Nucleotide excision repair is not induced in human embryonic lung fibroblasts treated with environmental pollutants.

The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (-S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (-S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced

Final concentration of benzo[a]pyrene (B[a]P) in culture media after treatment of the cells with various concentrations of EOM.

<p>Final concentration of benzo[a]pyrene (B[a]P) in culture media after treatment of the cells with various concentrations of EOM.</p

Relative levels of XPE, XPC and XPA mRNAs.

<p>The levels of mRNAs were analyzed after the 6 h treatment of HEL12469 cells with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) in the absence (–S9) and presence (+S9) of the microsomal S9 fraction. Mean ± SD values from three independent cell treatments are shown, asterisks denote a significant (p<0.05) increase/decrease of mRNA levels. The baseline mRNA level after treatment of the cells with DMSO is represented by a bold horizontal line.</p

Western blotting analyses of the levels of XPE, XPC and XPA proteins.

<p>Protein expression was measured after the 24 h treatment of HEL12469 cells with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) in the absence (–S9) and presence (+S9) of the microsomal S9 fraction. A representative result of two independent experiments is shown. Amido Black-stained proteins were used as a loading control.</p

PAH content in extractable organic matter (EOM) from individual samplings.

*<p>Significant differences (p<0.001) in PAH concentrations between EOMs from Prague-summer vs. Ostrava-summer and Prague-winter vs. Ostrava-winter.</p

Bulky DNA adduct levels/10⁸ nucleotides detected in DNA extracted from HEL12469 cells.

<p>The cells were treated for 24 h with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) in the absence (–S9) and presence (+S9) of the microsomal S9 fraction. P–W = Prague-winter, O–W = Ostrava-winter, P–S = Prague-summer, O–S = Ostrava-summer, N.D. – not detectable.</p

Relative levels of XPE, XPC and XPA proteins in lysates of HEL12469 cells.

<p>The cells were treated with B[a]P and EOMs for 24 h. The data represent mean protein levels relative to the control sample from two independent experiments. P–W = Prague-winter, O–W = Ostrava-winter, P–S = Prague-summer, O–S = Ostrava-summer.</p

Relative levels of unscheduled DNA synthesis (UDS).

<p>UDS in HEL12469 cells was studied after 24 h treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOMs) in the absence (–S9) and presence (+S9) of the S9 microsomal fraction. Mean ± SD UDS values relative to the DMSO-treated control are shown, asterisks denote a significant (p<0.05) increase/decrease in the activity of UDS. The baseline levels of UDS in cells treated with DMSO is represented by a bold horizontal line. Each mean UDS value is based on the analysis of 700–2500 cells.</p