Jiří Kohoutek

Aryl hydrocarbon receptor-mediated disruption of contact inhibition is associated with connexin43 downregulation and inhibition of gap junctional intercellular communication

The aryl hydrocarbon receptor (AhR) contributes to the control of cell-to-cell communication, cell adhesion, migration or proliferation. In the present study, we investigated the regulation of connexin43 (Cx43) and Cx43-mediated gap junctional intercellular communication (GJIC) during the AhR-dependent disruption of contact inhibition in non-tumorigenic liver epithelial cells. The contact inhibition of cell proliferation is a process restricting the cell division of confluent non-transformed cells, which is frequently abolished in cancer cells; however, the mechanisms contributing to its disruption are still only partially understood. Disruption of contact inhibition, which was induced by toxic AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or polycyclic aromatic hydrocarbons in epithelial WB-F344 cells, reduced Cx43 protein levels, possibly via enhanced proteasomal degradation, significantly decreased the amount of gap junction plaques and downregulated GJIC, in an AhR-dependent manner. Although both intracellular and membrane Cx43 pools were markedly reduced in cells released from contact inhibition by TCDD, siRNA-mediated Cx43 knock-down was not sufficient to stimulate proliferation in contact-inhibited cells. Our data suggest that downregulation of Cx43/GJIC in non-transformed epithelial cells is an inherent part of disruption of contact inhibition, which occurs at the post-transcriptional level. This process runs in parallel with alterations of other forms of cell-to-cell communication, thus suggesting that toxic AhR agonists may simultaneously abrogate contact inhibition and reduce GJIC, two essential mechanisms linked to deregulation of cell-to-cell communication during tumor promotion and progression.

Particle Size Distribution of Halogenated Flame Retardants and Implications for Atmospheric Deposition and Transport

This study investigates the distribution of polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD) and a group of novel flame retardants (NFRs) on atmospheric aerosols. Two high volume cascade impactors were used to collect particulate fractions of ambient air over a one year period at urban and rural sites. The majority of FRs were found on the finest aerosols (<0.95 μm). Concentrations of HBCD were higher than those of ΣPBDEs. Moreover, we noted seasonality and spatial differences in particle size distributions, yet a large portion of the observed differences were due to differences in particulate matter (PM) itself. When normalized by PM, the size distributions of the FRs exhibited much greater heterogeneity. Differences existed between the FR distributions by molecular weight, with the higher molecular weight FRs (e.g., BDE-209, Dechlorane Plus) distributed more uniformly across all particulate size fractions. The seasonal, spatial, and compound-specific differences are of crucial importance when estimating dry and wet deposition of FRs as smaller aerosols have longer atmospheric residence times. Estimated wet and dry deposition of four representative FRs (BDE-47, BDE-209, HBCD, and Dechlorane Plus) using size-segregated aerosol data resulted in lower deposition estimates than when bulk aerosol data were used. This has implications for estimates of long-range atmospheric transport and atmospheric residence times, as it suggests that without size-specific distributions, these parameters could be underestimated for FRs.

Air and seawater pollution and air-sea gas exchange of persistent toxic substances in the Aegean Sea: spatial trends of PAHs, PCBs, OCPs and PBDEs

Near-ground air (26 substances) and surface seawater (55 substances) concentrations of persistent toxic substances (PTS) were determined in July 2012 in a coordinated and coherent way around the Aegean Sea based on passive air (10 sites in 5 areas) and water (4 sites in 2 areas) sampling. The direction of air–sea exchange was determined for 18 PTS. Identical samplers were deployed at all sites and were analysed at one laboratory. hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs) as well as dichlorodiphenyltrichloroethane (DDT) and its degradation products are evenly distributed in the air of the whole region. Air concentrations of p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE) and o,p′-DDT and seawater concentrations of p,p′-DDE and p,p′-DDD were elevated in Thermaikos Gulf, northwestern Aegean Sea. The polychlorinated biphenyl (PCB) congener pattern in air is identical throughout the region, while polybrominated diphenylether (PBDE)patterns are obviously dissimilar between Greece and Turkey. Various pollutants, polycyclic aromatic hydrocarbons (PAHs), PCBs, DDE, and penta- and hexachlorobenzene are found close to phase equilibrium or net-volatilisational (upward flux), similarly at a remote site (on Crete) and in the more polluted Thermaikos Gulf. The results suggest that effective passive air sampling volumes may not be representative across sites when PAHs significantly partitioning to the particulate phase are included.

Source identification, spatio-temporal distribution and ecological risk of persistent organic pollutants in sediments from the upper Danube catchment

Riverine sediments, collected on a monthly basis during a period of one year, from five sites in a mixed land use region of the Czech Republic were analysed for chlorinated and brominated persistent organic pollutants (POPs). The region is located in the upper catchment of the Danube River. The POPs concentrations were as follows: 11-930 pg g(-1) polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/Fs), 170-980 pg g(-1) dioxin-like polychlorinated biphenyls (dl-PCBs), 34-13,700 pg g(-1) polychlorinated naphthalenes (PCNs), 5.7-29,200 pg g(-1) polybrominated diphenylethers (PBDEs) and 0.21-351 ng g(-1) hexabromocyclododecanes (HBCDs). Concentrations expressed as toxic equivalents (TEQs), for PCDD/F+dl-PCB+PCN (TEQPCDD/F+dl-PCB+PCN) ranged from 0.37 to 19 pg g(-1). The results revealed a clear spatial separation between sites based on concentration and congener profile. There were also some obvious temporal patterns of selected POPs, which were related to river flow (seasonality) and organic carbon (TOC) of the sediment. Potential sources of POPs include local municipalities (flame retardants), some diffuse sources (PCNs and PCDDs/Fs) and potential point sources (PBDEs). Risk assessment based on risk quotients (RQ) revealed limited to medium ecological risk from PBDEs. TEQPCDD/F+dl-PCB+PCN were low relative to other European rivers, hence the risk to aquatic organisms was considered to be low. PCNs contributed significantly to overall TEQ in several cases.

Upregulation of CYP1B1 expression by inflammatory cytokines is mediated by the p38 MAP kinase signal transduction pathway

Cytochrome P450 1B1 (CYP1B1) is an enzyme that has a unique tumor-specific pattern of expression and is capable of bioactivating a wide range of carcinogenic compounds. We have reported previously that coordinated upregulation of CYP1B1 by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and the aryl hydrocarbon receptor ligands, may increase bioactivation of promutagens, such as benzo[a]pyrene (BaP) in epithelial cells. Here, we extend those studies by describing a novel mechanism participating in the regulation of CYP1B1 expression, which involves activation of the p38 mitogen-activated protein kinase (p38) and mitogen- and stress-activated protein kinase 1 (MSK1). Using inhibitors of p38 and MSKs, as well as mouse embryonic cells derived from p38α-deficient and MSK1/2 double knockout mice, we show here that TNF-α potentiates CYP1B1 upregulation via the p38/MSK1 kinase cascade. Effects of this inflammatory cytokine on CYP1B1 expression further involve the positive transcription elongation factor b (P-TEFb). The inhibition of the P-TEFb subunit, cyclin-dependent kinase 9 (CDK9), which phosphorylates RNA polymerase II (RNAPII), prevented the enhanced CYP1B1 induction by a combination of BaP and inflammatory cytokine. Furthermore, using chromatin immunoprecipitation assays, we found that cotreatment of epithelial cells with TNF-α and BaP resulted in enhanced recruitment of both CDK9 and RNAPII to the Cyp1b1 gene promoter. Overall, these results have implications concerning the contribution of inflammatory factors to carcinogenesis, since enhanced CYP1B1 induction during inflammation may alter metabolism of exogenous carcinogens, as well as endogenous CYP1B1 substrates playing role in tumor development.

Sources and Distributions of Polycyclic Aromatic Hydrocarbons and Toxicity of Polluted Atmosphere Aerosols

Levels and sources, mass size and phase distributions of parent PAHs and the toxicity of ambient aerosols at urban and rural sites of central (Czech Republic) and south-eastern (Bosnia and Hercegovina) Europe, from 2006 to 2008, are investigated. PAH pollution levels are much higher in winter than in summer, obviously due to the seasonalities of emission strength, photochemical degradation and mixing. The levels are in the range of 10–100 ng m−3 at urban and rural sites, while strong concentration gradients exist towards background sites, in particular in summer, due to both dispersion and degradation during transport. Based on back-trajectory analysis of air masses travelling to a background site in the Czech Republic, regionally significant PAH source areas were localized in eastern and south-eastern Europe, while western European countries emit less. PAHs represent a mass fraction of ≈100–500 ppm of the inhalable particulate matter (i.e. <10 μm). Based on a size resolution of 6 fractions, unimodal PAH mass size distributions were found at urban and rural sites which peaked almost exclusively in the accumulation mode (0.1–1.0 μm). Mass median diameters were found higher for semivolatile PAHs than for non volatile PAHs, probably related to re-distribution of semivolatiles in the aerosol according to the surface size distribution. Genotoxicity and AhR-mediated (i.e. dioxin-like) activity were found in all size classes at urban and rural sites in similar magnitudes. Activities were found in general highest in the fine particulate matter (i.e. <1 μm). All biological effects tested were also found in extracts of the gas-phase. PAH TEQ and antiandrogenicity were even mostly associated with gaseous pollutants. The calculated TEQ mediated by parent PAHs corresponded by average to 7.5 and 95% of the dioxin-like activity in the particulate and gaseous fractions, respectively.

Retinoid X receptor suppresses transformation by the v-myb oncogene

The v‐myb oncogene of avian myeloblastosis virus causes acute monoblastic leukemia in vivo and transforms myelomonocytic cells in culture. Retinoids are potent regulators of proliferation and differentiation in various cell types, and they can initiate differentiation in certain types of leukemic cells. However, the BM2 v‐myb‐trans‐formed chicken monoblastic cell line is resistant to retinoic acid treatment. We found that overexpression of the retinoid X receptor confers sensitivity of BM2 cells to retinoic acid, resulting in induction of growth arrest and terminal differentiation. In contrast, the frequency of apoptosis was not affected by the retinoid X receptor in this cell type. We also demonstrated that suppression of transformation by v‐Myb results from the negative effect of retinoid X receptor on v‐Myb transactivation function, similar to that previously described for the retinoic acid receptor. The retinoid X receptor‐induced inhibition of transactivation by v‐Myb seems to be enhanced by a cell type‐specific factor(s), which is not required by retinoic acid receptor. J. Leukoc. Biol. 66: 1039–1048; 1999.

Project TOCOEN ‐ the fate of selected organic pollutants in the environment. Part XXII.‐the contents of PAHs, PCBs, PCDDs/Fs in soil from surroundings of brno municipal waste incinerator

The Brno municipal waste incinerator (MWI), a Project TOCOEN model source of PAHs, PCBs, PCDDs/Fs was observed and the soil contamination in its surroundings was determined. The total observed concentrations of PAHs, PCBs PCDDs/Fs were found in the ranges of 369.2 to 5,077.9 ng•g‐1, 2.0 to 111 ng•g‐1, and 0.018 to 0.140 pg•g‐1 TEQ, respectively.

Aryl hydrocarbon receptor negatively regulates expression of the plakoglobin gene (Jup)

Plakoglobin is an important component of intercellular junctions, including both desmosomes and adherens junctions, which is known as a tumor suppressor. Although mutations in the plakoglobin gene (Jup) and/or changes in its protein levels have been observed in various disease states, including cancer progression or cardiovascular defects, the information about endogenous or exogenous stimuli orchestrating Jup expression is limited. Here we show that the aryl hydrocarbon receptor (AhR) may regulate Jup expression in a cell-specific manner. We observed a significant suppressive effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a model toxic exogenous activator of the AhR signaling, on Jup expression in a variety of experimental models derived from rodent tissues, including contact-inhibited rat liver progenitor cells (where TCDD induces cell proliferation), rat and mouse hepatoma cell models (where TCDD inhibits cell cycle progression), cardiac cells derived from the mouse embryonic stem cells, or cardiomyocytes isolated from neonatal rat hearts. The small interfering RNA (siRNA)-mediated knockdown of AhR confirmed its role in both basal and TCDD-deregulated Jup expression. The analysis of genomic DNA located ~2.5kb upstream of rat Jup gene revealed a presence of evolutionarily conserved AhR binding motifs, which were confirmed upon their cloning into luciferase reporter construct. The siRNA-mediated knockdown of Jup expression affected both proliferation and attachment of liver progenitor cells. The present data indicate that the AhR may contribute to negative regulation of Jup gene expression in rodent cellular models, which may affect cell adherence and proliferation.

Novel metabolites in cyanobacterium Cylindrospermopsis raciborskii with potencies to inhibit gap junctional intercellular communication

Despite intensive research into toxic bloom-forming cyanobacteria, the majority of their metabolites remain unknown. The present study explored in detail a novel bioactivity identified in cyanobacteria, i.e. inhibition of gap junctional intercellular communication (GJIC), a marker of tumor promotion. The extracellular mixture (exudate) of the cyanobacterial strain Cylindrospermopsis raciborskii (SAG 1.97) was fractionated by semi-preparative reversed phase HPLC, and the fractions assessed for their potencies to inhibit GJIC. Two non-polar fractions that significantly inhibited GJIC were further fractionated, tested and analyzed using multiple mass spectrometric methods. Investigations led to the identification of a putative chemical compound (molecular formula C18H34O3, m/z 299.2581 for the [M+H](+) ion) responsible for observed bioactivities. Specific inhibitors of signaling pathways were used to screen for biochemical mechanisms beyond GJIC inhibition, and the results indicate the involvement of ERK1/2 kinases via a mechanism related to the action of epidermal growth factor EGF but clearly distinct from other anthropogenic tumor promoters like polychlorinated biphenyls or polycyclic aromatic hydrocarbons. The chemical and in vitro toxicological characterizations of the newly described metabolite provide important insights into the still poorly understood health impacts of complex toxic cyanobacterial blooms and indicate that currently applied monitoring practices may underestimate actual risks.