Biodegradation of benzo[a] pyrene with immobilized laccase: Genotoxicity of the products in HaCaT and A3 cells

Benzo[a]pyrene (BaP) is listed as a priority pollutant by the U.S. Environmental Protection Agency because it is one of the most potent carcinogens of all known polycyclic aromatic hydrocarbons (PAHs). The biodegradation of BaP is of interest as a means for mitigating its effects in polluted ecosystems. In the present study, BaP was oxidized with laccase from Trametes versicolor, which was immobilized on functionalized kaolinite particles, and the cytotoxicity and genotoxicity of BaP and its degradation intermediates were measured in human HaCaT keratinocytes and A3 T lymphocytes. Cytotoxicity was assessed by fluorescein diacetate (FDA) uptake, while the alkaline Comet assay measured genotoxicity, using tail moment, tail DNA content, and tail length as metrics for DNA damage. On the basis of first-order reaction kinetics, the half life (t(1/2)) for the oxidization of BaP by immobilized laccase was 58.5 hr. After 87 hr of oxidation, 20 mu M of BaP had decreased to 9.6 mu M. HPLC analysis identified 1,6-benzo[a]pyrene quinone (1,6-BaQ), 3,6-benzo[a]pyrene quinone (3,6-BaQ), and 6,12-benzo[a]pyrene quinone (6,12-BaQ) among the oxidation products. Most treatments of HaCaT cells and A3 lymphocytes with BaP or its quinone intermediates resulted in significant decreases in viability (P < 0.05); dose-dependent decreases in cell viability were detected at concentrations of 0.1, 1, and 5 mu M, but none of these treatments resulted in decreases of > 30%. While treatment of HaCaT cells with as little as 0.1 PM 6,12-BaQ caused significant DNA damage, DNA damage was detected in HaCaT cells only with 1 and 5 mu M 1,6-BaQ and 3,6-BaQ, and 5 mu M BaP. In Comet assays conducted with A3 lymphocytes, all three quinone intermediates caused significant increase in tail DNA content at 1 and 5 mu M. The results indicate that immobilized laccase is capable of degrading BaP, but several of those biodegradation products produce significant levels of DNA damage in human cells.