Search

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.

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.

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir&trade, 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&trade, 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&trade, In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes.

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.

Size-segregated particulate matter (PM) is frequently used in chemical and toxicological studies. Nevertheless, toxicological in vitro studies working with the whole particles often lack a proper evaluation of PM real size distribution and characterization of agglomeration under the experimental conditions. In this study, changes in particle size distributions during the PM sample manipulation and also semiquantitative elemental composition of single particles were evaluated. Coarse (1–10 μm), upper accumulation (0.5–1 μm), lower accumulation (0.17–0.5 μm), and ultrafine (

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.

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.

Identification of changes of phospholipid (PL) composition occurring during colorectal cancer (CRC) development may help us to better understand their roles in CRC cells. Here, we used LC-MS/MS-based PL profiling of cell lines derived from normal colon mucosa, or isolated at distinct stages of CRC development, in order to study alterations of PL species potentially linked with cell transformation. We found that a detailed evaluation of phosphatidylinositol (PI) and phosphatidylserine (PS) classes allowed us to cluster the studied epithelial cell lines according to their origin: i) cells originally derived from normal colon tissue (NCM460, FHC); ii) cell lines derived from colon adenoma or less advanced differentiating adenocarcinoma cells (AA/C1, HT-29); or, iii) cells obtained by in vitro transformation of adenoma cells and advanced colon adenocarcinoma cells (HCT-116, AA/C1/SB10, SW480, SW620). Although we tentatively identified several PS and PI species contributing to cell line clustering, full PI and PS profiles appeared to be a key to the successful cell line discrimination. In parallel, we compared PL composition of primary epithelial (EpCAM-positive) cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients, with PL profiles of cell lines derived from normal colon mucosa (NCM460) and from colon adenocarcinoma (HCT-116, SW480) cells, respectively. In general, higher total levels of all PL classes were observed in tumor cells. The overall PL profiles of the cell lines, when compared with the respective patient-derived cells, exhibited similarities. Nevertheless, there were also some notable differences in levels of individual PL species. This indicated that epithelial cell lines, derived either from normal colon tissue or from CRC cells, could be employed as models for functional lipidomic analyses of colon cells, albeit with some caution. The biological significance of the observed PL deregulation, or their potential links with specific CRC stages, deserve further investigation.

Organic pollutants associated with diesel exhaust particles (DEP), such as polycyclic aromatic hydrocarbons (PAHs) and their derivatives, may negatively impact human health. However, a comprehensive overview of their effects on endocrine nuclear receptor activities is still missing. Here, we evaluated the effects of extracts and chromatographic fractions (fractionated according to increasing polarity) of two standard reference materials derived from distinct types of diesel engines (SRM 2975, SRM 1650b), on activation of androgen receptor (AR), estrogen receptor alpha (ERα), peroxisome proliferator-activated receptor γ (PPARγ), glucocorticoid receptor (GR) and thyroid receptor α (TRα), using human cell-based reporter gene assays. Neither DEP standard modulated AR or GR activities. Crude extracts and fractions of SRM 1650b and SRM 2975 suppressed ERα-mediated activity in the ER-CALUX™ assay; however, this effect could be partly linked to their cytotoxicity in this cell line. We observed that only SRM 2975 extract and its fractions were partial PPARγ inducers, while SRM 1650b extract was not active towards this receptor. Importantly, we found that both extracts and polar fractions of SRM activated TRα and significantly potentiated the activity of endogenous TRα ligand, triiodothyronine. Based on a detailed chemical analysis of both extracts and their polar fractions, we identified several oxygenated PAH derivatives, that were present at relatively high levels in the analyzed DEP standards, including 3-nitrobenzanthrone (3-NBA), anthracene-9,10-dione, phenanthrene-9,10-dione, 9H-fluoren-9-one or benzo[a]anthracene-7,12-dione, to activate TRα activity. Nevertheless, these compounds provided only a minor contribution to the overall TRα activity identified in polar fractions. This suggests that yet unidentified polar polyaromatic compounds associated with DEP may, apart from their known impact on the aryl hydrocarbon receptor or steroid signaling, deregulate activities of additional nuclear receptors, in particular of TRα. This illustrates the need to better characterize endocrine disrupting activities of DEP.

Activation of the aryl hydrocarbon receptor (AhR)-mediated activity is one of key events in toxicity of polycyclic aromatic hydrocarbons (PAHs). Although various classes of AhR ligands may differentially activate human and rodent AhR, there is presently a lack of data on the human AhR-inducing relative potencies (REPs) of PAHs. Here, we focused on estimation of the AhR-mediated activities of a large set of environmental PAHs in human gene reporter AZ-AhR cell line, with an aim to develop the human AhR-based REP values with potential implications for risk assessment of PAHs. The previously identified weakly active PAHs mostly failed to activate the AhR in human cells. The order for REPs of individual PAHs in human cells largely corresponded with the available data from rodent-based experimental systems; nevertheless, we identified differences up to one order of magnitude in REP values of PAHs between human and rodent cells. Higher REP values were found in human cells for some important environmental contaminants or suspected carcinogens, such as indeno[1,2,3-cd]pyrene, benz[a]anthracene or benzo[b]fluoranthene, while lower REP values were determined for methyl-substituted PAHs. Our results also indicate that a different rate of metabolism for individual PAHs in human vs. rodent cells may affect estimation of REP values in human cell-based assay, and potentially alter toxicity of some compounds, such as benzofluoranthenes, in humans. We applied the AZ-AhR assay to evaluation of the AhR-mediated activity of complex mixtures of organic compounds associated with diesel exhaust particles, and we identified the polar compounds present in these mixtures as being particularly highly active in human cells, as compared with rodent cells. The present data suggest that differences may exist between the AhR-mediated potencies of PAHs in human and rodent cells, and that the AhR-mediated effects of polar PAH derivatives and metabolites in human cell models deserve further attention.

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.

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

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.

Modern vehicles equipped with Gasoline Direct Injection (GDI) engine have emerged as an important source of particulate emissions potentially harmful to human health. We collected and characterized gasoline exhaust particles (GEPs) produced by neat gasoline fuel (E0) and its blends with 15% ethanol (E15), 25% n-butanol (n-But25) and 25% isobutanol (i-But25). To study the toxic effects of organic compounds extracted from GEPs, we analyzed gene expression profiles in human lung BEAS-2B cells. Despite the lowest GEP mass, n-But25 extract contained the highest concentration of polycyclic aromatic hydrocarbons (PAHs), while i-But25 extract the lowest. Gene expression analysis identified activation of the DNA damage response and other subsequent events (cell cycle arrest, modulation of extracellular matrix, cell adhesion, inhibition of cholesterol biosynthesis) following 4 h exposure to all GEP extracts. The i-But25 extract induced the most distinctive gene expression pattern particularly after 24 h exposure. Whereas E0, E15 and n-But25 extract treatments resulted in persistent stress signaling including DNA damage response, MAPK signaling, oxidative stress, metabolism of PAHs or pro-inflammatory response, i-But25 induced changes related to the metabolism of the cellular nutrients required for cell recovery. Our results indicate that i-But25 extract possessed the weakest genotoxic potency possibly due to the low PAH content.

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.

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&ndash, 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.

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 mu g 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. Web of Science 17 11 art. no. 1833

Internal combustion engine emissions belong among the major anthropogenic sources of air pollution in urban areas. According to the International Agency for Research on Cancer, there is sufficient evidence of the carcinogenicity of diesel exhaust in human beings. Although alternative fuels, mainly biodiesel, have recently become popular, little is still known about the genotoxicity of emissions from these fuels. We analysed DNA damage expressed as the frequency of micronuclei (MN) in human bronchial epithelial cells (BEAS-2B), induced by extractable organic matter (EOM; tested concentrations: 1, 10 and 25 μg/ml) obtained from particle emissions from various blends of biodiesel with diesel fuels (including neat diesel fuel (B0), a blend of 70% B0 and 30% biodiesel (B30) and neat biodiesel (B100)). We also tested the effect of selected diesel exhaust organic/genotoxic components [benzo[a]pyrene (B[a]P) concentrations: 25, 100 and 200 μM; 1-nitropyrene (1-NP) concentrations: 1, 5 and 10 μM; 3-nitrobenzanthrone (3-NBA) concentrations: 1, 5 and 50 μM]. The cells were treated with the compounds for 28 and 48 hr. Our results showed that most of the tested compounds (except for the 25 μM B[a]P, 28-hr treatment) significantly increased MN frequency. The genotoxicity of EOMs from the engine emissions of diesel and biodiesel engines was comparable. Both nitro-PAH compounds demonstrated higher genotoxic potential in comparison with B[a]P. Considering our results and due to increasing popularity of alternative fuels, it is prudent that the potential genotoxic effects of various fuels are investigated across engine technologies and operating conditions in a relevant model system.

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.

ELISA is commonly used for the detection of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of whole body oxidative stress. However, the method has been criticized for high inter-laboratory variability and poor agreement with chromatographic techniques. We performed an inter-laboratory comparison of 8-oxodG assessed in 30 urine samples and a urine spiked with four different concentrations of 8-oxodG by ELISA using standardized experimental conditions, including: sample pretreatment with solid-phase extraction (SPE), performing analysis using a commercial kit from a single manufacturer and strict temperature control during the assay. We further compared the ELISA results with high-performance liquid chromatography-tandem mass spectrometry (HPLC MS/MS) and performed tentative identification of compounds that may contribute to the discrepancy between both methods. For all but one participating laboratory (Data 1) we observed consistent ELISA results lying mostly within 1 SD of the mean 8-oxodG concentration. Mean 8-oxodG levels assessed by ELISA correlated with the data obtained by HPLC MS/MS (R=0.679, p < 0.001). The correlation improved when Data 1 were excluded from the analysis (R=0.749, p < 0.001). We identified three outlying urine samples; one with an ELISA 8-oxodG concentration lower, and two with 8-oxodG levels higher, than those measured by HPLC MS/MS. Omitting these samples further improved inter-methodology agreement (R=0.869, p

Recent studies have shown urban dust particulate matter (UDPM) to decrease PAH-DNA adduct levels and cytochrome P450 (CYP) metabolic capacity in human mammary epithelial cell lines. The present study was designed to further elucidate the biochemical mechanisms involved in this inhibition process. We examined the effects of UDPM and its aromatic components on the metabolizing activities of human CYP1A1 and CYP1B1 enzymes as analyzed by the 7-ethoxyresorufin O-deethylation (EROD) assay. The data obtained indicate that the aromatic component of UDPM, derived from a neutral aromatic fraction of dichloromethane extract, inhibited the activity of CYP (no change in K M, decrease in k cat /K M ). Taken together, the decreases in the tumor initiating activity, PAH-DNA adduct formation and CYP1A1 and CYP1B1 activity by UDPM observed in vivo can be attributed to a noncompetitive inhibitory mechanism by the PAH component. Therefore, PAHs within a complex mixture could inhibit CYP metabolic capacity, thereby decreasin...

This study was focused on the fractionation of the mixture of polycyclic aromatic hydrocarbons and their derivatives, especially nitrated derivatives, found in soil and roadside dust extracts. Liquid chromatography with an open silica gel bed was used. Different volumes of hexane, hexane:dichloromethane (1:1 and 4:1, v/v), dichloromethane and methanol were used for the elution of individual fractions. Increasing volume of hexane was not sufficient for complete separation of a slightly polar fraction from the aromatic fraction. Increasing content of hexane in the elution mixture improved the separation of the group of target compounds. Soil and roadside dust extracts were obtained by using a pressurised solvent extractor with dichloromethane as the extraction solvent. Detection limits of the method ranged from 50 to 220 pg of individual nitro-PAHs injected into gas chromatograph. A significant amount of 1-nitropyrene was detected in the real samples collected close to the Brno centre. Este estudo focalizou o fracionamento da mistura de hidrocarbonetos policíclicos aromáticos e seus derivados, especialmente derivados nitrados, encontrados no solo e em extratos de poeira de beira de estrada. Foi utilizada cromatografia líquida com coluna de sílica gel aberta. Diferentes volumes de hexano, hexano:diclorometano (1:1 e 4:1, v/v), diclorometano e metanol foram usados para a eluição de frações individuais. O aumento do volume de hexano não foi suficiente para separar completamente uma pequena fração polar da fração aromática. O aumento do conteúdo de hexano na mistura de eluição melhorou a separação do grupo dos compostos alvos. Extratos de poeira de beira de estrada e do solo foram obtidos pelo uso de um solvente extrator pressurizado com diclorometano como solvente de extração. Os limites de detecção do método variaram de 50 a 220 pg para cada nitro-PAHs, injetado no cromatografo gasoso. Uma quantidade significante de 1-nitropireno foi detectada em amostras reais coletadas próximas ao centro de Brno.

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. 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. 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. 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.

Kinetics of photodegradation, half lifes and photodegradation products of two parent polycyclic aromatic hydrocarbons (anthracene and chrysene), their methyl-derivatives (9-methylanthracene and 6-methylchrysene) and selected nitro-derivatives (9-nitroanthracene and 6-nitrochrysene) were observed during exposition to UV irradiation in isooctane solutions. Additionally, influence of substituent position on photostability of PAH methyl-derivatives was observed in isooctane solutions. 1-Methylanthracene, 2-methylanthracene, 9-methylanthracene and 1-methylchrysene, 2-methylchrysene, 3-methylchrysene, 4-methylchrysene, 5-methylchrysene, 6-methylchrysene were chosen for the experiment. It was found that the effect of substituent type on photodegradation was very significant, especially in the case of nitrated polycyclic aromatic hydrocarbons. Decomposition of nitro-derivatives was easier than degradation of corresponding parent polycyclic aromatic hydrocarbons. On the other hand stability of methyl-derivatives w...

An analytical method based upon the pressurised solvent extraction and gas chromatography and mass spectrometry (GC-MS) for analysis of nitrated derivatives of polycyclic aromatic hydrocarbons (nitro-PAHs) in roadside dust has been developed. Five nitro-PAHs (1-nitronaphthalene, 1-nitropyrene, 9-nitroanthracene, 2-nitrofluorene, 6-nitrochrysene) were chosen for this study because of their variable physical properties and effect on human health. Experiments with spiked inert matrix were used for the best extraction conditions finding. Different extraction conditions (temperature, pressure, solvents and duration of extraction cycle) were examined. The highest recoveries were observed at the extraction pressure of 14 MPa, temperature of 100°C and two cycles of 5 min extraction time, when dichloromethane was used as an extraction solvent. These conditions were applied to the extraction of nitro-PAHs from roadside dust. After extraction the nitro-PAHs fraction was isolated by the silica gel column chromatograp...

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. ...

âupr P., M. ·karek, T. Barto‰, M. Ciganek, I. Holoubek: Assessment of Human Health Risk due to Inhalation Exposure in Cattle and Pig Farms in South Moravia. Acta Vet Brno 2005, 74: 305-312. The main topic of this study was human health risk assessment of defined inhalation exposure scenario in selected cattle and pig farms in south Moravia (Czech Republic). This exceptional evaluation of potential risks for farms manipulators was the main contribution of this study. Possible both human health risks, non-carcinogenic and carcinogenic (according to US EPA Human health risk assessment methodology), for feeders and other workers exposed to polluted indoor air in the farm stables were quantified in the selected pig and cattle farms with significantly increased concentrations mainly of carcinogenic PAHs and PCBs in the indoor air. No noncarcinogenic risks were determined in any of the localities, but also increased carcinogenic risks were observed. The highest carcinogenic health risk was found in the cattle stable (MAX IECR = 8.08·10 -6 ), the lowest one in the pig stable (MINIECR = 2.57·10 -6 ). Carcinogenic risk values in the farms under study were not extremely high, but those were approximately twice higher than a median value of the risk determined for research workers from Ko‰etice IECR = 5.96·10 -7 (years 1996 – 1999), Central European background monitoring station of EMEP.

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

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 24 h), 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 24 h 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 6 h exposure to PAHs led to a significantly higher AhR-mediated activity than the 24 h 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 6 h 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.

Monitoring river sediments contaminated predominantly with polyaromatic hydrocarbons by chemical and in vitro bioassay techniques

The photodecomposition of commercial lipophilic diaryliodonium hexafluoroantimonate SarCat ® SR-1012 was investigated in acetonitrile (ACN) and ACN/H 2 O mixed solvents by means of UV/Vis spectroscopy, an EPR in situ spin trapping technique and GC/MS analysis. The application of the spin traps nitrosodurene (ND) and 5,5-dimethyl-1-pyrroline N-oxide gave evidence for the production of radical intermediates corresponding to the breaking of the carbon–iodine bond, in addition to carbon–oxygen bonds in SR-1012 aryl substitution. The photolytic products analyzed in aprotic acetonitrile were iodobenzene, acetanilide, biphenyl and C 12 H 25 CH(OH)CH 3 , while in ACN/H 2 O (1:1, vol.) solution iodobenzene, biphenyl, C 12 H 25 CH(OH)CH 2 OC 6 H 4 OH and hydroxylated biphenyls were identified. The influence of oxygen on the product distribution in both solvent systems was inconsequential. The proton concentrations photogenerated in the irradiated SR-1012 ACN/H 2 O solutions were quantified spectrophotometrically using bromophenol blue indicator.

Road and barn dust samples were analysed for polycyclic aromatic hydrocarbons (PAHs) and nitrated polycyclic hydrocarbons (nitro‐PAHs). For this purpose, suitability of optimised HPLC with fluorimetric detection and GC/MS, both preceded by Soxhlet extraction of samples, gel permeation chromatography and HPLC with silica gel column for clean‐up, fractionation and preconcentration of the analytes, were compared. The detection limits of GC/MS for 16 PAHs ranged from 5 to 10 pg. The detection limits of GC/MS for 11 nitro‐PAHs ranged from 20 to 60 pg and those of on‐line reduction HPLC with fluorescence detection from 0.5 to 5 pg. Therefore, procedures with a higher separation efficiency and a more selective detection, such as GC/MS should be preferred when low concentrations of nitro‐PAHs are expected. PAH concentrations in road dust and barn dust samples ranged from 527 to 5680 μg/kg, and from 114 to 740 μg/kg, respectively. Nitro‐PAHs were detected in one sample of barn dust and two samples of road dust. Th...

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.

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.

Sediment extracts from three polluted sites of the river Elbe basin were fractionated using a novel on-line fractionation procedure. Resulting fractions were screened for mutagenic, aryl hydrocarbon (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 endpoints 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 like benzo[a]pyrene and benzofluoranthenes were identified in non-polar 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]anthracene-7-one, triclosan and 4-nonylphenol were identified as possible estrogenic and TTR-binding compounds. Thus, not only nonpolar compounds like PAHs, PCBs and PCDD/Fs but also the less characterized and investigated semipolar and polar substances should be considered as potent mutagenic, estrogenic, AhR-inducing, TTR-binding and GJIC inhibiting components for future studies. Effect-directed analysis of three sediment extracts and five in vitro endpoints suggests polar compounds as dominant substances for investigated effects., JRC.DDG.H.5-Rural, water and ecosystem resources

The objective of this study was to determine the concentrations of an extended series of polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs in outdoor air samples collected in low-contaminated urban areas, affected mainly by traffic emissions, and to estimate in vitro mutagenic and dioxin-like toxicity of extracts from these samples. Data on concentrations of PAHs and toxic in vitro potencies were compared in extracts obtained by different sampling methods. PAHs and their derivatives were analysed by high performance liquid chromatography with diode array and fluorescence detection, as well as gas chromatography with mass spectrometry. The total sum of 39 PAHs under study ranged from 6.7 to 62.7 ng.m(-3); of this, the sum of 16 U.S. EPA priority PAHs in urban air samples ranged from 3.2 to 6.2 ng.m(-3). Phenanthrene was the prevalent PAH in all air samples tested, with concentrations up to 17.6 ng.m(-3), followed by fluorene, fluoranthene and pyrene present mostly in the gaseous phase. Also, other low molecular weight PAHs (with MW up to 228) were distributed mostly in gaseous phase. The particulate phase contained mostly carcinogenic PAHs, among which, benzo[a]pyrene, indeno[1,2,3-cd]pyrene and benzofluoranthenes were predominant compounds (with benzo[a]pyrene reaching levels up to 1.57 ng.m(-3)). Traffic emissions were confirmed as the major source of PAHs in the airborne samples due to the presence of elevated concentrations of benzo[ghi]perylene and coronene. The most abundant nitrated PAH derivatives were nitronaphthalenes, which were present exclusively in the vapor phase; 9-nitroanthracene, 9-nitrophenantrene and 3-nitrofluoranthene were associated mostly with particulate matter (PM(10)). Bioassays for detection of the Ah receptor-mediated activity and mutagenicity in vitro were used as a screen of potential adverse effects of air pollutants emitted from traffic. The major part of mutagenic and aryl hydrocarbon receptor (AhR)-mediated activities was found to be present in the PM(10) fraction. Although the PM(10) sampling technique was found to be a suitable method regarding the subsequent determination of mutagenic and AhR-mediated activities in vitro, relative toxic potencies, associated with low molecular weight PAHs (such as tumor promotion and other adverse effects), could be underestimated.

Two methods to estimate distribution coefficients (K) between air and poly(dimethylsiloxane) (PDMS) coating of solid-phase microextraction (SPME) fibers for eight low molecular polycyclic aromatic hydrocarbons (PAHs) there are presented. The PDMS phases were used for determination of the coefficients according to equilibrium theory with help of a developed static calibration system (SCS). Another way to estimate the coefficients is based on the use of a linear relationship between the logarithm of the coefficients (log K) and linear temperature–programmed retention indexes (LTPRI) of the compounds without necessity to calibrate. The log K values for both of methods ranged from 5.2 (naphthalene) to 8.9 (pyrene) at 22 °C. Relative standard deviation (R.S.D.) of log K for each compound determined by static calibration was no more than 5.3%. R.S.D. of retention times for LTPRI indices did not exceed 0.28% for repeated injection. All experiments were implemented on a GC–MS system.

We investigate the issue of applicability of the solid phase microextraction (SPME) in the analysis of volatile organic compounds (VOCs) destruction products in the gliding arc discharge. Our research is focused on the measurements with the simple one stage gliding arc reactor, applied voltage was varied in the range of 3.5-4 kV. As a carrier gas, the dry air and its mixtures with nitrogen and oxygen, enriched by toluene, with flow rate of 1000-3500 ml/min was used. Total decomposition of toluene of 97 % was achieved at the oxygen content in carrier gas of 60 %. For measurements with air as a carrier gas, the highest efficiency was 95 %. We also tested the SPME technique suitability for the quantitative analysis of exhausts gases and if this technique can be used efficiently in the field to extract byproducts. Carbowax/divinylbenzene and Carboxen/polydimethylsiloxane/divinylbenzene fibres were chosen for sampling. Tens of various high-molecular substances were observed, especially a large number of oxygenous compounds and further several nitrogenous and CxHy compounds. The concentrations of various generated compounds strongly depend on the oxygen content in gas mixture composition. The results showed that the fiber coated by Carbowax/divinylbenzene can extract more products independently on the used VOC compound. The Carboxen/polydimethylsiloxane/divinylbenzene fiber is useful for the analysis of oxygenous compounds and its use will be recommended especially when the destruction is done in the oxygen rich atmosphere. With the higher ratio of oxygen in the carrier gas a distinctive decline of CxHy compounds amount have been observed. We also tried to describe the significant production of some compounds like benzyl alcohol, benzeneacetaldehyde, even in oxygen content is proximate 0 %. Experimental data demonstrated that it is necessary to use several SPME fibres for full-scale high-molecular products analysis.

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.

Reproductive dysfunction in wildlife populations can be a result of environmental contaminants binding to aryl hydrocarbon receptor (AhR) or estrogenic receptors. Signaling by both types of receptors can be affected by polycyclic aromatic hydrocarbons (PAHs), which are potential endocrine disruptors. However, our knowledge regarding the effects of oxygenated (oxy)-PAHs and azaarenes on AhR-mediated and estrogenic activities is incomplete. In the present study, we have identified 9-fluorenone, anthrone, anthraquinone, benzanthrone, benz[a]anthracene-7,12-dione, benz[c]acridine, and dibenz[a,h]acridine as prevalent oxy-PAHs and azaarenes found in river sediments. Their concentrations in sediment samples ranged from 2.1 to 165.2 ng g(-1) for oxy-PAHs and up to 27.3 ng g(-1) for azaarenes. Their relative AhR-inducing and estrogenic potencies were quantified in vitro using two cell lines that were stably transfected with a luciferase reporter gene system and expressed as induction equivalency factors (IEFs). The only oxy-PAHs with detectable levels of in vitro AhR-mediated activity were benzanthrone and benz[a]anthracene-7,12-dione. However, their IEFs were approximately three to four orders of magnitude lower than those of benzo[a]pyrene. On the other hand, azaarenes showed a strong AhR-mediated activity, with dibenzo[a,h]acridine being a far more potent inducer of activity than benzo[a]pyrene. Benzanthrone, benz[a]anthracene-7,12-dione, anthraquinone, and benz[a]acridine were weak inducers of in vitro estrogenic activity, with IEFs similar to that of benzo[a]pyrene. Based on concentrations and relative potencies, our results suggest that dibenzo[a,h]acridine can significantly contribute to the overall AhR-mediated activity in river sediments, whereas the remaining compounds do not. No studied compound was found to contribute significantly to estrogen receptor-mediated activity in vitro.

Extracts of sediment samples collected from the Morava River and its tributaries (Czech Republic) were examined for mutagenic, dioxin-like, and estrogenic activities. Moreover, the human leukemic HL-60 cell line was tested as a potential model for the detection of effects of environmental contaminants on cell proliferation and differentiation processes. Analytical data indicate that the sediments were contaminated predominantly with polycyclic aromatic hydrocarbons (PAHs) and phthalate esters. The sums of concentrations of 16 U.S. Environmental Protection Agency priority PAHs ranged from 0.8 to 13.2 micrograms/g and those of phthalates reached up to 3,000 ng/g, while only low levels of chlorinated hydrocarbons were found. The main goal of the present study was to determine effects of PAH prevalence on in vitro bioassays, with special emphasis on dioxin-like activity. The dioxin-like activity was tested using a reporter gene assay based on chemical-activated luciferase expression (the CALUX assay). Significant dioxin-like activity (2.6-40.1 micrograms/g benzo[a]pyrene equivalents and 5.9-48.2 ng/g 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents) was detected in all samples, and the results obtained with various exposure times or with both crude and PAH-deprived extracts indicate that the response was probably caused almost exclusively by the presence of high concentrations of PAHs. This corresponds with results of chemical analyses and indicates that various exposure times would allow a discrimination between dioxin-like activities of persistent compounds and easily metabolized aryl hydrocarbon (Ah) receptor inducers. Only sediment extracts containing the highest concentrations of PAHs were mutagenic, as determined by the umu assay. Estrogenic activity was found in several samples (4.75-22.61 pg/g estradiol equivalents) using cells stably transfected with an estrogen-responsive element linked to a luciferase promoter. Noncytotoxic doses of extracts had no effects on HL-60 cell proliferation, while two of the tested crude extracts significantly enhanced their all-trans retinoic acid-induced differentiation. These activities were not associated with phthalate esters and/or PAHs. Our results indicate that cellular and biochemical in vitro assays based on various specific modes of action may yield data complementary to results of mutagenicity tests and that they could be useful in environmental risk assessment. High levels of PAHs are apparently associated with dioxin-like and mutagenic activities rather than with estrogenic activity.