Availability and effects of n-TiO2 and PCB77 in fish in vitro models of the intestinal barrier and liver under single- and/or co-exposure scenarios.

Titanium dioxide nanoparticles (n-TiO2) and polychlorinated biphenyls (PCBs) can be present in the food of fish, leading to intestinal exposure uptake, and accumulation in inner organs. This study examined combination effects of n-TiO2 and PCB77 in vitro models of the fish intestinal epithelium and liver, i.e., RTgut-GC cell cultures grown in ThinCerts™ and RTL-W1 cell cultures grown in standard tissue culture plates. Mass spectrometry and microscopy techniques were used to obtain information on nanoparticle translocation across the intestinal barrier model. In addition, the substances' effect on intestinal barrier permeability, cell viability, expression of dioxin - and antioxidant response element -controlled genes, and induction of cytochrome P450 1a (Cyp1a)-dependent ethoxyresorufin-O-deethylase (EROD) activity were assessed. TiO2 nanoparticles were taken up by RTgut-GC cells and detected in the bottom compartment of the intestinal epithelial barrier model. It was not possible to conclude definitively if n-TiO2 translocation occurred via transcytosis or paracellular migration but observations of nanoparticles in the lateral space between adjacent epithelial cells were rare. PCB77 (1 and 10 µM, 24 h) did not affect barrier permeability, i.e., n-TiO2 translocation is probably not facilitated in case of co-exposure. Furthermore, previous and simultaneous exposure to n-TiO2 (1 and 10 mg/L, 24 h) did not have any influence on PCB77-induced Cyp1a mRNA and enzyme activity levels in RTL-W1 cells. Furthermore, there were no significant differences in expression of antioxidant response element-controlled genes comparing control, single substance, and mixture treatments, not even following long-term exposure (0.01-1 mg/L n-TiO2 + 1 nM PCB77, 4 weeks). While an underestimation of the effects of n-TiO2 and PCB77 cannot be fully excluded as concentration losses due to sorption to cell culture plastics were not measured, the results suggest that the test substances probably have a low potential to exhibit combination effects on the assessed endpoints when co-existing in fish tissues.

Influence of TiO2 nanocomposite UV filter surface chemistry and their interactions with organic UV filters on uptake and toxicity toward cultured fish gill cells.

Aquatic environments have been found to be contaminated with a variety of inorganic and organic UV filters. This includes novel nano-sized titanium dioxide (TiO2) composite particles, which have been increasingly developed and incorporated into commercial sunscreens in recent years. So far, relatively little is known about the effects of this novel class of UV filters on aquatic life. Therefore, this study aimed to determine and compare the toxicity of three such nanoparticulate TiO2 UV filters with different surface coatings, namely Eusolex® T-Avo (SiO2-coated), T-Lite™ SF (Al(OH)3/PDMS-coated), and Eusolex® T-S (Al2O3/stearic acid-coated) either alone, or in the presence of selected organic UV filters (octinoxate, avobenzone, octocrylene), toward fish using RTgill-W1 cell cultures as an in vitro experimental model. Besides standard exposure protocols, alternative approaches (i.e., exposure to water accommodated fractions (WAFs), hanging-drop exposure) were explored to account for nanoparticle (NP)-specific fate in the medium and obtain additional/complementary information on their toxicity in different conditions. The AlamarBlue, CFDA-AM and Neutral Red Retention (NR) assays were used to measure effects on different cellular endpoints. Transmission electron microscopy (TEM) was used to examine NP uptake. Our results showed that none of the TiO2 NP UV filters were cytotoxic at the concentrations tested (0.1-10 µg/mL; 24 h) but there were differences in their uptake by the cells. Thus, only the hydrophilic T-AVO was detected inside cells, but the hydrophobic T-Lite SF and T-S were not. In addition, our results show that the presence of NPs (or the used dispersant) tended to decrease organic UV filter toxicity. The level of combination effect depended on both NP-type (surface chemistry) and concentration, suggesting that the reduced toxicity resulted from reduced availability of the organic UV filters due to their adsorption to the NP surface. Thus, mixtures of TiO2 NP UV filters and organic UV filters may have a different toxicological profile compared to the single substances, but probably do not pose an increased hazard.

Mixture toxicity effects and uptake of titanium dioxide (TiO2) nanoparticles and 3,3',4,4'-tetrachlorobiphenyl (PCB77) in juvenile brown trout following co-exposure via the diet.

Titanium dioxide nanoparticles (n-TiO2) are among the man-made nanomaterials that are predicted to be found at high concentrations in the aquatic environment. There, they likely co-exist with other chemical pollutants. Thus, n-TiO2 and other chemical pollutants can be taken up together or accumulate independently from each other in prey organisms of fish. This can lead to dietary exposure of fish to n-TiO2-chemical pollutant mixtures. In this study, we examine if simultaneous dietary exposure to n-TiO2 and 3,3',4,4'-Tetrachlorobiphenyl (PCB77) -used as a model compound for persistent organic pollutants with dioxin-like properties- can influence the uptake and toxicological response elicited by the respective other substance. Juvenile brown trout (Salmo trutta) were fed custom-made food pellets containing n-TiO2, PCB77 or n-TiO2+PCB77 mixtures for 15 days. Ti and PCB77 concentrations in the liver were measured by ICP-MS and GC-MS, respectively. Besides, n-TiO2 uptake was assessed using TEM. Combination effects on endpoints specific for PCB77 (i.e., cytochrome P450 1A (CYP1A) induction) and endpoints shared by both PCB77 and n-TiO2 (i.e., oxidative stress-related parameters) were measured in intestine and liver using RT-qPCR and enzyme activity assays. The results show that genes encoding for proteins/enzymes essential for tight junction function (zo-1) and ROS elimination (sod-1) were significantly upregulated in the intestine of fish exposed to n-TiO2 and PCB77 mixtures, but not in the single-substance treatments. Besides, n-TiO2 had a potentiating effect on PCB77-induced CYP1A and glutathione reductase (GR) expression/enzyme activity in the liver. This study shows that simultaneous dietary exposure to nanomaterials and traditional environmental pollutants might result in effects that are larger than observed for the substances alone, but that understanding the mechanistic basis of such effects remains challenging.

Behavioural toxicity assessment of silver ions and nanoparticles on zebrafish using a locomotion profiling approach.

Zebrafish (Danio rerio) is not only a widely used species in the Fish Embryo Toxicity (FET) test but also an emerging model in behavioural ecotoxicology. By using automatic behaviour tracking technology, locomotion of developing zebrafish (ZF) larvae can be accurately recorded and potentially used in an ecotoxicological context to detect toxicant-induced behavioural alterations. In this study, we explored if and how quantitative locomotion data can be used for sub-lethal toxicity testing within the FET framework. We exposed ZF embryos to silver ions and nanoparticles, which previously have been reported to cause neurodevelopmental toxicity and behavioural retardation in early-life stages of ZF. Exposure to a broad range of silver (Ag(+) and AgNPs) concentrations was conducted, and developmental toxicity was assessed using FET criteria. For behavioural toxicity assessment, locomotion of exposed ZF eleutheroembryos (120hpf) was quantified according to a customised behavioural assay in an automatic video tracking system. A set of repeated episodes of dark/light stimulation were used to artificially stress ZF and evoke photo-motor responses, which were consequently utilized for locomotion profiling. Our locomotion-based behaviour profiling approach consisted of (1) dose-response ranking for multiple and single locomotion variables; (2) quantitative assessment of locomotion structure; and (3) analysis of ZF responsiveness to darkness stimulation. We documented that both silver forms caused adverse effects on development and inhibited hatchability and, most importantly, altered locomotion. High Ag(+) and AgNPs exposures significantly suppressed locomotion and a clear shift in locomotion towards inactivity was reported. Additionally, we noted that low, environmentally relevant Ag(+) concentrations may cause subordinate locomotive changes (hyperactivity) in developing fish. Overall, it was concluded that our locomotion-based behaviour-testing scheme can be used jointly with FET and can provide endpoints for sub-lethal toxicity. When combined with multivariate data analysis, this approach facilitated new insights for handling and analysis of data generated by automatized behavioural tracking systems.

Mixture effects between different azoles and ß-naphthoflavone on the CYP1A biomarker in a fish cell line.

The cytochrome P450 1A (CYP1A) biomarker response was studied in the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line, which represents a good model for studies on aryl hydrocarbon receptor (AhR) - CYP1A signaling. The PLHC-1 cells were exposed to the prototypical CYP1A inducer and AhR agonist ß-naphthoflavone (BNF) in combination with different azoles. Two imidazoles (clotrimazole and prochloraz) and two benzimidazoles (nocodazole and omeprazole) were used. Exposure to clotrimazole, prochloraz and nocodazole resulted in 2-4 fold induction of the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities at 24 and 48h, whereas exposure to the omeprazole for 48h had no effect on the EROD activity. Clotrimazole, nocodazole and prochloraz also acted as inhibitors of EROD activities in situ in PLHC-1 cells (IC50=1.3-7.7µM), whereas omeprazole had no effect on this activity (IC50=72µM). Exposure to 10µM prochloraz resulted in 3-fold induction of CYP1A mRNA and exposure to 10µM nocodazole resulted in 16-fold induction of CYP1A mRNA levels at 24h compared to controls. In the mixture experiments, more-than-additive mixture effects between BNF and the azoles clotrimazole, prochloraz and nocodazole on EROD activities were evident, with nocodazole showing the strongest mixture effect. The presence of nocodazole increased the response to BNF up to 200-fold on CYP1A mRNA and up to 16-fold on EROD activities and prolonged the effect of BNF exposure on EROD activities by 24h or longer. This suggests that azoles that are inhibitors and/or competing substrates for the CYP1A enzymes can cause increased sensitivity to exposures to chemicals that depend on CYP1A metabolism for their elimination in situations of mixed chemical exposures. The results also suggest that the EROD biomarker response can be significantly affected in azole-contaminated areas. The responsiveness of the EROD biomarker to BNF exposure was studied in PLHC-1 that had been pre-treated with nocodazole for 5 or 24h at concentrations that are known to disassemble microtubules at 24h in these cells. Pre-treatment of PLHC-1 cells with nocodazole for either 5 or 24h had no effect on the responsiveness to BNF exposure, which implies that the EROD activity can be induced in cells with disassembled microtubules.

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor.

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.

Interactions of pharmaceuticals and other xenobiotics on key detoxification mechanisms and cytoskeleton in Poeciliopsis lucida hepatocellular carcinoma, PLHC-1 cell line.

Fish are exposed to chemicals, including pharmaceuticals, in their natural habitat. This study focuses on effects of chemicals, including nine classes of pharmaceuticals, on key detoxification mechanisms in a fish liver cell-line (PLHC-1). Chemical interactions were investigated on efflux pumps, P-glycoprotein (Pgp) and multidrug resistance associated proteins (MRP1/MRP2), and on biotransformation enzymes, cytochrome P450 (CYP1A/CYP3A). Diclofenac and troleandomycin inhibited efflux activities, whereas ethinylestradiol activated efflux function. Exposure to troleandomycin and ß-naphthoflavone induced MRP2 mRNA levels, but no effects were seen on MRP1 or Pgp expressions. Inhibition of CYP1A activities were seen in cells exposed to α-naphthoflavone, ß-naphthoflavone, clotrimazole, nocodazole, ketoconazole, omeprazole, ethinylestradiol, lithocholic acid, rifampicin and troleandomycin. Exposure to fulvestrant, clotrimazole and nocodazole resulted in induction of CYP1A mRNA levels. Although, exposure to nocodazole resulted in disassembled microtubules. A CYP3A-like cDNA sequence was isolated from PLHC-1, but basal expression and activities were low and the gene was not responsive to prototypical CYP3A inducers. Exposure to ibuprofen, lithocholic acid and omeprazole resulted in fragmentation of microtubules. This study revealed multiple interactions on key detoxification systems, which illustrates the importance of study effects on regulation combined with functional studies to provide a better picture of the dynamics of the chemical defense system.

One-way inhibiting cross-talk between arylhydrocarbon receptor (AhR) and estrogen receptor (ER) signaling in primary cultures of rainbow trout hepatocytes.

The aryl hydrocarbon receptor (AhR) and the estrogen receptor (ER) are ligand-activated transcription factors, both of which can be activated by environmental pollutants. The AhR regulates cytochrome P450 1A (CYP1A) expression and can be induced by aromatic hydrocarbons. The ER regulates vitellogenin (VTG) expression and can be induced by estrogenic substances. Both receptor responses are established biomarkers used to assess the effects of pollutants in the aquatic environment. The receptors can also be affected in situations of mixed exposure. Cross-talk between these receptor pathways has been suggested, although there are conflicting data in the literature. We investigated cross-talk between ER-VTG and AhR-CYP1A signaling pathways in primary cultures of rainbow trout hepatocytes, using quantitative PCR (qPCR) for mRNA analyses and studies of CYP1A catalytic function and protein expression. The model agonists ß-naphthoflavone (BNF) and 17α-ethinylestradiol (EE(2)) were used for AhR and ER activation, respectively. Combined exposure to BNF and EE(2) reduced the EE(2)-mediated induction of VTG mRNA levels by about 40%, but had no effect on the BNF-mediated CYP1A mRNA levels, indicative of a one-way inhibiting AhR-ER cross-talk. However, basal levels of CYP1A mRNA were reduced 40% upon exposure to EE(2) alone, implying different cross-talk mechanism between basal and induced CYP1A mRNA levels. The mammalian ER antagonist fulvestrant (ICI) is commonly described as an absolute ER antagonist. However, ICI failed to reverse the ER activation caused by EE(2) in the present study. The CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activity was reduced by 80% in cells co-treated with BNF and EE(2), compared to cells exposed to BNF alone. In vitro inhibiting studies suggests that this reduction was a result of inhibition of the CYP1A catalyst by EE(2) since EE(2) acted as a potent inhibitor (IC(50): 4.6µM) of the EROD activity. In addition, ICI also acted as a potent inhibitor of the EROD enzyme (IC(50): 0.6µM). Taken together, our data supports a one-way inhibiting AhR-ER cross-talk in rainbow trout hepatocytes exposed to a mixture of BNF and EE(2).

Interactions of pharmaceuticals and other xenobiotics on hepatic pregnane X receptor and cytochrome P450 3A signaling pathway in rainbow trout (Oncorhynchus mykiss).

The pregnane X receptor (PXR) belongs to the nuclear hormone receptor (NR) superfamily and is commonly described as a xenophore or a pharmacophore, as it can be activated by a wide array of xenobiotics, including numerous pharmaceuticals and other environmental pollutants. The PXR regulates expression of e.g. cytochrome P450 3A (CYP3A) and the P-glycoprotein (P-gp) that are involved in excretion of lipophilic xenobiotics and endobiotics. A full length PXR cDNA was isolated from rainbow trout liver and it was expressed in a descending order of magnitude in liver>intestine>kidney>heart. A rainbow trout PXR reporter assay was developed and a suite of pharmaceuticals and other xenobiotics were screened. However, no specific activation of rainbow trout PXR was observed with the substances tested. Interactions of prototypical PXR agonists on PXR signaling in rainbow trout were further investigated in cells of hepatic origin exposed in vitro and in juvenile rainbow trout exposed in vivo. The rainbow trout hepatoma cell line (RTH-149), displayed 600 times lower expression of CYP3A mRNA compared to primary cultures of hepatocytes, and did not respond to treatment with either pregnenolone 16α-carbonitrile (PCN), ketoconazole (KCZ) or rifampicin (RIF), which implies a non-functional PXR in this cell line. Exposure of hepatocytes to PCN and lithocholic acid (LA), resulted in a weak concentration-dependent induction of CYP3A and P-gp mRNA levels, though, exposure to the higher concentration of LA (50 µM) decreased PXR mRNA levels. Exposure to dexamethasone (DEX) resulted in a decrease in PXR mRNA, without affecting CYP3A mRNA levels in hepatocytes in vitro. Injections of rainbow trout in vivo with 1 mg LA/kg fish resulted in a slight (albeit not significant) increase in CYP3A mRNA levels without affecting PXR mRNA levels. Although, injection with 10mg omeprazole (OME)/kg fish had no effect on PXR and CYP3A mRNA levels, a 60% inhibition of CYP3A enzyme activities was evident. An in vitro screening of the chemicals used showed that OME and RIF acted as weak CYP3A inhibitors whereas LA and DEX did not affect the CYP3A activity. In contrast, PCN acted as an activator of the CYP3A enzyme activity in vitro. Taken together, these data show that some prototypical PXR agonists weakly affect PXR activation in rainbow trout. Besides, some of these agonists have a stronger effect on the CYP3A catalyst. This study demonstrates the importance of investigation effects of pharmaceuticals on the PXR signaling pathway in non-target animals such as fish.

Ketoconazole, an antifungal imidazole, increases the sensitivity of rainbow trout to 17alpha-ethynylestradiol exposure.

This study focuses on effects of two classes of xenobiotics, azole fungicides and xenoestrogens, both of which have been detected in the aquatic environment. We hypothesize that azoles and estrogenic compounds are metabolized by cytochrome P450 (CYP) enzymes, and in particular CYP1A and CYP3A, to more readily excreted metabolites. We exposed rainbow trout (Oncorhynchus mykiss) to two different pharmaceutical representatives of theses two classes, such as the imidazole ketoconazole and the synthetic estrogen analogue, 17alpha-ethynylestradiol (EE(2)). Juvenile rainbow trout were i.p. injected with a single low dose of EE(2) (2.5 microg/kg), alone or in combination with ketoconazole (100mg/kg). Hepatic microsomal CYP1A and CYP3A protein expressions were analyzed in Western blots using polyclonal antibodies (PAb) and enhanced cheminoluminescence. CYP1A activities were analyzed using the ethoxyresorufin-O-deethylase (EROD) assay and CYP3A activities were analyzed using the benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD) assay. Plasma vitellogenin (vtg) and sex steroid hormones (i.e. 17beta-estradiol, testosterone and 11-keto-testosterone) were analyzed using commercially available ELISA-kits. The vtg mRNA expression was analyzed using quantitative (Q)-PCR. The dose of EE(2) selected had little or no effect on the estrogen receptor (ER) mediated vtg induction. However, in combination with ketoconazole this threshold-dose of EE(2) resulted in significantly elevated plasma vtg levels, 6 days post injection. Exposure to ketoconazole resulted in up to nine-fold induction of CYP1A after 3 days. However, this nine-fold induction was not reflected on the CYP1A catalytic activity, where exposure to ketoconazole resulted only in a two-fold increase in activity. Ketoconazole increased CYP3A protein levels 1.5-fold and decreased BFCOD activities by 80% at days 3 and 6. Treatment with ketoconazole and EE(2) alone and in combination had no significant effect on sex steroid hormones, compared to vehicle-treated fish. This study demonstrates that exposure to ketoconazole compromises the function of key enzymes involved in metabolic clearance of xenobiotics and steroids, and increases the sensitivity to EE(2) exposure in juvenile rainbow trout.