Evaluation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) adduct levels and DNA strand breaks in human peripheral blood lymphocytes exposed in vitro to polycyclic aromatic hydrocarbons with or without animal metabolic activation

Abstract

The polycyclic aromatic hydrocarbons (PAHs) dibenzo(a,h)anthracene, benzo(ghi)perylene, benzo(b)fluoranthene and benzo(a)pyrene have been identified in urban air from Mexico City and some of them are classified as human carcinogens. In the present study, human peripheral blood lymphocytes were exposed in vitro to different concentrations of PAHs with (+S9) or without (−S9) metabolic activation. The genotoxic and cytotoxic effects of each PAH were examined with an alkaline comet assay and trypan blue dye exclusion, and oxidative DNA damage was determined via the detection of 8-hydroxy-2′-deoxyguanosine (8-OhdG) adduct levels by enzyme-linked immunosorbent assay (ELISA). The DNA damage was evaluated with two genotoxicity parameters: the frequency of comets and the comet tail length. Concentrations of 20, 40, 80, 160 and 320 µM DB(a,h)A−S9; 20, 40, 80, 160 and 240 µM B(ghi)P−S9; 20, 30, 40, 60 and 80 µM B(b)F−S9; and 80 µM B(a)P−S9 for 24 h induced a small but significant increase in the means of comet frequency, in the tail length and in the 8-oHDg levels in relation to the control (0.5% DMSO−S9). However, all PAHs+S9 produced a more significant increase in DNA strand breaks and the level of 8-OHdG compared with the control (0.5% DMSO+S9), with a concentration-effect relationship. The viability of lymphocytes exposed to all PAHs−S9 and PAHs+S9 was not modified compared with the control. The results of this study demonstrate that the comet and ELISA are rapid, suitable and sensitive methods to detect in vitro PAH-induced DNA damage in human peripheral lymphocytes.

Keywords::

• Polycyclic aromatic hydrocarbons
• human peripheral lymphocytes
• oxidative DNA damage
• DNA strand breaks
• 8-OHdG

Acknowledgments

The authors thank CONACyT (Consejo Nacional de Ciencia y Tecnología), Apoyos Complementarios a Investigadores en Fase de Consolidación SNI Nivel I, PAPIIT-DGAPA-UNAM IN213908, and Posgrado en Ciencias Biológicas Universidad Nacional Autónoma de México for the support to carry out this project. We are grateful to the chemist Ana Luisa Alarcón Jiménez of the Laboratorio de Contaminación Ambiental del Centro de Ciencias de la Atmósfera for her technical assistance in the alkaline comet assay.

Declaration of interest

The authors declare no conflicts of interest.