Complex Alterations of Fatty Acid Metabolism and Phospholipidome Uncovered in Isolated Colon Cancer Epithelial Cells.

The development of colon cancer, one of the most common malignancies, is accompanied with numerous lipid alterations. However, analyses of whole tumor samples may not always provide an accurate description of specific changes occurring directly in tumor epithelial cells. Here, we analyzed in detail the phospholipid (PL), lysophospholipid (lysoPL), and fatty acid (FA) profiles of purified EpCAM+ cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients. We found that a number of FAs increased significantly in isolated tumor cells, which also included a number of long polyunsaturated FAs. Higher levels of FAs were associated with increased expression of FA synthesis genes, as well as with altered expression of enzymes involved in FA elongation and desaturation, including particularly fatty acid synthase, stearoyl-CoA desaturase, fatty acid desaturase 2 and ELOVL5 fatty acid elongase 5 We identified significant changes in ratios of specific lysoPLs and corresponding PLs. A number of lysophosphatidylcholine and lysophosphatidylethanolamine species, containing long-chain and very-long chain FAs, often with high numbers of double bonds, were significantly upregulated in tumor cells. Increased de novo synthesis of very long-chain FAs, or, altered uptake or incorporation of these FAs into specific lysoPLs in tumor cells, may thus contribute to reprogramming of cellular phospholipidome and membrane alterations observed in colon cancer.

Ordinary Gasoline Emissions Induce a Toxic Response in Bronchial Cells Grown at Air-Liquid Interface.

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir™, a three-dimensional (3D) model of the human airway, and BEAS-2B, cells originating from the human bronchial epithelium, grown at the air-liquid interface to assess the toxicity of ordinary gasoline exhaust produced by a direct injection spark ignition engine. The transepithelial electrical resistance (TEER), production of mucin, and lactate dehydrogenase (LDH) and adenylate kinase (AK) activities were analyzed after one day and five days of exposure. The induction of double-stranded DNA breaks was measured by the detection of histone H2AX phosphorylation. Next-generation sequencing was used to analyze the modulation of expression of the relevant 370 genes. The exposure to gasoline emissions affected the integrity, as well as LDH and AK leakage in the 3D model, particularly after longer exposure periods. Mucin production was mostly decreased with the exception of longer BEAS-2B treatment, for which a significant increase was detected. DNA damage was detected after five days of exposure in the 3D model, but not in BEAS-2B cells. The expression of CYP1A1 and GSTA3 was modulated in MucilAir™ tissues after 5 days of treatment. In BEAS-2B cells, the expression of 39 mRNAs was affected after short exposure, most of them were upregulated. The five days of exposure modulated the expression of 11 genes in this cell line. In conclusion, the ordinary gasoline emissions induced a toxic response in MucilAir™. In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes.

The processes associated with lipid peroxidation in human embryonic lung fibroblasts, treated with polycyclic aromatic hydrocarbons and organic extract from particulate matter.

Polycyclic aromatic hydrocarbons (PAHs) may cause lipid peroxidation via reactive oxygen species generation. 15-F2t-isoprostane (IsoP), an oxidative stress marker, is formed from arachidonic acid (AA) by a free-radical induced oxidation. AA may also be converted to prostaglandins (PG) by prostaglandin-endoperoxide synthase (PTGS) induced by NF-κB. We treated human embryonic lung fibroblasts (HEL12469) with benzo[a]pyrene (B[a]P), 3-nitrobenzanthrone (3-NBA) and extractable organic matter (EOM) from ambient air particulate matter <2.5 µm for 4 and 24 h. B[a]P and 3-NBA induced expression of PAH metabolising, but not antioxidant enzymes. The concentrations of IsoP decreased, whereas the levels of AA tended to increase. Although the activity of NF-κB was not detected, the tested compounds affected the expression of prostaglandin-endoperoxide synthase 2 (PTGS2). The levels of prostaglandin E2 (PGE2) decreased following exposure to B[a]P, whereas 3-NBA exposure tended to increase PGE2 concentration. A distinct response was observed after EOM exposure: expression of PAH-metabolising enzymes was induced, IsoP levels increased after 24-h treatment but AA concentration was not affected. The activity of NF-κB increased after both exposure periods, and a significant induction of PTGS2 expression was found following 4-h treatment. Similarly to PAHs, the EOM exposure was associated with a decrease of PGE2 levels. In summary, exposure to PAHs with low pro-oxidant potential results in a decrease of IsoP levels implying 'antioxidant' properties. For such compounds, IsoP may not be a suitable marker of lipid peroxidation.

The Biological Effects of Complete Gasoline Engine Emissions Exposure in a 3D Human Airway Model (MucilAirTM) and in Human Bronchial Epithelial Cells (BEAS-2B).

The biological effects induced by complete engine emissions in a 3D model of the human airway (MucilAirTM) and in human bronchial epithelial cells (BEAS-2B) grown at the air-liquid interface were compared. The cells were exposed for one or five days to emissions generated by a Euro 5 direct injection spark ignition engine. The general condition of the cells was assessed by the measurement of transepithelial electrical resistance and mucin production. The cytotoxic effects were evaluated by adenylate kinase (AK) and lactate dehydrogenase (LDH) activity. Phosphorylation of histone H2AX was used to detect double-stranded DNA breaks. The expression of the selected 370 relevant genes was analyzed using next-generation sequencing. The exposure had minimal effects on integrity and AK leakage in both cell models. LDH activity and mucin production in BEAS-2B cells significantly increased after longer exposures; DNA breaks were also detected. The exposure affected CYP1A1 and HSPA5 expression in MucilAirTM. There were no effects of this kind observed in BEAS-2B cells; in this system gene expression was rather affected by the time of treatment. The type of cell model was the most important factor modulating gene expression. In summary, the biological effects of complete emissions exposure were weak. In the specific conditions used in this study, the effects observed in BEAS-2B cells were induced by the exposure protocol rather than by emissions and thus this cell line seems to be less suitable for analyses of longer treatment than the 3D model.

Butyrate and docosahexaenoic acid interact in alterations of specific lipid classes in differentiating colon cancer cells.

Docosahexaenoic acid (DHA) and sodium butyrate (NaBt) exhibit a number of interactive effects on colon cancer cell growth, differentiation, or apoptosis; however, the molecular mechanisms responsible for these interactions and their impact on cellular lipidome are still not fully clear. Here, we show that both dietary agents together induce dynamic alterations of lipid metabolism, specific cellular lipid classes, and fatty acid composition. In HT-29 cell line, a model of differentiating colon carcinoma cells, NaBt supported incorporation of free DHA into non-polar lipids and their accumulation in cytoplasmic lipid droplets. DHA itself was not incorporated into sphingolipids; however, it significantly altered representation of individual ceramide (Cer) classes, in particular in combination with NaBt (DHA/NaBt). We observed altered expression of enzymes involved in Cer metabolism in cells treated with NaBt or DHA/NaBt, and exogenous Cer 16:0 was found to promote induction of apoptosis in differentiating HT-29 cells. NaBt, together with DHA, increased n-3 fatty acid synthesis and attenuated metabolism of monounsaturated fatty acids. Finally, DHA and/or NaBt altered expression of proteins involved in synthesis of fatty acids, including elongase 5, stearoyl CoA desaturase 1, or fatty acid synthase, with NaBt increasing expression of caveolin-1 and CD36 transporter, which may further promote DHA incorporation and its impact on cellular lipidome. In conclusion, our results indicate that interactions of DHA and NaBt exert complex changes in cellular lipidome, which may contribute to the alterations of colon cancer cell differentiation/apoptotic responses. The present data extend our knowledge about the nature of interactive effects of dietary fatty acids.

Dietary fatty acids specifically modulate phospholipid pattern in colon cells with distinct differentiation capacities.

PURPOSE: Although beneficial effects of the dietary n-3 docosahexaenoic acid (DHA) or butyrate in colon carcinogenesis have been implicated, the mechanisms of their action are not fully clear. Here, we investigated modulations of composition of individual phospholipid (PL) classes, with a particular emphasis on cardiolipins (CLs), in colon cells treated with DHA, sodium butyrate (NaBt), or their combination (DHA/NaBt), and we evaluated possible associations between lipid changes and cell fate after fatty acid treatment. METHODS: In two distinct human colon cell models, foetal colon (FHC) and adenocarcinoma (HCT-116) cells, we compared patterns and composition of individual PL classes following the fatty acid treatment by HPLC-MS/MS. In parallel, we measured the parameters reflecting cell proliferation, differentiation and death. RESULTS: In FHC cells, NaBt induced primarily differentiation, while co-treatment with DHA shifted their response towards cell death. In contrast, NaBt induced apoptosis in HCT-116 cells, which was not further affected by DHA. DHA was incorporated in all main PL types, increasing their unsaturation, while NaBt did not additionally modulate these effects in either cell model. Nevertheless, we identified an unusually wide range of CL species to be highly increased by NaBt and particularly by DHA/NaBt, and these effects were more pronounced in HCT-116 cells. DHA and DHA/NaBt enhanced levels of high molecular weight and more unsaturated CL species, containing DHA, which was specific for either differentiation or apoptotic responses. CONCLUSIONS: We identified a wide range of CL species in the colon cells which composition was significantly modified after DHA and NaBt treatment. These specific CL modulations might contribute to distinct cellular differentiation or apoptotic responses.

Comparative Analysis of Toxic Responses of Organic Extracts from Diesel and Selected Alternative Fuels Engine Emissions in Human Lung BEAS-2B Cells.

This study used toxicogenomics to identify the complex biological response of human lung BEAS-2B cells treated with organic components of particulate matter in the exhaust of a diesel engine. First, we characterized particles from standard diesel (B0), biodiesel (methylesters of rapeseed oil) in its neat form (B100) and 30% by volume blend with diesel fuel (B30), and neat hydrotreated vegetable oil (NEXBTL100). The concentration of polycyclic aromatic hydrocarbons (PAHs) and their derivatives in organic extracts was the lowest for NEXBTL100 and higher for biodiesel. We further analyzed global gene expression changes in BEAS-2B cells following 4 h and 24 h treatment with extracts. The concentrations of 50 µg extract/mL induced a similar molecular response. The common processes induced after 4 h treatment included antioxidant defense, metabolism of xenobiotics and lipids, suppression of pro-apoptotic stimuli, or induction of plasminogen activating cascade; 24 h treatment affected fewer processes, particularly those involved in detoxification of xenobiotics, including PAHs. The majority of distinctively deregulated genes detected after both 4 h and 24 h treatment were induced by NEXBTL100; the deregulated genes included, e.g., those involved in antioxidant defense and cell cycle regulation and proliferation. B100 extract, with the highest PAH concentrations, additionally affected several cell cycle regulatory genes and p38 signaling.

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

α-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.

Benzo[b]naphtho[d]thiophenes and naphthylbenzo[b]thiophenes: Their aryl hydrocarbon receptor-mediated activities and environmental presence.

Polycyclic aromatic sulfur heterocyclic compounds (PASHs) belong among ubiquitous environmental pollutants; however, their toxic effects remain poorly understood. Here, we studied the aryl hydrocarbon receptor (AhR)-mediated activity of dibenzothiophene, benzo[b]naphtho[d]thiophenes, and naphthylbenzo[b]thiophenes, as well as their presence in two types of environmental matrices: river sediments collected from both rural and urban areas, and in airborne particulate matter (PM2.5) sampled in cities with different levels and sources of pollution. Benzo[b]naphtho[2,1-d]thiophene, benzo[b]naphtho[2,3-d]thiophene, 2,2-naphthylbenzo[b]thiophene, and 2,1-naphthylbenzo[b]thiophene were newly identified as efficient AhR agonists in both rat and human AhR-based reporter gene assays, with 2,2-naphthylbenzo[b]thiophene being the most potent compound identified in both species. Benzo[b]naphtho[1,2-d]thiophene and 3,2-naphthylbenzo[b]thiophene elicited AhR-mediated activity only in the rat liver cell model, while dibenzothiophene and 3,1-naphthylbenzo[b]thiophene were inactive in either cell type. Independently of their ability to activate the AhR, benzo[b]naphtho[1,2-d]thiophene, 2,1-naphthylbenzo[b]thiophene, 3,1-naphthylbenzo[b]thiophene, and 3,2-naphthylbenzo[b]thiophene inhibited gap junctional intercellular communication in a model of rat liver epithelial cells. Benzo[b]naphtho[d]thiophenes were dominant PASHs present in both PM2.5 and sediment samples, with benzo[b]naphtho[2,1-d]thiophene being the most abundant one, followed by benzo[b]naphtho[2,3-d]thiophene. The levels of naphthylbenzo[b]thiophenes were mostly low or below detection limit. Benzo[b]naphtho[2,1-d]thiophene and benzo[b]naphtho[2,3-d]thiophene were identified as the most significant contributors to the AhR-mediated activity in the environmental samples evaluated in this study. Both induced nuclear translocation of the AhR, and they induced CYP1A1 expression in a time-dependent manner, suggesting that their AhR-mediated activity may depend on the rate of their intracellular metabolism. In conclusion, some PASHs could be significant contributors to the overall AhR-mediated toxicity of complex environmental samples suggesting that more attention should be paid to the potential health impacts of this group of environmental pollutants.

Characterization of elements, PAHs, AhR-activity and pro-inflammatory responses of road tunnel-derived particulate matter in human hepatocyte-like and bronchial epithelial cells.

The aims were to characterize the content of elements and polycyclic aromatic hydrocarbons (PAHs) in size-separated particulate matter (PM) sampled in a road tunnel, estimate the contribution of PAHs to the toxic potential, and measure the pro-inflammatory potential of PM samples and extracts with increasing polarity. Several elements/metals previously associated with cytokine responses were found. Based on PAHs levels and published PAHs potency, the calculated mutagenic and carcinogenic activities of size-separated samples were somewhat lower for coarse than fine and ultrafine PM. The AhR-activity of the corresponding PM extracts measured in an AhR-luciferase reporter model (human hepatocytes) were more similar. The highest AhR-activity was found in the neutral (parent and alkylated PAHs) and polar (oxy-PAHs) fractions, while the semi-polar fractions (mono-nitrated-PAHs) had only weak activity. The neutral and polar aromatic fractions from coarse and fine PM were also found to induce higher pro-inflammatory responses and CYP1A1 expression in human bronchial epithelial cells (HBEC3-KT) than the semi-polar fractions. Fine PM induced higher pro-inflammatory responses than coarse PM. AhR-inhibition reduced cytokine responses induced by parent PM and extracts of both size fractions. Contributors to the toxic potentials include PAHs and oxy-PAHs, but substantial contributions from other organic compounds and/or metals are likely.

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

BACKGROUND: Recently, 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. METHOD: For 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. RESULTS: Dose-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 Goeman's 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-ß 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). CONCLUSION: The microarray data suggested a prominent role of activation of aryl hydrocarbon receptor-dependent gene expression.

Aryl Hydrocarbon Receptor (AhR) Limits the Inflammatory Responses in Human Lung Adenocarcinoma A549 Cells via Interference with NF-κB Signaling.

Apart from its role in the metabolism of carcinogens, the aryl hydrocarbon receptor (AhR) has been suggested to be involved in the control of inflammatory responses within the respiratory tract. However, the mechanisms responsible for this are only partially known. In this study, we used A549 cell line, as a human model of lung alveolar type II (ATII)-like cells, to study the functional role of the AhR in control of inflammatory responses. Using IL-1ß as an inflammation inducer, we found that the induction of cyclooxygenase-2 and secretion of prostaglandins, as well as expression and release of pro-inflammatory cytokines, were significantly higher in the AhR-deficient A549 cells. This was linked with an increased nuclear factor-κB (NF-κB) activity, and significantly enhanced phosphorylation of its regulators, IKKα/ß, and their target IκBα, in the AhR-deficient A549 cells. In line with this, when we mimicked the exposure to a complex mixture of airborne pollutants, using an organic extract of reference diesel exhaust particle mixture, an exacerbated inflammatory response was observed in the AhR-deficient cells, as compared with wild-type A549 cells. Together, the present results indicate that the AhR may act as a negative regulator of the inflammatory response in the A549 model, via a direct modulation of NF-κB signaling. Its role(s) in the control of inflammation within the lung alveoli exposed to airborne pollutants, especially those which simultaneously activate the AhR, thus deserve further attention.

The impact of extractable organic matter from gasoline and alternative fuel emissions on bronchial cell models (BEAS-2B, MucilAir™).

Air pollution caused by road traffic has an unfavorable impact on the environment and also on human health. It has previously been shown, that complete gasoline emissions lead to toxic effects in cell models originating from human airways. Here we focused on extractable organic matter (EOM) from particulate matter, collected from gasoline emissions from fuels with different ethanol content. We performed cytotoxicity evaluation, quantification of mucin and extracellular reactive oxygen species (ROS) production, DNA breaks detection, and selected gene deregulation analysis, after one and five days of exposure of human bronchial epithelial model (BEAS-2B) and a 3D model of the human airway (MucilAir™). Our data suggest that the longer exposure had more pronounced effects on the parameters of cytotoxicity and mucin production, while the impacts on ROS generation and DNA integrity were limited. In both cell models the expression of CYP1A1 was induced, regardless of the exposure period or EOM tested. Several other genes, including FMO2, IL1A, or TNF, were deregulated depending on the exposure time. In conclusion, ethanol content in the fuels did not significantly impact the toxicity of EOM. Biological effects were mostly linked to xenobiotics metabolism and inflammatory response. BEAS-2B cells were more sensitive to the treatment.

Inhibition of Aryl Hydrocarbon Receptor (AhR) Expression Disrupts Cell Proliferation and Alters Energy Metabolism and Fatty Acid Synthesis in Colon Cancer Cells.

The aryl hydrocarbon receptor (AhR) plays a wide range of physiological roles in cellular processes such as proliferation, migration or control of immune responses. Several studies have also indicated that AhR might contribute to the regulation of energy balance or cellular metabolism. We observed that the AhR is upregulated in tumor epithelial cells derived from colon cancer patients. Using wild-type and the corresponding AhR knockout (AhR KO) variants of human colon cancer cell lines HCT116 and HT-29, we analyzed possible role(s) of the AhR in cell proliferation and metabolism, with a focus on regulation of the synthesis of fatty acids (FAs). We observed a decreased proliferation rate in the AhR KO cells, which was accompanied with altered cell cycle progression, as well as a decreased ATP production. We also found reduced mRNA levels of key enzymes of the FA biosynthetic pathway in AhR KO colon cancer cells, in particular of stearoyl-CoA desaturase 1 (SCD1). The loss of AhR was also associated with reduced expression and/or activity of components of the PI3K/Akt pathway, which controls lipid metabolism, and other lipogenic transcriptional regulators, such as sterol regulatory element binding transcription factor 1 (SREBP1). Together, our data indicate that disruption of AhR activity in colon tumor cells may, likely in a cell-specific manner, limit their proliferation, which could be linked with a suppressive effect on their endogenous FA metabolism. More attention should be paid to potential mechanistic links between overexpressed AhR and colon tumor cell metabolism.

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.

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, 17ß-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.

Transcription profiles in BEAS-2B cells exposed to organic extracts from particulate emissions produced by a port-fuel injection vehicle, fueled with conventional fossil gasoline and gasoline-ethanol blend.

Emissions from road traffic are among the major contributors to air pollution worldwide and represent a serious environmental health risk. Although traffic-related pollution has been most commonly associated with diesel engines, increasing evidence suggests that gasoline engines also produce a considerable amount of potentially hazardous particulate matter (PM). The primary objective of this study was to compare the intrinsic toxic properties of the organic components of PM, generated by a conventional gasoline engine fueled with neat gasoline (E0), or gasoline-ethanol blend (15 % ethanol, v/v, E15). Our results showed that while E15 has produced, compared to gasoline and per kg of fuel, comparable particle mass (µg PM/kg fuel) and slightly more particles by number, the organic extract from the particulate matter produced by E15 contained a larger amount of harmful polycyclic aromatic hydrocarbons (PAHs), as determined by the chemical analysis. To examine the toxicity, we monitored genome-wide gene expression changes in human lung BEAS-2B cells, exposed for 4 h and 24 h to a subtoxic dose of each PM extract. After 4 h exposure, numerous dysregulated genes and processes such as oxidative stress, lipid and steroid metabolism, PPARα signaling and immune response, were found to be common for both extract treatments. On the other hand, 24 h exposure resulted in more distinctive gene expression patterns. Although we identified several common modulated processes indicating the metabolism of PAHs and activation of aryl hydrocarbon receptor (AhR), E15 specifically dysregulated a variety of other genes and pathways related to cancer promotion and progression. Overall, our findings suggest that the ethanol addition to gasoline changed the intrinsic properties of PM emissions and increased the PAH content in PM organic extract, thus contributing to a more extensive toxic response particularly after 24 h exposure in BEAS-2B cells.

Markers of lipid oxidation and inflammation in bronchial cells exposed to complete gasoline emissions and their organic extracts.

Road traffic emissions consist of gaseous components, particles of various sizes, and chemical compounds that are bound to them. Exposure to vehicle emissions is implicated in the etiology of inflammatory respiratory disorders. We investigated the inflammation-related markers in human bronchial epithelial cells (BEAS-2B) and a 3D model of the human airways (MucilAir™), after exposure to complete emissions and extractable organic matter (EOM) from particles generated by ordinary gasoline (E5), and a gasoline-ethanol blend (E20; ethanol content 20% v/v). The production of 22 lipid oxidation products (derivatives of linoleic and arachidonic acid, AA) and 45 inflammatory molecules (cytokines, chemokines, growth factors) was assessed after days 1 and 5 of exposure, using LC-MS/MS and a multiplex immunoassay, respectively. The response observed in MucilAir™ exposed to E5 gasoline emissions, characterized by elevated levels of pro-inflammatory AA metabolites (prostaglandins) and inflammatory markers, was the most pronounced. E20 EOM exposure was associated with increased levels of AA metabolites with anti-inflammatory effects in this cell model. The exposure of BEAS-2B cells to complete emissions reduced lipid oxidation, while E20 EOM tended to increase concentrations of AA metabolite and chemokine production; the impacts on other inflammatory markers were limited. In summary, complete E5 emission exposure of MucilAir™ induces the processes associated with the pro-inflammatory response. This observation highlights the potential negative health impacts of ordinary gasoline, while the effects of alternative fuel are relatively weak.

Environmental six-ring polycyclic aromatic hydrocarbons are potent inducers of the AhR-dependent signaling in human cells.

The toxicities of many environmental polycyclic aromatic hydrocarbons (PAHs), in particular those of high-molecular-weight PAHs (with MW higher than 300), remain poorly characterized. The objective of this study was to evaluate the ability of selected environmentally relevant PAHs with MW 302 (MW302 PAHs) to activate the aryl hydrocarbon receptor (AhR), since this represents a major toxic mode of action of PAHs. A large number of the evaluated compounds exhibited strong AhR-mediated activities, in particular in human models. The studied MW302 PAHs also significantly contributed to the overall calculated AhR activities of complex environmental mixtures, including both defined standard reference materials and collected diesel exhaust particles. The high AhR-mediated activities of representative MW302 PAHs, e.g. naphtho[1,2-k]fluoranthene, corresponded with the modulation of expression of relevant AhR target genes in a human lung cell model, or with the AhR-dependent suppression of cell cycle progression/proliferation in estrogen-sensitive cells. This was in a marked contrast with the limited genotoxicity of the same compound(s). Given the substantial levels of the AhR-activating MW302 PAHs in combustion particles, it seems important to continue to investigate the toxic modes of action of this large group of PAHs associated with airborne particulate matter.