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Exposure of humans to metal oxide nanoparticles (NPs) occurs mainly via air, and inhaled metal oxide NPs may generate inflammation. The aim of this study was to investigate the proinflammatory potential of six metal oxide NPs (CeO2, Mn2O3, CuO, ZnO, Co3O4 and WO3; 27–108 μg ml−1) using human primary 3-dimensional airway epithelium (MucilAir™) and dendritic cell (DC) models. Metal oxide NPs were mainly aggregated/agglomerated in the cell media, as determined by dynamic light scattering, scanning electron microscopy and differential centrifugal sedimentation. WO3 and ZnO were highly soluble, both with and without respiratory mucus. Proinflammatory signalling by the epithelium was evaluated after a 24 hour exposure by increased interleukin-6 and -8 and monocyte chemoattractant protein 1 cytokine release, which occurred only for CuO. Moreover, maturation of immature human DCs, which play a key role in the lung immune system, were evaluated by expression of surface markers HLA-DR, CD80, CD83 and CD86 after a 48 hour exposure. Only Mn2O3 consistently upregulated DC maturation markers. Furthermore, by addition of medium from metal oxide NP-exposed 3-dimensional airway cultures to metal oxide NP-exposed DC cultures, the interplay between lung epithelium and DCs was studied. Such an interplay was again only observed for Mn2O3 and in one of five DC donors. Our results show that, even when using dosages that represent very high in vivo exposure levels, up to 27 hours of constant human airway exposure, metal oxide NPs cause minimal proinflammatory effects and that epithelial cells not necessarily interfere with DC maturation upon metal oxide NP exposure. The present approach exemplifies a relevant translation towards human safety assessment. Copyright © 2017 John Wiley & Sons, Ltd.

Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI.

Chronic kidney disease (CKD) is accompanied by accumulating levels of uremic solutes in the circulation. Changes in the size and composition of the bile acid pool have also been observed. We investigated via which mechanisms uremic solutes may interfere with hepatocyte function and thus contribute to altered bile acid handling. We studied interference on the level of bile acid synthesis by cytochrome P450 7A1 (CYP7A1), explored effects on hepatic bile acid transporters, and investigated effects on mitochondrial function. In HEK293 cells overexpressing bile salt transporters, we observed that p-cresyl sulfate inhibited Na+-taurocholate cotransporting polypeptide (NTCP)-mediated uptake of taurocholic acid (TCA), whereas organic anion-transporting polypeptide 1B1 (OATP1B1)-mediated TCA uptake was increased. Assays in transporter-overexpressing membrane vesicles revealed that kynurenic acid inhibited TCA transport via the bile salt efflux pump (BSEP), whereas p-cresyl glucuronide and hippuric acid increased TCA efflux via multidrug resistance-associated protein 3 (MRP3). Moreover, indoxyl sulfate decreased mRNA expression of NTCP, OATP1B3 and CYP7A1 in primary human hepatocytes. Transport studies confirmed a decreased TCA uptake in indoxyl sulfate-exposed hepatocytes. Decreased hepatocyte viability was found for all seven uremic solutes tested, whereas five out of seven also decreased intracellular ATP levels and mitochondrial membrane potential. In conclusion, uremic solutes affect hepatic bile acid transport and mitochondrial function. This can contribute to the altered bile acid homeostasis observed in CKD patients.

Contains fulltext : 108532.pdf (Publisher’s version ) (Open Access) Breast cancer resistance protein (BCRP) is known for its protective function against the toxic effects of exogenous compounds. In addition to this, a role in the transport of endogenous compounds has been described. Since BCRP in the plasma membrane was shown to be regulated by sex steroids, we investigated the presence and possible role of BCRP in steroid hormone-producing organs. Therefore, the presence and localization of Bcrp was investigated in endocrine organs of wild-type mice. Furthermore, the interaction of various steroid hormones with human BCRP activity was studied. Quantitative PCR revealed Bcrp mRNA in the pituitary and adrenal glands, pancreas, ovary, testis and adipose tissue. Immunohistochemistry revealed the presence of Bcrp in the cortex of the adrenal gland and in plasma membranes of adipocytes. In the pituitary gland, pancreas, ovary and testis, Bcrp was mainly located in the capillaries. The interaction between BCRP and 12 steroid hormones was studied using membrane vesicles of HEK293-BCRP cells. Estradiol, testosterone, progesterone and androstenedione inhibited BCRP-mediated uptake of (3)H-estrone sulphate (E(1)S) most potently, with calculated inhibitory constant (Ki) values of 5.0 +/- 0.2, 36 +/- 14, 14.7 +/- 1.3 and 217 +/- 13 muM, respectively. BCRP function was attenuated non-competitively, which implies an allosteric inhibition of BCRP-mediated E(1)S transport by these steroids. In conclusion, localization of Bcrp in endocrine organs together with the efficient allosteric inhibition of the efflux pump by steroid hormones are suggestive for a role for BCRP in steroid hormone regulation. 01 augustus 2012

Contains fulltext : 125412.pdf (Publisher’s version ) (Closed access) Endocrine-disrupting chemicals (EDCs) are considered to cause testicular toxicity primarily via interference with steroid hormone function. Alternatively, EDCs could possibly exert their effects by interaction with ATP-binding cassette (ABC) transporters that are expressed in the blood-testis barrier. In this study, we investigated the effects of bisphenol A (BPA), tetrabromobisphenol A (TBBPA), bis(2-ethylhexyl) phthalate, mono(2-ethylhexyl) phthalate, perfluorooctanoic acid (PFOA), and perfluorooctanesulfonic acid (PFOS) on breast cancer resistance protein (BCRP), multidrug resistance proteins 1 and 4 (MRP1 and MRP4), and P-glycoprotein (P-gp) using membrane vesicles overexpressing these transporters. BPA solely inhibited BCRP activity, whereas TBBPA, PFOA, and PFOS inhibited all transporters tested. No effect was observed for the phthalates. Using transporter-overexpressing Madin-Darby canine kidney cells, we show that BPA and PFOA, but not TBBPA, are transported by BCRP, whereas none of the compounds were transported by P-gp. To investigate the toxicological implications of these findings, testosterone secretion and expression of steroidogenic genes were determined in murine Leydig (MA-10) cells upon exposure to the selected EDCs. Only BPA and TBBPA concentration dependently increased testosterone secretion by MA-10 cells to 6- and 46-fold of control levels, respectively. Inhibition of the Mrp's by MK-571 completely blocked testosterone secretion elicited by TBBPA, which could not be explained by coinciding changes in expression of steroidogenic genes. Therefore, we hypothesize that transporter-mediated efflux of testosterone precursors out of MA-10 cells is inhibited by TBBPA resulting in higher availability for testosterone production. Our data show the toxicological and clinical relevance of ABC transporters in EDC risk assessment related to testicular toxicity.

Contains fulltext : 127304.pdf (Publisher’s version ) (Closed access) Hyperuricemia is related to a variety of pathologies, including chronic kidney disease (CKD). However, the pathophysiological mechanisms underlying disease development are not yet fully elucidated. Here, we studied the effect of hyperuricemia on tryptophan metabolism and the potential role herein of two important uric acid efflux transporters, multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP). Hyperuricemia was induced in mice by treatment with the uricase inhibitor oxonic acid, confirmed by the presence of urate crystals in the urine of treated animals. A transport assay, using membrane vesicles of cells overexpressing the transporters, revealed that uric acid inhibited substrate-specific transport by BCRP at clinically relevant concentrations (calculated IC50 value: 365+/-13muM), as was previously reported for MRP4. Moreover, we identified kynurenic acid as a novel substrate for MRP4 and BCRP. This finding was corroborated by increased plasma levels of kynurenic acid observed in Mrp4(-/-) (107+/-19nM; P=0.145) and Bcrp(-/-) mice (133+/-10nM; P=0.0007) compared to wild type animals (71+/-11nM). Hyperuricemia was associated with >1.5 fold increase in plasma kynurenine levels in all strains. Moreover, hyperuricemia led to elevated plasma kynurenic acid levels (128+/-13nM, P=0.005) in wild type mice but did not further increase kynurenic acid levels in knockout mice. Based on our results, we postulate that elevated uric acid levels hamper MRP4 and BCRP functioning, thereby promoting the retention of other potentially toxic substrates, including kynurenic acid, which could contribute to the development of CKD.

Contains fulltext : 88905.pdf (Publisher’s version ) (Closed access) Platelet-rich plasma (PRP), containing various growth factors, may speed up wound and bone healing. Using osteoconductive alloplastic materials in reconstructive surgery, the amount of autogenous bone needed can be reduced. The purpose of this experiment was to study the effect of PRP on a mixture of autogenous bone and deproteinized bovine bone mineral (Bio-Oss) particles in goats. Four, round, critical size defects were made in the foreheads of 20 goats. In all goats the defects were filled with a mixture of autogenous particulate cancellous bone and (Bio-Oss) particles, in which 1 ml of PRP was added in two of the four defects. The goats were allocated to four subgroups each containing five goats, which were killed after 1, 2, 6 and 12 weeks. The results of the histological and histomorphometric examination showed that early and late bone healing were not enhanced when PRP was used. 01 april 2010