Bile acids are surface-active steroid compounds with toxic effects for bacteria. Recently, the isolation and characterization of a bacterium, Pseudomonas sp. strain Chol1, growing with bile acids as the carbon and energy source was reported. In this study, initial reactions of the aerobic degradation pathway for the bile acid cholate were investigated on the biochemical and genetic level in strain Chol1. These reactions comprised A-ring oxidation, activation with coenzyme A (CoA), and ␤-oxidation of the acyl side chain with the C 19 -steroid dihydroxyandrostadienedione as the end product. A-ring oxidizing enzyme activities leading to ⌬ 1,4 -3-ketocholyl-CoA were detected in cell extracts and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cholate activation with CoA was demonstrated in cell extracts and confirmed with a chemically synthesized standard by LC-MS/MS. A transposon mutant with a block in oxidation of the acyl side chain accumulated a steroid compound in culture supernatants which was identified as 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) by nuclear magnetic resonance spectroscopy. The interrupted gene was identified as encoding a putative acyl-CoA-dehydrogenase (ACAD). DHOPDC activation with CoA in cell extracts of strain Chol1 was detected by LC-MS/MS. The growth defect of the transposon mutant could be complemented by the wild-type ACAD gene located on the plasmid pBBR1MCS-5. Based on these results, the initiating reactions of the cholate degradation pathway leading from cholate to dihydroxyandrostadienedione could be reconstructed. In addition, the first bacterial gene encoding an enzyme for a specific reaction step in side chain degradation of steroid compounds was identified, and it showed a high degree of similarity to genes in other steroid-degrading bacteria.

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Investigating the accumulation and translocation of titanium dioxide nanoparticles with different surface modifications in static and dynamic human placental transfer models. European Journal of Pharmaceutics and Biopharmaceutics, (25 pp.).

Aquifer samples from the precipitation zone of an in situ iron and manganese removal plant that was operated for 10 years were analyzed for iron and manganese minerals. Measurements were performed by various chemical extraction techniques (5 M HCI, 0.008 M Ti(III)-EDTA, 0.114 M ascorbic acid), X-ray diffraction and Mössbauer spectroscopy. Chemical extractions showed that iron was precipitated as ferric oxides, whereas manganese was not oxidized but deposited as Mn(II) probably within carbonates. The ferric oxides in particular accumulate preferentially in the smaller grain- size fractions. This tendency was observed to a lesser extent for manganese. X-ray diffraction and Mössbauer spectroscopy showed that the ferric oxides were mainly crystalline (goethite, 50% to 100% of the iron). Ferrihydrite was found as well, but only as a minor fraction (< or = 12%). Pure manganese minerals were not found by X-ray diffraction. The precipitated amounts of iron (5 to 27 micromol/g Fe as ferric oxide) and manganese (1 to 4 micromol/g Mn) during 10 years operation of the treatment plant agree with values that were estimated from operational parameters (9 to 31 micromol/g Fe and 3 to 6 micromol/g Mn). Considering the small amounts of precipitated iron and manganese, no long-term risks of clogging of the aquifer are expected.

In view of the steadily increasing number of chemical compounds used in various products and applications, high-throughput toxicity screening techniques can help meeting the needs of 21st century risk assessment. Zebrafish (Danio rerio), especially its early life stages, are increasingly used in such screening efforts. In contrast, cell lines derived from this model organism have received less attention so far. A conceivable reason is the limited knowledge about their overall capacity to biotransform chemicals and the spectrum of expressed biotransformation pathways. One important biotransformation route is the mercapturic acid pathway, which protects organisms from harmful electrophilic compounds. The fully functional pathway involves a succession of several enzymatic reactions. To investigate the mercapturic acid pathway performance in the zebrafish embryonic cell line, PAC2, we analyzed the biotransformation products of the reactions comprising this pathway in the cells exposed to a nontoxic concentration of the reference substrate, 1-chloro-2,4-dinitrobenzene (CDNB). Additionally, we used targeted proteomics to measure the expression of cytosolic glutathione S-transferases (GSTs), the enzyme family catalyzing the first reaction in this pathway. Our results reveal that the PAC2 cell line expresses a fully functional mercapturic acid pathway. All but one of the intermediate CDNB biotransformation products were identified. The presence of the active mercapturic acid pathway in this cell line was further supported by the expression of a large palette of GST enzyme classes. Although the enzymes of the class alpha, one of the dominant GST classes in the zebrafish embryo, were not detected, this did not seem to affect the capacity of the PAC2 cells to biotransform CDNB. Our data provide an important contribution toward using zebrafish cell lines, specifically PAC2, for animal-free high- throughput screening in toxicology and chemical hazard assessment.