Analysis of DNA Damage Biomarkers in Human Leukocytes by PAHs Exposure

Aims: To study the potential genotoxicity of two polynuclear aromatic hydrocarbons (PAHs), exposed cultured human leukocytes in vitro using two types of biomarkers genotoxicity: DNA strand breaks (DNA-SB) and adduct formation (DNA-PAHs).

Study Design: Human leukocytes were exposed to toxic cultures with different concentrations of anthracene (ANT), phenanthrene (PHE) and benzo(a)pyrene (B(a)P) for 24 hours. Four toxic test groups, PAHs, control group, analytic blank group and standard fluorescence group were considered.

Place and Duration of Study: Laboratorio de Citopatología Ambiental, of Departamento de Morfología at Escuela Nacional de Ciencias Biológicas, IPN; between february 2016 and july 2018.

Methodology: Human leukocytes cells were isolated and cell viability was previously verified by Trypan dye exclusion test. Firstly, the lethal concentrations with neutral red (NR50) assay for each one PAHs was obtained. Then sublethal concentrations range of these toxics for both biomarkers were used. In case of DNA fragmentation, a fluorochrome was used to mark DNA strandbreaks and isolation with alkaline solution finally determined with fluorescence spectroscopy. To test the formation of DNA-PAHs adducts, first of all they were isolated with a solvent system with polarity gradient and finally determined with fluorescence spectroscopy.

Results: PAHs with 3 aromatic rings showed lethal cytotoxicity, lower in case of the B(a)P with 5 aromatic rings. These results are in contrast with previously reported observations, in which non-adduct with DNA and DNA strandbreaks formation were detected with Anthracene and Phenanthrene while DNA adduction formation and DNA strandbreaks were produced in case of B(a)P.

Conclusion: Biomarkers may be used as suitable discriminants of genotoxic agents as well as of environmental pollutants with genotoxic potential and for application in studies of environmental risk assessment and in hazardous waste evaluation. The application of both genotoxic biomarkers, DNA strand breaks and production of adducts DNA-PAHs, as genotoxicity assays are quickly and accurate techniques for determining the carcinogenic potential of environmental samples.