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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., Competing Interests: The authors declare no conflict of interest.

The toxic equivalency concept used for the risk assessment of polychlorinated biphenyls (PCBs) is based on the aryl hydrocarbon receptor (AhR)-mediated toxicity of coplanar dioxin-like (DL) PCBs. Most PCBs in the environment, however, are non-dioxin-like (NDL) PCBs that cannot adopt a coplanar structure required for AhR activation. For NDL-PCBs, no generally accepted risk concept is available because their toxicity is insufficiently characterized. Here, we systematically determined in vitro toxicity profiles for 24 PCBs regarding 10 different mechanisms of action. Prior to testing, NDL-PCB standards were purified to remove traces of DL compounds. All NDL-PCBs antagonized androgen receptor activation and inhibited gap junctional intercellular communication (GJIC). Lower chlorinated NDL-PCBs were weak estrogen receptor (ER) agonists, whereas higher chlorinated NDL-PCBs were weak ER antagonists. Several NDL-PCBs inhibited estradiol-sulfotransferase activity and bound to transthyretin (TTR) but with much weaker potencies than reported for hydroxylated PCB metabolites. AhR-mediated expression of uridine-glucuronyl transferase isozyme UGT1A6 was induced by DL-PCBs only. Hierarchical cluster analysis of the toxicity profiles yielded three separate clusters of NDL-PCBs and a fourth cluster of reference DL-PCBs. Due to small differences in relative potency among congeners, the highly abundant indicator PCBs 28, 52, 101, 118, 138, 153, and 180 also contributed most to the antiandrogenic, (anti)estrogenic, antithyroidal, tumor-promoting, and neurotoxic potencies calculated for PCB mixtures reported in human samples, whereas the most potent AhR-activating DL-PCB-126 contributed at maximum 0.2% to any of these calculated potencies. PCB-168 is recommended as an additional indicator congener, given its relatively high abundance and antiandrogenic, TTR-binding, and GJIC-inhibiting potencies.

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

Tumour-associated expression of CA IX (carbonic anhydrase IX) is to a major extent regulated by HIF-1 (hypoxia-inducible factor-1) which is important for transcriptional activation and consists of the oxygen-regulated subunit HIF-1alpha and the partner factor ARNT [AhR (aryl hydrocarbon receptor) nuclear translocator]. We have previously observed that HIF-1alpha competes with the AhR for interaction with ARNT under conditions when both conditionally regulated factors are activated. We have therefore investigated whether TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced activation of the AhR pathway might interfere with CA IX expression. The results from the present study suggest that TCDD treatment reduces hypoxic induction of both CA IX mRNA and protein expression. Moreover, the transcriptional activity of the CA9 promoter was significantly reduced by expression of CAAhR (constitutively active AhR), which activates transcription in a ligand-independent manner. Finally, we found that ARNT is critical for both hypoxic induction and the TCDD-mediated inhibition of CA9 expression.