Miroslav Ciganek

Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay.

Activation of aryl hydrocarbon receptor (AhR) by 30 polycyclic aromatic hydrocarbons (PAHs) was determined in the chemical-activated luciferase expression (CALUX) assay, using two exposure times (6 and 24h), in order to reflect the metabolization of PAHs. AhR-inducing potencies of PAHs were expressed as induction equivalency factors (IEFs) relative to benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In 24h exposure assay, the highest IEFs were found for benzo[k]fluoranthene, dibenzo[a,h]anthracene and dibenzo[a,k]fluoranthene (approximately three orders of magnitude lower than TCDD) followed by dibenzo[a,j]anthracene, benzo[j]fluoranthene, indeno[1,2,3-cd]pyrene, and naphtho[2,3-a]pyrene. The 6h exposure to PAHs led to a significantly higher AhR-mediated activity than the 24h exposure (generally by two orders of magnitude), probably due to the high rate of PAH metabolism. The strongest AhR inducers showed IEFs approaching that of TCDD. Several PAHs, including some strong mutagens, such as dibenzo[a,l]pyrene, cyclopenta[cd]pyrene, and benzo[a]perylene, elicited only partial agonist activity. Calculation of IEFs based on EC25 values and/or 6h exposure data is suggested as an alternative approach to estimation of toxic potencies of PAHs with high metabolic rates and/or the weak AhR agonists. The IEFs, together with the recently reported relative mutagenic potencies of PAHs [Mutat. Res. 371 (1996) 123; Mutat. Res. 446 (1999) 1] were combined with data on concentrations of PAHs in extracts of model environmental samples (river sediments) to calculate AhR-mediated induction equivalents and mutagenic equivalents. The highest AhR-mediated induction equivalents were found for benzo[k]fluoranthene and benzo[j]fluoranthene, followed by indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[a]pyrene, dibenzo[a,j]anthracene, chrysene, and benzo[b]fluoranthene. High mutagenic equivalents in the river sediments were found for benzo[a]pyrene, dibenzo[a,e]pyrene, and naphtho[2,3-a]pyrene and to a lesser extent also for benzo[a]anthracene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[j]fluoranthene, dibenzo[a,e]fluoranthene and dibenzo[a,i]pyrene. These data illustrate that AhR-mediated activity of PAHs, including the highly mutagenic compounds, occurring in the environment but not routinely monitored, could significantly contribute to their adverse effects.

Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles

BackgroundRecently, we used cell-free assays to demonstrate the toxic effects of complex mixtures of organic extracts from urban air particles (PM2.5) collected in four localities of the Czech Republic (Ostrava-Bartovice, Ostrava-Poruba, Karvina and Trebon) which differed in the extent and sources of air pollution. To obtain further insight into the biological mechanisms of action of the extractable organic matter (EOM) from ambient air particles, human embryonic lung fibroblasts (HEL12469) were treated with the same four EOMs to assess changes in the genome-wide expression profiles compared to DMSO treated controls.MethodFor this purpose, HEL cells were incubated with subtoxic EOM concentrations of 10, 30, and 60 μg EOM/ml for 24 hours and global gene expression changes were analyzed using human whole genome microarrays (Illumina). The expression of selected genes was verified by quantitative real-time PCR.ResultsDose-dependent increases in the number of significantly deregulated transcripts as well as dose-response relationships in the levels of individual transcripts were observed. The transcriptomic data did not differ substantially between the localities, suggesting that the air pollution originating mainly from various sources may have similar biological effects. This was further confirmed by the analysis of deregulated pathways and by identification of the most contributing gene modulations. The number of significantly deregulated KEGG pathways, as identified by Goemans global test, varied, depending on the locality, between 12 to 29. The Metabolism of xenobiotics by cytochrome P450 exhibited the strongest upregulation in all 4 localities and CYP1B1 had a major contribution to the upregulation of this pathway. Other important deregulated pathways in all 4 localities were ABC transporters (involved in the translocation of exogenous and endogenous metabolites across membranes and DNA repair), the Wnt and TGF-β signaling pathways (associated particularly with tumor promotion and progression), Steroid hormone biosynthesis (involved in the endocrine-disrupting activity of chemicals), and Glycerolipid metabolism (pathways involving the lipids with a glycerol backbone including lipid signaling molecules).ConclusionThe microarray data suggested a prominent role of activation of aryl hydrocarbon receptor-dependent gene expression.

Polar Compounds Dominate in Vitro Effects of Sediment Extracts

Sediment extracts from three polluted sites of the river Elbe basin were fractionated using a novel online fractionation procedure. Resulting fractions were screened for mutagenic, aryl hydrocarbon receptor (AhR)-mediated, transthyretin (TTR)-binding, and estrogenic activities and their potency to inhibit gap junctional intercellular communication (GJIC) to compare toxicity patterns and identify priority fractions. Additionally, more than 200 compounds and compound classes were identified using GC-MS/MS, LC-MS/MS, and HPLC-DAD methods. For all investigated end points, major activities were found in polar fractions, which are defined here as fractions containing dominantly compounds with at least one polar functional group. Nonpolar PAH fractions contributed to mutagenic and AhR-mediated activities while inhibition of GJIC and estrogenic and TTR-binding activities were exclusively observed in the polar fractions. Known mutagens in polar fractions included nitro- and dinitro-PAHs, azaarenes, and keto-PAHs, while parent and monomethylated PAHs such as benzo[a]pyrene and benzofluoranthenes were identified in nonpolar fractions. Additionally, for one sample, high AhR-mediated activities were determined in one fraction characterized by PCDD/Fs, PCBs, and PCNs. Estrone, 17β-estradiol, 9H-benz[de]anthracen-7-one, and 4-nonylphenol were identified as possible estrogenic and TTR-binding compounds. Thus, not only nonpolar compounds such as PAHs, PCBs, and PCDD/Fs but also the less characterized and investigated more polar substances should be considered as potent mutagenic, estrogenic, AhR-inducing, TTR-binding, and GJIC-inhibiting components for future studies.

Activation of the aryl hydrocarbon receptor is the major toxic mode of action of an organic extract of a reference urban dust particulate matter mixture: The role of polycyclic aromatic hydrocarbons

Many of the toxic and carcinogenic effects of urban air pollution have been linked to polycyclic aromatic hydrocarbons (PAHs) adsorbed to airborne particulate matter (PM). The carcinogenic properties of PAHs in complex organic mixtures derived from PM have been chiefly attributed to their mutagenicity. Nevertheless, PAHs are also potent activators of the aryl hydrocarbon receptor (AhR), which may contribute to their nongenotoxic effects, including tumor promotion. As the genotoxicity of carcinogenic PAHs in complex mixtures derived from urban PM is often inhibited by other mixture constituents, the AhR-mediated activity of urban PM extracts might significantly contribute to the carcinogenic activity of such mixtures. In the present study, we used an organic extract of the urban dust standard reference material, SRM1649a, as a model mixture to study a range of toxic effects related to DNA damage and AhR activation. Both the organic extract and its neutral aromatic fraction formed a low number of DNA adducts per nucleotide in the liver epithelial WB-F344 cells model, without inducing DNA damage response, such as tumor suppressor p53 activation and apoptosis. In contrast, we found that this extract, as well as its neutral and polar fractions, were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events occurred at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls were only minor contributors to the overall AhR-mediated activity. Taken together, our data suggest that more attention should be paid to the AhR-dependent nongenotoxic events elicited by urban PM constituents, especially PAHs and their derivatives.

Concentrations of methylated naphthalenes, anthracenes, and phenanthrenes occurring in Czech river sediments and their effects on toxic events associated with carcinogenesis in rat liver cell lines.

Alkylated polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants. In the present study, we determined levels of monomethylated naphthalenes (MeNap), phenanthrenes (MePhe), and anthracenes (MeAnt) in Czech river sediments. The levels of MePhe generally were lower than the concentrations of phenanthrene. In contrast, both MeNap and MeAnt were found at levels higher than their respective parent compounds in the majority of sampling sites. We then investigated their aryl hydrocarbon receptor (AhR)-mediated activity, accumulation of phosphorylated p53 protein, induction of expression of cytochrome P450 1A1 (CYP1A1), inhibition of gap junctional intercellular communication (GJIC), and effects on cell proliferation in rat liver cell models to evaluate the relative importance of these toxicity mechanisms of low-molecular-weight methylated PAHs. Methylated phenanthrene and anthracene compounds were weak inducers of AhR-mediated activity as determined both in a reporter gene assay system and by detection of the endogenous gene (Cyp1a1) induction. 2-Methylphenanthrene was the most potent AhR ligand. Contribution of MeAnt and MePhe to overall AhR-inducing potencies should be taken into account in PAH-contaminated environments. Nevertheless, their effects on AhR were not sufficient to modulate cell proliferation in a normal rat liver progenitor cell model system. These PAHs only had a marginal effect on p53 phosphorylation at high doses of 1-, 3-, and 9-MePhe as well as 1 MeAnt. On the other hand, both 2- and 9-MeAnt as well as all the MePhe under study were efficient inhibitors of GJIC, suggesting that these compounds might act as tumor promoters. In summary, inhibition of GJIC and partial activation of AhR seem to be the most prominent toxic effects of the methylated PAHs in the present study.

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.

The aryl hydrocarbon receptor-mediated and genotoxic effects of fractionated extract of standard reference diesel exhaust particle material in pulmonary, liver and prostate cells

Diesel exhaust particles (DEP) and the associated complex mixtures of organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), or their derivatives, have been suggested to exert deleterious effects on human health. We used a set of defined cellular models representing liver, lung and prostate tissues, in order to compare non-genotoxic and genotoxic effects of crude and fractionated extract of a standard reference DEP material - SRM 1650b. We focused on the aryl hydrocarbon receptor (AhR)-mediated activity, modulation of cell proliferation, formation of DNA adducts, oxidative DNA damage, and induction of DNA damage responses, including evaluation of apoptosis, and phosphorylation of p53 tumor suppressor and checkpoint kinases (Chk). Both PAHs and the polar aromatic compounds contributed to the AhR-mediated activity of DEP-associated organic pollutants. The principal identified AhR agonists included benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene, chrysene and several non-priority PAHs, including benzochrysenes and methylated PAHs. In contrast to PAHs, polar compounds contributed more significantly to overall formation of DNA adducts associated with phosphorylation of p53, Chk1 or Chk2, and partly with apoptosis. Therefore, more attention should be paid to identification of DEP-associated polar organic compounds, contributing to the AhR activation and cytotoxic/genotoxic effects of complex airborne mixtures of organic contaminants produced by diesel engines.

PHOTOSTABILITY OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) AND NITRATED POLYCYCLIC AROMATIC HYDROCARBONS (NPAHs) IN DICHLOROMETHANE AND ISOOCTANE SOLUTIONS

Abstract Kinetics of photodegradation of eight PAHs (naphthalene, anthracene, phenanthrene, pyrene, biphenyl, chrysene, benz[a]anthracene and tetracene) and five NPAHs (1–nitronaphthalene, 2–nitrofluorene, 9–nitroanthracene, 1–nitropyrene and 6–nitrochrysene) was studied in isooctane and dichloromethane solutions standing in a laboratory and in a light simulator. Samples were stored in closed glass vials and fused silica cuvettes. PAHs and NPAHs were divided into three groups according to their life times. Degradation of all PAHs and NPAHs was faster in dichloromethane compared with isooctane. Decomposition of NPAHs was easier than degradation of corresponding parent PAHs. The most frequent photoproducts were quinones, benzoic acid and other derivatives with hydroxyl, carbonyl and carboxyl groups for both PAHs and NPAHs. Chlorinated compounds were identified in dichloromethane. Degradation intermediates underwent further oxidation, fragmentation, reduction or radical chlorine addition in dichloromethane. Final degradation products of all PAHs were n–alkanes. Based on GC–MS data the mechanism of photoproducts formation was proposed.

Mitochondrial targeting overcomes ABCA1-dependent resistance of lung carcinoma to α-tocopheryl succinate

Abstractα-Tocopheryl succinate (α-TOS) is a promising anti-cancer agent due to its selectivity for cancer cells. It is important to understand whether long-term exposure of tumour cells to the agent will render them resistant to the treatment. Exposure of the non-small cell lung carcinoma H1299 cells to escalating doses of α-TOS made them resistant to the agent due to the upregulation of the ABCA1 protein, which caused its efflux. Full susceptibility of the cells to α-TOS was restored by knocking down the ABCA1 protein. Similar resistance including ABCA1 gene upregulation was observed in the A549 lung cancer cells exposed to α-TOS. The resistance of the cells to α-TOS was overcome by its mitochondrially targeted analogue, MitoVES, that is taken up on the basis of the membrane potential, bypassing the enhanced expression of the ABCA1 protein. The in vitro results were replicated in mouse models of tumours derived from parental and resistant H1299 cells. We conclude that long-term exposure of cancer cells to α-TOS causes their resistance to the drug, which can be overcome by its mitochondrially targeted counterpart. This finding should be taken into consideration when planning clinical trials with vitamin E analogues.

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-β 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.