Purification and Recovery of Bulky Hydrophobic DNA Adducts

Norwood, C.B.

doi:10.1006/abio.1999.4161

Cited by 8 publications

(5 citation statements)

References 40 publications

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“…Therefore, separation of the modified and the unmodified nucleotides and within the modified nucleotides offers the method much needed selectivity. Nowadays, capillary liquid chromatography (LC) [22], nano-LC [23] and capillary zone electrophoresis (CZE) [14,21,24,25] are the most used separation techniques for the analysis of DNA adducts and have proven to allow a very sensitive analysis procedure, e.g., in conjunction with mass spectrometric detection. ESI is currently the most used ionization technique for interfacing LC or CZE separations to MS.…”

Section: Introductionmentioning

confidence: 99%

Analysis of benzo[a]pyrene diol epoxide‐DNA adducts by capillary zone electrophoresis‐ electrospray ionization‐mass spectrometry in conjunction with sample stacking

et al. 2002

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Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)-ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)(-) at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)(-) at m/z 632] and a BPDE-dCMP adduct [(M-H)(-) at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH(3) from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH(2) position of the nucleobase. So, the three adducts could be identified as BPDE-N(2)-dGMP, BPDE-N(6)-dAMP and BPDE-N(4)-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.

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Section: Introductionmentioning

confidence: 99%

Analysis of benzo[a]pyrene diol epoxide‐DNA adducts by capillary zone electrophoresis‐ electrospray ionization‐mass spectrometry in conjunction with sample stacking

et al. 2002

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“…Although it may appear that such recoveries are low similar values were found by Norwood in his detailed study. 9 This relatively small recovery values could be due to some degradation of the adduct with its manipulation. Nevertheless, it is clear that the SPE procedure alone contribute less to the RSD than the two steps combined, i.e., digestion of DNA and the SPE clean-up.…”

Section: Resultsmentioning

confidence: 99%

“…Capillary electrophoresis (CE) is a technique that provides fast analysis time with superior separation efficiencies, ease of use, and low analysis cost. Several DNA adducts, especially PAH derivates such as BPDE, have been separated and analyzed by CE using UV, 9 mass spectrometry (MS), 10 and laser-induced fluorescence (LIF) detection. 11 Fluorescence detection methods are known to be very sensitive and when this detection mode is used together with CE low adduct levels can be detected.…”

Section: Introductionmentioning

confidence: 99%

dGMP-BPDE DNA adduct investigation in environmentally exposed rural workers by capillary electrophoresis with laser-induced fluorescence detection

Carrilho

Formenton-Catai

Lanças

et al. 2005

J. Braz. Chem. Soc.

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Um método alternativo à pos-marcação com 32 P radioativo foi proposta para a determinação do aduto de desoxiguanosina monofosfato com epóxido de benzo [a]pirenodiol (dGMP-BPDE), um biomarcador para exposição humana à hidrocarbonetos policíclicos aromáticos (PAH) carcinogênicos, usando eletroforese capilar com fluorescência induzida a laser (CE-LIF). O instrumento CE-LIF modificado foi ajustado para operar com um laser UV de He/Cd (325 nm) para detecção da fluorescência nativa dos adutos de BPDE. O método foi linear por mais de três ordens de grandeza e apresentou limites de detecção de 2,5 × 10 -9 mol L -1 com relação sinal/ruído igual a 3 após diluições sucessivas do padrão de dGMP-BPDE. Neste nível de concentração, a recuperação foi de 1 aduto para cada 10 7 bases não modificadas. Os valores de CV% para ensaios inter-e intra-dias foi melhor que 7% e os estudos de recuperação em três níveis diferentes renderam valores em torno de 50%. Este método foi validado e aplicado pela primeira vez a determinação de dGMP-BPDE em amostras de sangue provenientes de trabalhadores rurais Brasileiros, os quais foram expostos a PAH na colheita de cana-de-açucar e fornos de produção de carvão vegetal.An alternative method to 32 P-postlabeling has been proposed for sensitive detection and quantitation of deoxyguanosine monophosphate -benzo[a]pyrenediol epoxide (dGMP-BPDE), a biomarker for human exposure to carcinogenic polycyclic aromatic hydrocarbons (PAH), using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A modified CE-LIF instrument was adjusted to operate with a He/Cd UV laser (325 nm) for native fluorescence detection from BPDE adducts. The method was linear over three decades in concentration, with the detection limit of 2.5 × 10 -9 mol L -1 at the signal-to-noise ratio of three after consecutive dilution of the dGMP-BPDE standard. At this level, recovery of 1 adduct per 10 7 normal nucleotides was possible. The RSD values for inter-and intra-day determination were better than 7% and recovery studies at three different levels yielded values around 50%. This method has been validated and for the first time applied to determination of dGMP-BPDE in blood samples from Brazilian rural workers, which were exposed to PAH in sugar-cane plantation harvesting and charcoal-production ovens.

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“…However, this technique requires the use of radiolabeled compounds that can cause health risks, therefore more sensitive alternatives are desired for adduct detection. The separation and analysis of BPDE adducts by capillary electrophoresis (CE) using bare fused-silica capillaries with UV [9,10,11], mass spectrometry (MS) [12,13], and laser-induced fluorescence (LIF) [14,15] detection has been reported. It is well known that CE lacks of high sensitivity when using UV absorption detection and MS requires on-column sample enrichment injection procedures such as sample stacking [12,13].…”

Section: Introductionmentioning

confidence: 99%

Applications of capillary electrophoresis with laser-induced fluorescence for analysis of dGMP–BPDE adduct

Formenton-Catai

Carrilho

2003

Anal Bioanal Chem

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DNA adducts are thought to be crucial to the initiation of mutational and carcinogenic processes. Polycyclic aromatic hydrocarbons (PAHs) have been identified as one major source of carcinogenic risk since they can bind to DNA thus forming an adduct. Quantification of this adduct is important because it may correlate to the risk for cancer development. In this study, the adduct formed between 2'-deoxyguanosine 5'-monophosphate and benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) was analyzed by capillary electrophoresis. Both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) modes with laser-induced fluorescence detection were used for the separation and analysis of DNA adducts. The exploration of capillary electrophoresis in several modes provided different separation mechanisms in which the stereochemical forms of the adduct could be separated. The best result obtained was using a coated fused-silica capillary in Tris-TAPS buffer, which provided high sensitivity with a detection limit of 2.5x10(-9) mol L(-1). MECC separation of the BPDE adduct, although less sensitive, provided an efficient enantioselective separation option.

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Purification and Recovery of Bulky Hydrophobic DNA Adducts

Cited by 8 publications

References 40 publications

Analysis of benzo[a]pyrene diol epoxide‐DNA adducts by capillary zone electrophoresis‐ electrospray ionization‐mass spectrometry in conjunction with sample stacking

Analysis of benzo[a]pyrene diol epoxide‐DNA adducts by capillary zone electrophoresis‐ electrospray ionization‐mass spectrometry in conjunction with sample stacking

dGMP-BPDE DNA adduct investigation in environmentally exposed rural workers by capillary electrophoresis with laser-induced fluorescence detection

Applications of capillary electrophoresis with laser-induced fluorescence for analysis of dGMP–BPDE adduct

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