Genotoxicity but not the AhR-mediated activity of PAHs is inhibited by other components of complex mixtures of ambient air pollutants.

In this study, we compared the genotoxicity and aryl hydrocarbon receptor (AhR)-dependent transcriptional changes of selected target genes in human lung epithelial A549 cells incubated for 24 h, either with extractable organic matter (EOMs) from airborne particles <2.5 µm (PM2.5) collected at four localities from heavily polluted areas of the Czech Republic or two representative toxic polycyclic aromatic hydrocarbons (PAHs) present in EOMs, benzo[a]pyrene (B[a]P) and benzo[k]fluoranthene (B[k]F). Genotoxic effects were determined using DNA adduct analysis or analysis of expression of selected AhR-related genes involved in bioactivation of PAHs (CYP1A1, CYP1B1) and transcriptional repression (TIPARP). Sampled localities differing in the extent and source of air pollution did not exhibit substantially different genotoxicity. DNA adduct levels induced by three subtoxic EOM concentrations were relatively low (1-5 adducts/10(8) nucleotides), compared to levels induced by similar concentrations of B[a]P, while B[k]F gave very low DNA adduct levels. Here, we compared genotoxicity and gene deregulation induced by complex mixtures containing PAHs with the effects of the comparable concentrations of individual PAHs. Our results suggested inhibition of formation of B[a]P-induced DNA adducts compared to individual B[a]P, probably attributable to competitive inhibition by other non-genotoxic EOM components. In contrast, induction of AhR target genes appeared not to be antagonized by the components of complex mixtures, as induction of CYP1A1, CYP1B1 and TIPARP transcripts reached maximum levels induced by PAHs.

Analysis of gene expression changes in A549 cells induced by organic compounds from respirable air particles.

A number of toxic effects of respirable ambient air particles (genotoxic effects, inflammation, oxidative damage) have been attributed to organic compounds bound onto the particle surface. In this study, we analyzed global gene expression changes caused by the extractable organic matters (EOMs) from respirable airborne particles <2.5µm (PM2.5), collected at 3 localities from heavily polluted areas of the Czech Republic and a control locality with low pollution levels, in human lung epithelial A549 cells. Although the sampled localities differed in both extent and sources of air pollution, EOMs did not induce substantially different gene expression profiles. The number of transcripts deregulated in A549 cells treated with the lowest EOM concentration (10µg/ml) ranged from 65 to 85 in 4 sampling localities compared to the number of transcripts deregulated after 30µg/ml and 60µg/ml of EOMs, which ranged from 90 to 109, and from 149 to 452, respectively. We found numerous commonly deregulated genes and pathways related to activation of the aryl hydrocarbon receptor (AhR) and metabolism of xenobiotics and endogenous compounds. We further identified deregulation of expression of the genes involved in pro-inflammatory processes, oxidative stress response and in cancer and developmental pathways, such as TGF-ß and Wnt signaling pathways. No cell cycle arrest, DNA repair or pro-apoptotic responses were identified at the transcriptional level after the treatment of A549 cells with EOMs. In conclusion, numerous processes and pathways deregulated in response to EOMs suggest a significant role of activated AhR. Interestingly, we did not observe substantial gene expression changes related to DNA damage response, possibly due to the antagonistic effect of non-genotoxic EOM components. Moreover, a comparison of EOM effects with other available data on modulation of global gene expression suggests possible overlap among the effects of PM2.5, EOMs and various types of AhR agonists.

Toxic effects of methylated benzo[a]pyrenes in rat liver stem-like cells.

The methylated benzo[a]pyrenes (MeBaPs) are present at significant levels in the environment, especially in the sediments contaminated by petrogenic PAHs. However, the existing data on their toxic effects in vitro and/or in vivo are still largely incomplete. Transcription factor AhR plays a key role in the metabolic activation of PAHs to genotoxic metabolites, but the AhR activation may also contribute to the tumor promoting effects of PAHs. In this study, the AhR-mediated activity of five selected MeBaP isomers was estimated in the DR-CALUX reporter gene assay performed in rat hepatoma cells. Detection of other effects, including induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs, DNA adduct formation, production of reactive oxygen species, oxidation of deoxyguanosine, and cell cycle modulation and apoptosis, was performed in the rat liver epithelial WB-F344 cell line, a model of liver progenitor cells. We identified 1-MeBaP as the most potent inducer of AhR activation, stable DNA adduct formation, checkpoint kinase 1 and p53 phosphorylation, and apoptosis. These effects suggest that 1-MeBaP is a potent genotoxin eliciting a typical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), and cell death. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in the angular ring, elicited only low levels DNA adduct formation and apoptosis. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. MeBaPs induced a weak production of ROS, which did not lead to significant oxidative DNA damage. Importantly, 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their tumor promotion and carcinogenicity.