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.

Department of Genetic Ecotoxicology Institute of Experimental Medicine AS CR Prague Czech Republic

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Schlesinger RB (2007) The health impact of common inorganic components of fine particulate matter (PM2.5) in ambient air: a critical review. Inhal Toxicol 19: 811–832. PubMed

Valavanidis A, Fiotakis K, Vlachogianni T (2008) Airborne particulate matter and human health: toxicological assessment and importance of size and composition of particles for oxidative damage and carcinogenic mechanisms. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 26: 339–362. PubMed

Pelucchi C, Negri E, Gallus S, Boffetta P, Tramacere I, et al. (2009) Long-term particulate matter exposure and mortality: a review of European epidemiological studies. BMC Public Health 9: 453. PubMed PMC

Harrison RM, Smith DJ, Kibble AJ (2004) What is responsible for the carcinogenicity of PM2.5? Occup Environ Med 61: 799–805. PubMed PMC

Xue W, Warshawsky D (2005) Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicol Appl Pharmacol 206: 73–93. PubMed

IARC (2012) IARC monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Chemical Agents and Related Occupations. Lyon, France: IARC Publications. PubMed PMC

Baird WM, Hooven LA, Mahadevan B (2005) Carcinogenic polycyclic aromatic hydrocarbon-DNA adducts and mechanism of action. Environ Mol Mutagen 45: 106–114. PubMed

Michalopoulos G, Sattler GL, O’Connor L, Pitot HC (1978) Unscheduled DNA synthesis induced by procarcinogens in suspensions and primary cultures of hepatocytes on collagen membranes. Cancer Res 38: 1866–1871. PubMed

Limsirichaikul S, Niimi A, Fawcett H, Lehmann A, Yamashita S, et al. (2009) A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU). Nucleic Acids Res 37: e31. PubMed PMC

Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105: 2415–2420. PubMed PMC

Hanzalova K, Rossner P Jr, Sram RJ (2010) Oxidative damage induced by carcinogenic polycyclic aromatic hydrocarbons and organic extracts from urban air particulate matter. Mutat Res 696: 114–121. PubMed

Binkova B, Giguere Y, Rossner P Jr, Dostal M, Sram RJ (2000) The effect of dibenzo[a,l]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution. Mutat Res 471: 57–70. PubMed

Binkova B, Chvatalova I, Lnenickova Z, Milcova A, Tulupova E, et al. (2007) PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms. Mutat Res 620: 49–61. PubMed

Phillips DH, Castegnaro M (1999) Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols. Mutagenesis 14: 301–315. PubMed

Reddy MV, Randerath K (1986) Nuclease P1-mediated enhancement of sensitivity of [32P]- postlabeling test for structurally diverse DNA adducts. Carcinogenesis 7: 1543–1551. PubMed

Rossner P Jr, Uhlirova K, Beskid O, Rossnerova A, Svecova V, et al. (2011) Expression of XRCC5 in peripheral blood lymphocytes is upregulated in subjects from a heavily polluted region in the Czech Republic. Mutat Res 713: 76–82. PubMed

Rossner P Jr, Binkova B, Chvatalova I, Sram RJ (2002) Acrylonitrile exposure: the effect on p53 and p21(WAF1) protein levels in the blood plasma of occupationally exposed workers and in vitro in human diploid lung fibroblasts. MutatRes 517: 239–250. PubMed

Rossner P Jr, Binkova B, Sram RJ (2003) Heat shock proteins hsp32 and hsp70 as biomarkers of an early response? In vitro induction of heat shock proteins after exposure of cell culture to carcinogenic compounds and their real mixtures. Mutat Res 542: 105–116. PubMed

Binkova B, Sram RJ (2004) The genotoxic effect of carcinogenic PAHs, their artificial and environmental mixtures (EOM) on human diploid lung fibroblasts. Mutat Res 547: 109–121. PubMed

Sevastyanova O, Binkova B, Topinka J, Sram RJ, Kalina I, et al. (2007) In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part II: human cell lines. Mutat Res 620: 123–134. PubMed

Brockmann R, Beyer A, Heinisch JJ, Wilhelm T (2007) Posttranscriptional expression regulation: what determines translation rates? PLoS Comput Biol 3: e57. PubMed PMC

Hargrove JL, Hulsey MG, Beale EG (1991) The kinetics of mammalian gene expression. BioEssays 13: 667–674. PubMed

Lagerwerf S, Vrouwe MG, Overmeer RM, Fousteri MI, Mullenders LH (2011) DNA damage response and transcription. DNA repair 10: 743–750. PubMed

Volker M, Mone MJ, Karmakar P, van Hoffen A, Schul W, et al. (2001) Sequential assembly of the nucleotide excision repair factors in vivo. Mol Cell 8: 213–224. PubMed

Luijsterburg MS, Goedhart J, Moser J, Kool H, Geverts B, et al. (2007) Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC. J Cell Sci 120: 2706–2716. PubMed

Payne A, Chu G (1994) Xeroderma pigmentosum group E binding factor recognizes a broad spectrum of DNA damage. Mutat Res 310: 89–102. PubMed

Wittschieben BO, Iwai S, Wood RD (2005) DDB1-DDB2 (xeroderma pigmentosum group E) protein complex recognizes a cyclobutane pyrimidine dimer, mismatches, apurinic/apyrimidinic sites, and compound lesions in DNA. J Biol Chem 280: 39982–39989. PubMed

Croteau DL, Peng Y, Van Houten B (2008) DNA repair gets physical: mapping an XPA-binding site on ERCC1. DNA Repair (Amst) 7: 819–826. PubMed PMC

Shell SM, Zou Y (2008) Other proteins interacting with XP proteins. Adv Exp Mol Biol 637: 103–112. PubMed PMC

Sugasawa K (2010) Regulation of damage recognition in mammalian global genomic nucleotide excision repair. Mutat Res 685: 29–37. PubMed

Yang J, Liu X, Niu P, Zou Y, Gong Z, et al. (2007) Dynamic changes of XPA, XPC, XPF, XPG and ERCC1 protein expression and their correlations with levels of DNA damage in human bronchial epithelia cells exposed to benzo[a]pyrene. Toxicol Lett 174: 10–17. PubMed

Chen JK, Wu ZL, Liu YG, Lei YX (2000) Effects of metabolites of benzo(a)pyrene on unschedule DNA synthesis in BALB/3T3 cell line. Chemosphere 41: 139–142. PubMed

Adimoolam S, Ford JM (2003) p53 and regulation of DNA damage recognition during nucleotide excision repair. DNA Repair (Amst) 2: 947–954. PubMed

Feng Z, Hu W, Tang MS (2004) Trans-4-hydroxy-2-nonenal inhibits nucleotide excision repair in human cells: a possible mechanism for lipid peroxidation-induced carcinogenesis. Proc Natl Acad Sci U S A 101: 8598–8602. PubMed PMC

Feng Z, Hu W, Marnett LJ, Tang MS (2006) Malondialdehyde, a major endogenous lipid peroxidation product, sensitizes human cells to UV- and BPDE-induced killing and mutagenesis through inhibition of nucleotide excision repair. Mutat Res 601: 125–136. PubMed

Zhao X, Wan Z, Chen G, Zhu H, Jiang S, et al. (2002) Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China. Mutat Res 514: 177–192. PubMed

Libalova H, Uhlirova K, Klema J, Machala M, Sram RJ, et al. (2012) Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles. Part Fibre Toxicol 9: 1. PubMed PMC