Differential expression of CYP1A1 and CYP1A2 genes in H4IIE rat hepatoma cells exposed to TCDD and PAHs.

Rat hepatoma cells H4IIE were treated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) (dibenz(a,h)anthracene, benzo(a)pyrene, benz(a)anthracene, chrysene), low-concentration mixtures of PAHs and TCDD, and environmental mixtures contaminated by PAHs and their derivatives. Expression of the gene battery comprising cytochrome P450 Cyp1a1, Cyp1a2, Cyp1b1, and glutathione-s-transferase Gsta2 and Gstp was investigated using quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results revealed that TCDD induce Cyp1a1>Cyp1a2>Cyp1b1, while PAHs and PAH-containing environmental mixtures induce Cyp1a2>Cyp1a1>Cyp1b1 gene expression pattern. While low-concentration mixtures elicited a more pronounced response in comparison to single treatments, the typical gene expression patterns were not observed. In all samples, Gsta2 was predominantly expressed relative to Gstp. These findings indicate that differential Cyp1a1 and Cyp1a2 expression in the H4IIE cells might be used for detection of PAHs in highly contaminated environmental mixtures, but not in low-concentration mixtures of these compounds.

Endocrine disrupting, mutagenic, and teratogenic effects of upper Danube River sediments using effect-directed analysis.

Effect-directed analysis (EDA) can be useful in identifying and evaluating potential toxic chemicals in matrixes. Previous investigations of extracts of sediments from the upper Danube River in Germany revealed acute nonspecific and mechanism-specific toxicity as determined by several bioassays. In the present study, EDA was used to further characterize these sediments and identify groups of potentially toxic chemicals. Four extracts of sediments were subjected to a novel fractionation scheme coupled with identification of chemicals to characterize their ability to disrupt steroidogenesis or cause mutagenic and/or teratogenic effects. All four whole extracts of sediment caused significant alteration of steroidogenesis and were mutagenic as well as teratogenic. The whole extracts of sediments were separated into 18 fractions and these fractions were then subjected to the same bioassays as the whole extracts. Fractions 7 to 15 of all four extracts were consistently more potent in both the Ames fluctuation and H295R assays. Much of this toxicity could be attributed to polycyclic aromatic hydrocarbons, sterols, and in fraction 7-naphthoic acids. Because the fraction containing polychlorinated biphenyls, polychlorodibenzodioxin/furan, dichlorodiphenyltrichloroethane, and several organophosphates did not cause any observable effects on hormone production or a mutagenic response, or were not detected in any of the samples, these compounds could be eliminated as causative agents for the observed effects. These results demonstrate the value of using EDA, which uses multiple bioassays and new fractionation techniques to assess toxicity. Furthermore, to our knowledge this is the first study using the recently developed H295R assay within EDA strategies.

Identification and quantitative confirmation of dinitropyrenes and 3-nitrobenzanthrone as major mutagens in contaminated sediments.

Polar fractions of a sediment extract of the industrial area of Bitterfeld, Germany, have been subjected for effect-directed identification of mutagens using the Ames fluctuation assay with TA98. Mutagenicity could be well recovered in several secondary and tertiary fractions. Dinitropyrenes and 3-nitrobenzanthrone could be confirmed to contribute great shares of the observed mutagenicity. In addition, a multitude of polar polycyclic aromatic compounds has been tentatively identified in mutagenic fractions including nitro-PAHs, azaarenes, ketones, quinones, hydroxy-compounds, lactones and carboxylic acids although their contribution to mutagenicity could not be quantified due to a lack of standards. Diagnostic Salmonella strains YG1024 and YG1041 were applied to confirm the contribution of nitro-aromatic compounds. We suggest the inclusion of dinitropyrenes and 3-nitrobenzanthrone into sediment monitoring in order to minimize the mutagenic risk to aquatic organisms and to human health.

Effects-directed analysis of sediments from polluted marine sites in Norway.

The environmental status of two polluted marine sites in Norway was investigated by a combination of target chemical analysis and effect-directed analysis (EDA). The two selected sites, the Grenland area and Oslo harbor, in addition to two reference sites, were classified according to the Norwegian environmental classification system based upon results of the target chemical analyses. The polluted sites were characterized by high levels of metals, polycyclic aromatic hydrocarbons (PAH), and polychlorinated biphenyls (PCB). High levels of organotin compounds were also detected in Oslo harbor. The aryl hydrocarbon receptor (AhR) agonist activity in extracts of sediments from marine sites close to Oslo, Oslo harbor, and Grenland were investigated using the CALUX (chemical-activated luciferase expression) assay, which showed elevated levels of activity. As expected from the history of dioxin release into the Grenland area, the results were highest in this area. The presence of estrogen receptor (ER) and androgen receptor (AR) antagonists was also detected in the sediment extracts. Following fractionation of the sediment extracts, EDA was used to tentatively identify the AhR agonists. The compounds responsible for AhR agonist activity in samples from Oslo harbor were isolated in fraction 13, and to a lesser extent in fractions 9-11. In Grenland, the main activity was found in the more polar fractions, namely fractions 14-18. The AhR agonists identified in Oslo harbor were mainly PAH, while in the Grenland area the compounds identified were mainly nitrogen/oxygen-containing polyaromatic compounds (N/O-PAC).

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.

HPLC/APCI-FTICR-MS as a tool for identification of partial polar mutagenic compounds in effect-directed analysis.

Identification of unknown compounds remains one of the biggest challenges for the assignment of adverse effects of sediment contamination and other complex environmental mixtures to responsible toxicants by effect-directed analysis (EDA). The identification depends on information gained from biotesting, chromatographic separation, and mass spectrometric detection. Thus, a methodology is provided for non-target identification of partial polar mutagenic polyaromatic compounds in sediment extracts by using polymeric reversed-phase HPLC column, high-resolution mass spectrometry and PubChem database. After visualization and processing the chromatogram constituents by using deconvolution software, the unambiguous elemental compositions generated were used as input in PubChem database to find a possible identity for the suspected species. The retrieved structures from the database search were refined by characterized chromatographic and mass spectrometric classifiers based on 55 model compounds comprising eight different classes representing mutagenic substructures. The applicability of the method was demonstrated by positive and tentative identification of constituents of mutagenic sediment fractions similar to selected model compounds.

Partitioning-based dosing: an approach to include bioavailability in the effect-directed analysis of contaminated sediment samples.

One of the major challenges of effect-directed identification of hazardous chemicals in sediments is the appropriate consideration of bioavailability for a hazard-based prioritization of fractions and toxicants. The equilibrium partitioning approach is frequently used to estimate the bioavailability of sediment contaminants. To simulate partitioning ofthe constituents of complex sediment contaminations in the sediment-water-biota system, a new dosing technique based on silicone elastomer stirrer bars (silicone rods) for use in a growth inhibition test with the green algae Scenedesmus vacuolatus was developed. The dosing technique has a high loading capacity and a fast achievement of equilibrium (5-50 min) for a broad range of model compounds. The subsequent delivery of compounds stored by the silicone during the bioassay compensates possible losses of compounds and keeps the concentrations constant. Furthermore, a dose-dependent response is achieved for single compounds and complex mixtures from sediment extracts. The extracts of three sediment samples from the Elbe River Basin were fractionated using a multistep HPLC method and investigated in the bioassay using conventional DMSO dosing and loaded silicone rods. The results of the toxicity test clearly show that the toxicity patterns are quite different for the two dosing techniques, whereas the differences between the sampling locations are less pronounced. We suggestthat partitioning-based dosing better reflects exposure in sediments and thus provides a more realistic identification of key toxicants.

Effect-directed analysis of sediment-associated algal toxicants at selected hot spots in the river Elbe basin with a special focus on bioaccessibility.

Benthic algae are the dominant primary producers in many rivers and may be at risk of being affected by sediment-associated toxicants. Toxicity as well as bioaccessibility should be considered for the identification of major toxicants affecting benthic algae. Thus, effect-directed analysis (EDA) combining biotesting, fractionation, and chemical analysis was combined with both exhaustive and bioaccessibility-directed extraction to identify the major toxicants in sediments at three industrial sites in the Elbe River basin (Bitterfeld, Most, and Prelouc). Exhaustive accelerated solvent extraction was used together with bioaccessibility-directed extraction with TENAX in aqueous suspension. Growth inhibition of the green alga Scenedesmus vacuolatus identified polycyclic aromatic compounds as important algal toxicants at all three sites, while in Bitterfeld and Prelouc additional toxicants, such as N-phenyl-2-naphthylamine and 7H-benzo[de]anthracen-7-one, played an important role. Confirmation of the identified compounds was based on the index of confirmation quality (ICQ) comparing the effect of an artificial mixture to that of the original sample. The results suggest a successful confirmation of identified toxicants for most fractions with ICQ values between 0.5 and 2. The bioaccessibility of toxicants decreased in the order Prelouc > Bitterfeld > Most as determined by the investigation of TENAX samples. Similar total concentrations of toxicants in Prelouc and Most sediment result in bioaccessible concentrations in Prelouc that exceed those in Most by a factor of 10 to 20. The extraction with TENAX with a subsequent EDA proved to be a powerful tool for prioritizing contaminants and contaminated sites with respect to the hazard of sediment-associated toxicants to green algae.

Automated fractionation procedure for polycyclic aromatic compounds in sediment extracts on three coupled normal-phase high-performance liquid chromatography columns.

Polychlorinated biphenyls, naphthalenes, dibenzo-p-dioxins and furans, as well as polyaromatic hydrocarbons (PAHs), hydroxy-, keto- and nitro-PAHs and sulphur, oxygen and nitrogen heterocycles represent major groups of toxicants in contaminated sediments. The identification of toxic substances in effect-directed analysis is often based on a group-specific fractionation of these polycyclic aromatic compounds (PACs). Several fractionation steps using different methods, solvent exchange and concentration steps are generally necessary to achieve this aim increasing the risk of losses and artefact formation. In order to avoid these disadvantages and to enhance the throughput, an automated on-line fractionation method was developed using coupled and automatically connected columns, including cyanopropyl- and nitrophenylpropyl-bonded silica and porous graphitised carbon stationary phases. Exploiting the potential of each column, compounds are separated mainly according to their polarity, number of aromatic carbons and planarity. Excellent group-specific resolution, high reproducibility and good recoveries suggest that this method is suitable for the fractionation of a multitude of major sediment contaminants in one HPLC-run.