chemistry can be employed to distinguish between several possible structures for an organic molecule. Students also gained experience with managing and analyzing multiple sets of data.…”

Formação de adutos exocíclicos com bases de DNA: implicações em mutagênese e carcinogênese

“…Os agentes que levam a esses danos incluem produtos normais do metabolismo celular, como espécies reativas de oxigênio, produtos da peroxidação lipídica, agentes alquilantes, estrógenos, espécies reativas de nitrogênio, agentes clorinantes e certos intermediários de algumas vias metabólicas 22 , e fontes exógenas inevitáveis, tais como radiação UV, radiação ionizante, radioisótopos de ocorrência natural e numerosas substâncias químicas genotóxicas presentes naturalmente ou como contaminantes na dieta e no ar 23,24 . O desenvolvimento de métodos analíticos suficientemente sensíveis para a detecção dos danos basais em DNA foi de fundamental importância para que pudesse ser avaliada a contribuição desses danos para a carcinogênese [25][26][27][28][29][30] . Muitos dos danos endógenos observados no DNA correspondem a oxidações de bases 31,32 [45][46][47] .…”

Application of Gas Chromatography/Electron Capture Negative Chemical Ionization High-Resolution Mass Spectrometry for Analysis of DNA and Protein Adducts

“…EG, one of several adducts resulting from exposure to vinyl chloride, is promutagenic and has been shown to cause a high frequency of misincorporation during DNA replication (24,25). HEV (26) and 7-HEG (27) are adducts generated by the exposure to ethylene oxide.…”