Sociedade Brasileira de Espectrometria de Massas – BrMASS
MET – Metabolome & Lipidome
Mass spectrometry as a tool to detect footprints of genomic
changes
Tiago Franco de Oliveira1, Ana Paula de Melo Loureiro1
tito@usp.br
1
Departamento de Análises Clínicas e Toxicológicas - FCF/USP
A current challenge is the measurement of low levels of DNA adducts that are
formed upon normal exposure situations to genotoxic agents. DNA enzymatic
methylation, on the other hand, is one of the main epigenetic mechanisms controlling
gene expression. The objective here was to design a sensitive analytical approach to
detect genotoxic and epigenetic changes in a same DNA sample.
An isotope dilution HPLC-ESI-MS/MS-MRM method with SPE online was
validated for quantitation of three DNA lesions: 8-oxodG, 1,N6-εdA and 1,N2-εdG. The
isotopic internal standards [15N5]8-oxodG, [15N5]1,N6-εdA and [15N5]1,N2-εdG were
synthesized, purified, and used for accurate quantitation, compensating for analyte loss
during sample preparation and matrix effects. An HPLC/DAD method was in turn
validated for 5-methyl-dC quantitation. Accuracy and precision were determined by
adding 8-oxodG (50, 100, 200 fmol), etheno adducts (10, 20, 40 fmol), and 5-methyldC (50, 100, 200 pmol) to DNA before enzymatic hydrolysis. Normal deoxynucleosides
were quantitated by HPLC/DAD. Excellent agreement was verified between added and
quantitated amounts for all analytes (R2 = 0,9998), with an average coefficient of
variation of 4,98%. The limits of quantitation on column were: 8-oxodG, 10 fmol; 1,N2εdG, 1 fmol; 1,N6-εdA, 0,1 fmol; 5-methyl-dC, 1 pmol. The global DNA methylation
could be measured using DNA amounts as low as 2 µg.
The method was applied to DNA samples obtained from cultured human HepG2
cell line exposed for 24 - 48 hours to 5, 10, 50, and 100 µM of purified CI Disperse
Blue 291 (DB291; m/z 509/511), a dinitro-bromo-phenylazo dye widely used that
contributes to the mutagenic activity of textile industry effluents. Additionally, we
investigated the formation of oxygen reactive species (ROS) by measuring intracellular
2',7'-dichlorofluorescein (DCF) fluorescence, cell cycle changes, DNA fragmentation,
mitochondrial membrane potential (m), intracellular calcium, and DB291
biotransformation. Increased m and intracellular calcium levels were observed after
cell incubation with 50 µM of the dye. Intracellular ROS generation was observed with
increasing dye concentrations. Cytotoxicity (IC50 = 74 µM) was accompanied by DNA
fragmentation, increased DNA levels of 8-oxodG and cell cycle arrest in G0/G1 and
G2/M. The DNA methylation pattern was also altered after DB291 exposure. DB291
biotransformation occurred in the first 24 hours of cell incubation, generating mainly a
product with m/z 435/437, which may be the result of nitro groups reduction and loss.
The biotransformation pathway may be related to DB291 toxic effects. Results point to
toxicity of the DB291 dye through mitochondrial changes and oxidative damage, which
can lead to mutagenicity, cell cycle arrest and cell death.
Acknowledgements: FAPESP; CNPq; PRP / USP, Prof. Paolo Di Mascio / IQUSP,
Profa. Gisela A. Umbuzeiro/UNICAMP.
4º Congresso BrMass – 10 a 13 de Dezembro de 2011