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.

Synergistic interaction caused to human gingival fibroblasts from dental monomers.

OBJECTIVES: The toxicity of monomers like bisphenol-A-glycidyldimethacrylate (BisGMA) and urethane-dimethacrylate (UDMA) to cells is well studied. In these studies solubilizers, which have a toxic potential, are used to dissolve the basic monomers in the aqueous medium. In these experiments it is not possible to confidently exclude a synergistic effect of basic monomers and solubilizers in cells. Moreover, less is known about the synergistic interaction between basic- and comonomers (triethyleneglycoldimethacrylate (TEGDMA); 2-hydroxyethylmethacrylate (HEMA)) in cells. We dissolved the basic monomers in the comonomers and incubated human gingival fibroblasts (HGFs) with these binary mixtures in different concentrations. METHODS: Proliferating HGFs monolayers were cultured in the absence or presence of mixtures of BisGMA/TEGDMA, BisGMA/HEMA, UDMA/TEGDMA and UDMA/HEMA. Twenty-four hours later XTT was added and the formazan formation was quantified. EC(50) values were obtained at half-maximum-effect concentrations from fitted curves. RESULTS: EC(50) values were (mmol/l; mean±sem; n=5): 0.01 BisGMA/0.48 TEGDMA; 0.04 BisGMA/4.99 HEMA; 0.04 UDMA/1.60 TEGDMA and 0.02 UDMA/2.26 HEMA. All tested mixtures induced a dose-dependent loss of viability in HGFs after 24h. SIGNIFICANCE: The EC(50) values of binary mixtures were significantly (p<0.05) lower compared to the EC(50) values of the pure substances indicating a synergistic interaction of the mixtures on the HGFs. The widely used (co)monomers BisGMA and TEGDMA have the lowest EC(50) values. The highest decrease of EC(50) values, compared with the pure substances, were found in the mixture UDMA/HEMA. Worst case calculations show that the EC(50) values from binary mixtures are at least 6 fold lower compared with known amounts of elutable (co)monomers from polymerized composites.

Cytokine release from human leukocytes exposed to silorane- and methacrylate-based dental materials.

OBJECTIVES: Silorane-based dental monomers contain an epoxy functional group. Less is known about the toxicological and inflammatory potential of silorane-based composites. Therefore we compared the release of 24 cytokines from human leukocytes after incubation with silorane-based Filtek™ Silorane (Silo) and methacrylate-based TetricEvo Flow® (TC). METHODS: Leukocytes from nine healthy test persons (P) were incubated with Silo or TC for up to 72h. All 24h cytokines were quantified with a magnetic bead assay. RESULTS: Silo stimulates the leukocytes to higher release of cytokines when compared to TC. 72h after beginning the experiment, leukocytes from P6 incubated with Silo secreted more than an 18-fold amount of interleukin (IL)-6 when compared with leukocytes incubated with TC (771.8 vs 42.1pg/ml). Only leukocytes from P8 incubated with Silo release up to 14.4pg/ml IL-2 after 72h. SIGNIFICANCE: The significantly higher induction of cytokines with Silo in comparison to TC is test person independent. This indicates a higher sensitization potential for Silo. Because of the cytokine release pattern (especially the release of T-cell dependent IL-2) from leukocytes from P8 after incubation with Silo it is likely that P8 can develop an allergic Type IV sensitization to Silo. Therefore the cytokine release assay is a helpful tool for providing information about possible immunological reactions to dental resins in individual cases as well as for a general risk assessment and comparison between different dental materials.