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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.

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Our current knowledge about the biotransformation enzymes in wild ruminants is limited. The present study aimed to compare basic levels and specific activities of cytochrome P450 isoforms (CYP1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A) in males of red deer ( Cervus elaphus), fallow deer ( Dama dama), roe deer ( Capreolus capreolus) and mouflon ( Ovis musimon). The proteins from the major cytochrome P450 (CYP) subfamilies were detected in all ruminant species by Western blotting, using polyclonal antibodies raised against rat or human CYP enzymes. The immunochemical data seem to suggest that humans and wild ruminants share some similar hepatic CYP enzymes corresponding to members of subfamilies 2 and 3; ruminant liver samples also contained two proteins cross-reacting with anti-rat CYP1A antibodies. High activities of CYP1A enzymes found in liver microsomes of male fallow deer and roe deer are indicative of increased susceptibility of these species towards promutagens that are metabolically activated by these CYPs. On the other hand, low activities of CYP1A-dependent alkoxyresorufin O-dealkylase activities were detected in male mouflons. Oxidative metabolism of testosterone was significantly higher in wild ruminants than the values previously reported from bulls. Androstene-3,17-dione and 6beta-hydroxytestosterone were the most important products of testosterone oxidation in liver microsomes of all the ruminant species under study. The highest CYP3A-dependent testosterone 6beta-hydroxylase activity was found in mouflons and fallow deer. A different pattern of CYP activities towards testosterone was found in roe deer, which showed high activities of testosterone 2beta-hydroxylase and lower production of androstene-3,17-dione. An increased activity of CYP4A-dependent laurate 12-hydroxylase found in roe deer and mouflons might indicate a higher metabolic turnover of fatty acids. The data on CYP activities indicated that high metabolic rates of steroids, fatty acids, and xenobiotics may occur in male wild ruminants. The highest hepatic activities specific for CYP3A, CYP2C, CYP2D, and CYP2E enzymes were found in mouflon, suggesting that this species has the highest biotransformation capacity.

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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.