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

Abstract

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.

Solid-Phase purification of deoxyguanosine-benzo[a]pyrene diol epoxide adducts from genomic DNA adduct synthesis

“…The development of such methodology was fundamental for quantitative determination of dG-BPDE adducts, as biomarkers, in order to evaluate the exposure to PAHs of rural workers in the sugar cane harvesting. 26 Capillary electrophoresis analysis with LIF detection provided excellent results for step-bystep monitoring of the synthesis and the purification yielding shorter analysis time, smaller sample volume, lower detection levels, and no solvent consumption when compared to HPLC analysis.…”

Progress in capillary electrophoresis of biomarkers and metabolites between 2002 and 2005

“…The use of ammonium acetate buffer in ACN makes it possible to couple CE with MS [67]. Recently the CE method performed using the PVA-coated capillary and Tris-TAPS buffer (pH 8.3) was applied for the determination of dGMP-BPDE in blood samples from Brazilian rural workers, who were exposed to the PAH in sugar cane plantation harvesting and charcoal productions ovens [68].…”