Effects of pharmaceuticals and personal care products (PPCPs) on multixenobiotic resistance (MXR) related efflux transporter activity in zebrafish (Danio rerio) embryos.

Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish (Danio rerio). Concentration dependent effects of test compounds were recorded with the dye accumulation assay using zebrafish embryos and in ATPase assays with recombinant zebrafish Abcb4. In the dye accumulation assay embryos at 24h post fertilisation (hpf) were exposed to 8µm rhodamine 123 along with test compounds for 2h. The rhodamine 123 tissue levels upon the exposure served as a measure for MXR transporter efflux activity of the embryo (low rhodamine levels - high activity; high levels - low activity). The known ABC protein inhibitors MK571, vinblastine and verapamil served as positive controls. All tested PPCPs affected rhodamine 123 accumulation in embryos. For seven compounds rhodamine tissue levels were either both decreased and increased depending on the compound concentration indicating both stimulation and inhibition of rhodamine 123 efflux by those compounds, only increased (inhibition, six compounds) or only decreased (stimulation, one compound). Recombinant zebrafish Abcb4 was obtained with the baculovirus expression system and PPCPs were tested for stimulation/inhibition of basal transporter ATPase activity and for inhibition of the transporter ATPase activity stimulated with verapamil. Eight of the tested PPCPs showed effects on Abcb4 ATPase activity indicating that their effects in the dye accumulation assay may have indeed resulted from interference with Abcb4-mediated rhodamine 123 efflux. Slight stimulatory effects were found for musk xylene, nerol, isoeugenol, α-amylcinnamaldehyde, α-hexylcinnamaldehyde and simvastatin indicating Abcb4 substrate/competitive inhibitor properties of those compounds. Likewise, decreases of the verapamil-stimulated Abcb4 ATPase activity by diclofenac and fluoxetine may indicate competitive transporter inhibition. Sertraline inhibited the basal and verapamil-stimulated Abcb4 ATPase activities suggesting its property as non-competitive Abcb4 inhibitor. Taken together, our finding that chemically diverse PPCPs interfere with MXR efflux activity of zebrafish indicates that (1) efflux transporters may influence bioaccumulation of many PPCPs in fish and that (2) many PPCPs may act as chemosensitisers. Furthermore, it appears that interference of PPCPs with efflux activity in zebrafish embryos is not only from effects on Abcb4 but also on other efflux transporter subtypes.

Expression patterns and organization of the hsp70 genes correlate with thermotolerance in two congener endemic amphipod species (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal.

We studied various aspects of heat-shock response with special emphasis on the expression of heat-shock protein 70 (hsp70) genes at various levels in two congener species of littoral endemic amphipods (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal which show striking differences in their vertical distribution and thermal tolerance. Although both the species studied demonstrate high constitutive levels of Hsp70, the thermotolerant E. cyaneus exhibited a 5-fold higher basal level of Hsp70 proteins under normal physiological conditions (7 °C) and significantly lower induction of Hsp70 after temperature elevation compared with the more thermosensitive E. verrucosus. We isolated the hsp70 genes from both species and analysed their sequences. Two isoforms of the cytosolic Hsp70/Hsc70 proteins were detected in both species under normal physiological conditions and encoded by two distinct hsp/hsc70 family members. While both Hsp70 isoforms were synthesized without heat shock, only one of them was induced by temperature elevation. The observed differences in the Hsp70 expression patterns, including the dynamics of Hsp70 synthesis and threshold of induction, suggest that the increased thermotolerance in E. cyaneus (compared with E. verrucosus) is associated with a complex structural and functional rearrangement of the hsp70 gene family and favoured the involvement of Hsp70 in adaptation to fluctuating thermal conditions. This study provides insights into the molecular mechanisms underlying the thermal adaptation of Baikal amphipods and represents the first report describing the structure and function of the hsp70 genes of endemic Baikal species dwelling in thermally contrasting habitats.

Multiple receptors mediate apoJ-dependent clearance of cellular debris into nonprofessional phagocytes.

Phagocytosis of apoptotic, senescent, and dying cells by macrophages is a well characterized process. More recently it has been shown that in addition to macrophages vital neighboring cells in the affected tissue participate in the cellular clearance. While scavenger receptors have been shown to mediate uptake into macrophages, it is poorly understood how cellular debris is internalized by nonprofessional phagocytes. We here analyze the endocytic activity of vital fibroblasts and epithelial cells exposed to cellular debris and membrane remnants. We show a mutual stimulation in the endocytosis of debris and apolipoproteinJ (clusterin) in these cells. Experiments using RAP (receptor-associated protein) to block ligand binding to LRP and megalin as well as studies in LRP- and megalin-deficient cells suggest that the uptake of apoJ and cellular debris is mediated by megalin, LRP, and yet unidentified internalization mechanisms.

Toxicity of waters from two streams to early life stages of brown trout (Salmo trutta f. fario L.), tested under semi-field conditions.

The development of brown trout (Salmo trutta f. fario L.) in water of two differently polluted streams and in a control situation was monitored in order to get insights into the impact of anthropogenic chemical stressors on the reproductive success of this fish species indigenous to both streams. The test streams, situated in the south of Stuttgart, Germany, were the complexly polluted Körsch stream and the less polluted Krähenbach stream. Bypass systems connected to the streams and a laboratory control system were used for continuous exposure of early brown trout stages shortly after fertilisation up to the end of the embryonic development. Temperature and oxygen conditions were standardised in all test series in order to minimise unspecific effects. The examined endpoints were: (1) mortality, (2) developmental rate, (3) time course of hatching, (4) malformations, and (5) growth. A retarded development, reduced growth rates and higher mortality rates of Körsch stream water exposed embryos indicated an embryotoxic potential for the more polluted stream. High infection-related mortality rates of embryos suggested the presence of confounding factors also in the less polluted Krähenbach stream. In parallel to the exposure experiment, physicochemical and limnochemical parameters as well as concentrations of organic contaminants and heavy metals were monitored. Analytical data confirm the different degrees of pollution of both streams.

[Opacity and opacity changes in composites]. / Opazität und Opazitätsveränderungen von Kompositen.

The opacity of 5 self-curing and 11 light-curing composite resins was measured 24 hours after sample/preparation and after 6 months of storage (dry or in water) by means of an UV-visible spectrophotometer. The opacity of the investigated hybrid composite resins was between 47.3 and 62.4%. The opacity of the microfilled composite resins ranged from 48 to 64.4%. The opacity was not influenced by the polymerization time. The spectral opacity decreased with greater wavelengths, particularly in microfilled composite resins. All materials except Durafill, which became more translucent, were more opaque after 180 days of storage in water. Storage in water for 180 days caused greater changes of the opacity than dry storage for the same time. The opacity changes resulting from storage in water can be reduced by increasing the polymerization time.