Trout Hepatocytes Research Articles

Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures.

Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation. Trout hepatocyte spheroids are a promising in vitro model for this assessment. In this investigation, the gene expression and function for xenobiotic metabolism and cellular efflux were characterised. Using fluorescence, transport and real time PCR analysis, the expression and functionality of a variety of genes related to xenobiotic metabolism and drug efflux were assessed in a range of trout hepatocyte culture preparations. Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques. A transient decline in the expression of genes related to both xenobiotic metabolism and transport was determined during spheroid development, with a subsequent recovery in older spheroids. The most mature spheroids also exhibited an expression profile most comparable to that reported in vivo. Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors. In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux. Together with the enhanced gene expression and longevity of the model, hepatocytes in spheroid culture may prove to be an accurate alternative model to study the mechanisms of these processes in fish liver and provide an assay to determine the bioaccumulation potential of environmental contaminants.

Evaluation of the sensitivity, responsiveness and reproducibility of primary rainbow trout hepatocyte vitellogenin expression as a screening assay for estrogen mimics

Vitellogenin (Vtg) induction in primary fish hepatocytes has been proposed as an in vitro screening assay for ER agonists and antagonists, but has not yet been used extensively as a high-throughput screening tool due to poor reproducibility, sensitivity and overall feasibility. The present work has evaluated the role of seasonal variation, normalization, optimal culture and assay conditions on the sensitivity, responsiveness and reproducibility of in vitro vtg gene mRNA and protein expression in rainbow trout (Oncorhynchus mykiss) primary hepatocytes using the xenoestrogen 17α-ethynylestradiol (EE2) as a test chemical. The results show that primary hepatocytes display a relatively high individual and seasonal variation in both Vtg mRNA and protein induction potential, although less variance was observed in assay sensitivity. Data normalization of assay response to maximum (3nM EE2) and minimum (DMSO) Vtg production dramatically reduced this variance and led to improved assay reproducibility. A time-dependent response was observed both for mRNA and protein expression, reaching maximum Vtg induction after 96h of exposure, although reproducible concentration response curves for both Vtg mRNA and protein could be obtained already after 48h. A need for chemical re-exposure of the hepatocytes was identified to be important for sustaining exposure concentrations in extended studies (>48h), whereas different plate formats (96, 24 or 6 wells) did not affect the bioassay performance. In conclusion, standardization of hepatocyte bioassay and test conditions as well as data-normalization procedures are proposed to be instrumental for more consistent and comparable results in future use of this in vitro assay.

Reactive oxygen species and cytotoxicity in rainbow trout hepatocytes: effects of medium and incubation time.

This study evaluated the effects of exposure medium and culture age on intracellular reactive oxygen species (ROS) development and cytotoxicity in fish hepatocytes following exposure to copper (Cu). ROS was quantified using the fluorescent probes DHR 123 and CM-H2DCFDA following exposure to Cu in Leibovitz' medium (L-15) or Tris-buffered saline (TBS). Similarly, culture age effects were investigated using 1-, 2- and 4-day-old cultured hepatocytes by exposing them to Cu in TBS. The exposure in L-15 resulted in significantly higher ROS compared to TBS using CM-H2DCFDA, but not DHR 123. The age of the primary cultures significantly affected the development of ROS for both probes. None of the exposures caused cytotoxicity in the hepatocytes. The results showed that both factors may affect responses to stressors, and suggested that the use of a simple medium such as TBS may be preferable for some applications. It is also preferable to use 1-day-old primary hepatocyte cultures.

Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17β-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together with cryopreserved trout hepatocytes for screening estrogenic chemicals, resulting in a reduction of the time required to perform the assay and enabling greater access to the model system through the approach of cryopreservation.

Comparison of cellular and molecular cytotoxic mechanisms of Cochlodinium polykrikoides in isolated trout and rat hepatocytes

Harmful algal blooms produced by the marine ichthyotoxic dinoflagellate Cochlodinium polykrikoides are responsible for mass mortalities of wild and farmed fish globally. This study compared the cytotoxic mechanisms of C. polykrikoides total extract on both trout and rat liver hepatocytes. Trout hepatocytes were more sensitive than rat hepatocytes against C. polykrikoides extract. The effective concentration 50 after 3 hour incubation (EC503hr) concentrations found for C. polykrikoides extract in trout and rat hepatocytes (i.e., 50% membrane lysis in 3 hr) were Eq. 1 cell/ml and Eq. 240 cell/ml, respectively. C. polykrikoides extract exposure in both isolated trout and rat hepatocytes resulted in membrane lysis, reactive oxygen species formation, glutathione depletion, collapse of mitochondrial membrane potential, ATP depletion, increase in adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio, cytochrome c release into the hepatocyte cytosol, and activation of caspases cascade. Trout hepatocyte toxicity was also associated with lysosomal membrane injury. Mitochondrial permeability transition in both trout and rat hepatocytes produced cytochrome c release from the mitochondrial intramembrane space into the cytosol. Thus, the cytochrome c release triggered activation of caspase-3 and apoptosis. Finally, data demonstrated that C. polykrikoides extract may induce more apoptotic phenotype in rat than trout hepatocytes, which in the latter favored predominantly necrotic mode of cell death.

Intra- and interlaboratory reliability of a cryopreserved trout hepatocyte assay for the prediction of chemical bioaccumulation potential.

Measured rates of intrinsic clearance determined using cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of improving modeled bioaccumulation predictions for fish. To date, however, the intra- and interlaboratory reliability of this procedure has not been determined. In the present study, three laboratories determined in vitro intrinsic clearance of six reference compounds (benzo[a]pyrene, 4-nonylphenol, di-tert-butyl phenol, fenthion, methoxychlor and o-terphenyl) by conducting substrate depletion experiments with cryopreserved trout hepatocytes from a single source. O-terphenyl was excluded from the final analysis due to nonfirst-order depletion kinetics and significant loss from denatured controls. For the other five compounds, intralaboratory variability (% CV) in measured in vitro intrinsic clearance values ranged from 4.1 to 30%, while interlaboratory variability ranged from 27 to 61%. Predicted bioconcentration factors based on in vitro clearance values exhibited a reduced level of interlaboratory variability (5.3-38% CV). The results of this study demonstrate that cryopreserved trout hepatocytes can be used to reliably obtain in vitro intrinsic clearance of xenobiotics, which provides support for the application of this in vitro method in a weight-of-evidence approach to chemical bioaccumulation assessment.

Effects of clofibric acid alone and in combination with 17β-estradiol on mRNA abundance in primary hepatocytes isolated from rainbow trout

Clofibric acid (CA) is the active substance of lipid lowering drugs. It is resistant to degradation, polar in nature, and has been found ubiquitously in the aquatic environment. Though CA is classified as a peroxisomal proliferator in rodents and is considered as a potential endocrine disruptor, little information exists on the effects of CA in aquatic organisms, such as fish. In the present study, we examined the mRNA levels of peroxisome proliferator- and estrogen-sensitive genes on the exposure of primary rainbow trout (Oncorhynchus mykiss) hepatocytes to CA alone and in combination with the natural female sex hormone, 17β-estradiol (E2). Our results demonstrate that rainbow trout hepatocytes are relatively refractory to the effects of CA on the PPAR signaling pathway and lipid metabolism. Moreover, CA did not show recognizable estrogenic activity, but after the induction of vitellogenesis by E2, CA significantly reduced vitellogenin (VTG) mRNA abundance. Apparently, the indirect repression of VTG transcription, independent of estrogen receptors, occurred. The mechanism is not yet clearly understood but may involve disruption of the stabilization of VTG mRNA known to be induced by E2.

Genotoxic potential of several naphthenic acids and a synthetic oil sands process-affected water in rainbow trout (Oncorhynchus mykiss)

The exploitation of oil sands has raised major environmental concerns, particularly regarding the presence of high concentration in contaminants such as polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) in oil sands process-affected water (OSPW). The purpose of this study was, first to evaluate the genotoxic impact of OSPW-related compounds such as NAs and PAHs in a salmonid species and secondly to assess if OSPW exposure leads to genotoxicity. For this purpose, rainbow trout hepatocytes were exposed in vitro to environmentally relevant concentrations of synthetic NAs, naphtalene, benzo(a)pyrene, and extracts of synthetic OSPW (generated by a laboratory bitumen extraction) and of oil sands leaching water (OSLW, mimicking leaching of oil sands in river water). Primary DNA damage was assessed by the formamidopyrimidine-DNA glycolyase (Fpg)-modified comet assay. Genotoxicity was observed in hepatocytes exposed to several NAs, mixture of them, OSPW and OSLW extracts. The chemical structure of NAs influences the genotoxicity potential: among the NAs tested, the most cyclic NA was the most genotoxic. It also appears that genotoxicity was more marked for OSPW than for OSLW. Because exposure to OSPW led to oxidative DNA damage, while after exposure to several NAs, these types of DNA damage were limited, the NAs tested in this study could not be qualified as the only major contaminants responsible for OSPW genotoxicity. Notwithstanding, it should be noteworthy that exposure to NAs resulted in genotoxic impact at concentrations lower than those documented by literature for fresh OSPW. Further research is needed to explore the relationships between the chemical structure of NAs and their genotoxicity in the light of the distribution of NAs in fresh OSPW samples as well as in surface waters.

Silver nanoparticles stimulate glycogenolysis in rainbow trout (Oncorhynchus mykiss) hepatocytes

Silver nanoparticles (AgNPs) are found in many consumer products yet their biological effects on non-target aquatic organisms are yet to be fully understood. This research aimed to investigate the effects of AgNPs on cell signaling in rainbow trout (Oncorhynchus mykiss) hepatocytes. We focused on the β-adrenoreceptor (AR), which mediates glycogenolysis, and the glucocorticoid receptor (GCR), which mediates gluconeogenesis. These two receptors have been extensively studied in trout hepatocytes due to their key roles during the stress response to increase glucose availability (among other things), allowing the organisms to cope with the stressor. We show for the first time that AgNPs at a concentration of 1μg/mL did not interfere with the function of either the β-AR or the GCR systems in rainbow trout hepatocytes, but at the concentration of 10μg/mL AgNPs stimulated glycogenolysis which was apparently receptor-independent. This study suggests that AgNPs could affect hormone-regulated cell signaling pathways at a concentration of 10μg/mL.

Ecotoxicological assessment of pulsed ultraviolet light‐treated water containing microbial species and Cryptosporidium parvum using a microbiotest test battery

AbstractDevelopment of a pulsed ultraviolet (PUV) light system as an alternative disinfection method for water treatment has been investigated, yet little is known about the impact of applying such emerging technologies on the natural aquatic environment. This study reports on the use of a battery of ecotoxicological tests representative of different ecological trophic levels to determine the safety and potability of PUV‐treated water containing known pathogens. Water samples containing Cryptosporidium oocysts and Escherichia coli were treated with PUV light, and the ecotoxicological safety was determined by assessing toxicity in a range of representative microbiotests and the Rainbow Trout Hepatocyte (RTH)‐149 fish cell line. Results show that PUV rapidly inactivated Cryptosporidium and the selected microbial species; greater rates of inactivation were observed at higher ultraviolet doses. PUV‐treated samples did not elicit ecotoxicological effects in the trophic levels studied. This study highlights that PUV is an effective means of disinfecting water containing pathogens such as Cryptosporidium.

Inhibition of P-glycoprotein in the blood–brain barrier alters avermectin neurotoxicity and swimming performance in rainbow trout

The importance of the blood brain barrier (BBB) and the contribution to its function by the efflux transporter P-glycoprotein (P-gp) in teleosts were examined using the P-gp substrates and central nervous system neurotoxins ivermectin (22,23-dihydroavermectin B1a+22,23-dihydroavermectin B1b) [IVM]) and emamectin benzoate (4″-deoxy-49″epimethylaminoavermectin B1 benzoate [EB]). Trout were injected intraperitoneally with 0.01–1.0 and 1–50mg/kg of IVM or EB, respectively either alone or in combination with cyclosporin A (CsA: a P-gp substrate) at 1mg/kg. IVM affected the swimming performance (critical swimming speed, burst swimming distance, and schooling) at significantly lower concentrations than EB. When fish were exposed to IVM or EB in the presence of CsA, alterations to swimming were increased, suggesting that competition for P-gp in the BBB by CsA increased IVM and EB penetration into the CNS and decreased swimming capabilities. The effect of co-administration of CsA on swimming-related toxicity was different between IVM and EB-treated fish; EB toxicity was increased to a greater extent than IVM toxicity. The greater chemosensitization effect of EB vs. IVM was examined using a P-gp competitive inhibition assay in isolated trout hepatocytes with rhodamine 123 as a substrate. At the cellular level, IVM was a more potent inhibitor of P-gp than EB, which allowed for a greater accumulation of R123 in hepatocytes. These results provide evidence for a role of P-gp in the BBB of fish, and suggest that this protein protects fish from environmental neurotoxins.

Optimizing the use of rainbow trout hepatocytes for bioaccumulation assessments with fish

Biotransformation rates measured using cryopreserved trout hepatocytes can be extrapolated to the whole animal to predict metabolism impacts on chemical bioaccumulation. Future use of these methods within a regulatory context requires, however, that they be optimized and standardized. Specifically, questions exist concerning gender differences in metabolism, cryopreservability of cells, and the accuracy of in vitro–in vivo scaling factors.2. In this study, we evaluated hepatocytes from juvenile male and female trout. No gender differences in cell size, protein abundance, cytochrome P450 content, ethoxyresorufin-O-deethylase activity, uridine diphosphate glucuronosyltransferase activity or intrinsic clearance of pyrene were observed for freshly isolated hepatocytes. There was a small difference in measured glutathione-S-transferase activity (<25%; males > females).3. Cells were cryopreserved by two methods: direct placement into liquid N2 vapor and controlled, slow-rate freezing. Comparable live recovery and enzymatic activity were observed regardless of freezing method or gender. Cells cryopreserved in liquid N2 vapor exhibited activity levels similar to those of freshly isolated cells, although there were small but significant differences in pyrene clearance and glutathione-S-transferase activity (frozen < fresh). Hepatocellularity values did not differ by sex.4. These results suggest that hepatocytes from male and female juvenile trout may be used interchangeably for in vitro–in vivo metabolism extrapolations.

Nanosilver cytotoxicity in rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes

Silver nanoparticles (AgNPs) are present in a multitude of consumer and medical products; however, the toxicity of AgNPs is not fully understood. This research aimed to elucidate the relationship between AgNP cytotoxicity and oxidative stress and damage in rainbow trout (Oncorhynchus mykiss) hepatocytes and erythrocytes in comparison to silver ions (Ag+). Generally the cytotoxicity of AgNPs and Ag+ was similar, such that both silver types generated reactive oxygen species, decreased glutathione levels, and decreased activities of glutathione reductase and glutathione-S-transferase. Nonetheless, the two silver types had different cellular targets; AgNPs increased lipid peroxidation without apparent uptake into the cells whereas Ag+ increased DNA damage. Furthermore, the toxicity of both silver types was generally decreased in cells treated with cysteine while treatment with buthionine sulfoximine increased the toxicity of both silver types.

Effects of dietary supplementation of coriander oil, in canola oil diets, on the metabolism of [1-14C] 18:3n-3 and [1-14C] 18:2n-6 in rainbow trout hepatocytes

The aim of this study was to investigate the effects of petroselinic acid, found in coriander oil, on the ability of rainbow trout hepatocytes to increase the production of eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) from [1-(14)C] α-linolenic acid (18:3n-3; ALA) and to reduce the production of arachidonic acid (20:4n-6; ARA) from [1-(14)C] 18:2n-6. Addition of coriander oil increased the production of 22:6n-3, from [1-(14)C] 18:3n-3, at the 0.5 and 1.0% inclusion levels and reduced the conversion of [1-(14)C] 18:2n-6 to 20:4n-6. β-Oxidation was significantly increased at the 1.5% inclusion level for [1-(14)C] 18:2n-6, however β-oxidation for [1-(14)C] 18:3n-3 only showed an increasing trend. Acetate, a main breakdown product of fatty acids (FA) via peroxisomal β-oxidation, decreased three-fold for [1-(14)C] 18:2n-6 and nearly doubled for [1-(14)C] 18:3n-3 when coriander was added at a 1.5% inclusion level. Acyl coenzyme A oxidase (ACO) enzyme activity showed no significant differences between treatments. Relative gene expression of ∆6 desaturase decreased with addition of coriander oil compared to the control. The addition of petroselinic acid via coriander oil to vegetable oil (VO) based diets containing no fishmeal (FM) or fish oil (FO), significantly increased the production of anti-inflammatory precursor 22:6n-3 (P=0.011) and decreased pro-inflammatory precursor 20:4n-6 (P=0.023) in radiolabelled hepatocytes of rainbow trout.

Understanding glucose uptake during methionine deprivation in incubated rainbow trout (Oncorhynchus mykiss) hepatocytes using a non-radioactive method

The role of methionine supplementation in fish metabolism remains largely unexplored. This study investigates the effects of methionine deprivation (MD) on glucose uptake in rainbow trout (Oncorhynchus mykiss) hepatocytes. To this end, primary hepatocytes were incubated in the presence (+M) or the absence (-M) of methionine for 48h and glucose uptake was assessed using a novel non-radioactive, fluorescent-linked enzymatic assay. Evidence indicated that glucose uptake increased under methionine deprivation, primarily due to the increased abundance of membrane bound sodium-glucose transporter 2 (SGLT2), which was likely facilitated by the cellular reduction in [ATP] resulting from increased mitochondrial uncoupling, as supported by elevated transcript levels of uncoupling protein 2a (UCP2a). This study is the first to suggest that the mechanisms responsible for the rapid glucose uptake associated with MR are facilitated by the greater abundance of SGLT2 glucose transporter and mitochondrial uncoupling.

Transgenic (cyp19a1b-GFP) zebrafish embryos as a tool for assessing combined effects of oestrogenic chemicals

Endocrine disrupting chemicals and especially oestrogen receptor (ER) agonists have been extensively studied over the years due to their potential effects on sexual development and reproduction in vertebrates, notably fish. As ER agonists can exist as complex mixtures in the aquatic environment, evaluating the impact of combined exposure on oestrogenic effects has become increasingly important. Use of predictive models such as concentration addition (CA) and independent action (IA) has allowed assessment of combined estrogenic effects of complex multi-compound mixtures of ER agonists in various fish in vitro and in vivo experimental models. The present work makes use of a transgenic zebrafish strain, tg(cyp19a1b-GFP), which expresses the green fluorescent protein (GFP) under the control of the cyp19a1b (brain aromatase or aromatase B) gene to determine the oestrogenic potency of ER agonists alone or in mixtures. In these studies, tg(cyp19a1b-GFP) zebrafish embryos were exposed for four days (from one to five days post fertilization) to five different oestrogenic chemicals; 17α-ethinylestradiol (EE2), 17β-estradiol (E2), estrone (E1), bisphenol A (BPA) and 4-tert-octylphenol (OP), and three mixtures of up to four of these compounds. The mixture of BPA, OP and E2 was also tested with primary cultures of rainbow trout hepatocytes by analysing the ER-mediated induction of the oestrogenic biomarker vitellogenin in order to compare the performance of the two methods for assessing oestrogenic effects of complex mixtures. The three tested mixtures were predominantly acting in an additive manner on the expression of GFP. Additivity was indicated by the overlap of the 95% confidence interval of the concentration response curves for the observed data with the CA and IA prediction models, and model deviation ratios within a factor of two for a majority of the mixture concentrations. However, minor deviations determined as more than additive effects for the mixture of EE2, E1 and E2 and less than additive effects for the mixture of BPA, OP, EE2 and E1 were observed at the higher mixture concentrations tested. The successful prediction of additivity by CA and IA in tg(cyp19a1b-GFP) zebrafish embryos and deviations at high mixture concentrations seemed to correspond well to results obtained in the rainbow trout hepatocyte assay. The present results clearly show the usefulness of combining predictive modelling and use of in vitro bioassays for rapid screening of oestrogenic effects of complex mixtures and environmental samples.

Assessment of energetic costs of AhR activation by β-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis

Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist β-naphthoflavone (βNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na+/K+-ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1μM βNF for 48h did not alter hepatocyte AEC, protein synthesis, or Na+/K+-ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies.

Oxidative mechanisms of fish hepatocyte toxicity by the harmful dinoflagellate Cochlodinium polykrikoides

Harmful Algal Blooms caused by the marine ichthyotoxic dinoflagellate Cochlodinium polykrikoides are responsible for mass mortalities of wild and farmed fish worldwide. In this research, we investigated the cytotoxic mechanisms of aqueous extract of C. polykrikoides on isolated Rainbow trout (Oncorhynchus mykiss) liver hepatocytes. Algal extract exposure with isolated trout hepatocytes caused hepatocyte membrane lysis, reactive oxygen species (ROS) formation, glutathione depletion, lysosomal membrane rupture, collapse of mitochondrial membrane potential, ATP depletion and increase in ADP/ATP ratio, cytochrome C release into the hepatocyte cytosol, and activation of caspases cascade. Anti-oxidants, free radical scavengers, mitochondrial permeability transition (MPT) pore sealing agents, microsomal oxidases inhibitors, ATP generators and lysosomotropic agents protected fish hepatocytes against C. polykrikoides. Fish hepatocyte toxicity was also associated with mitochondrial and lysosomal membrane injury. These events caused cytochrome C release from the mitochondrial intra-membrane space into cytosol. The cytochrome C release could trigger activation of caspase-3 and apoptosis.

A Comparative Toxicogenomic Investigation of Oil Sand Water and Processed Water in Rainbow Trout Hepatocytes

The purpose of this study was to compare the expression of gene transcripts involved in toxic stress in rainbow trout hepatocytes exposed to oil sand water (OSW), lixiviate (OSLW), and processed water (OSPW). We pose the hypothesis that the changes in gene expression responses in cells exposed to a simulated oil sand extraction procedure (OSPW) differ from the gene expression responses of OSLW and OS. Rainbow trout hepatocytes were exposed to increasing concentrations of OSW, OSLW, and OSPW for 48 h at 15 °C. Cell viability was assessed by measuring membrane permeability, total RNA levels, and gene expression using an array of 16 genes involved in xenobiotic biotransformation (GST, CYP1A1, CYP3A4, MDR), metal homeostasis and oxidative stress (MT, SOD, and CAT), estrogenicity (VTG, ERβ), DNA repair (LIG, APEX, UNG, and OGG), cell growth (GADD45 and PCNA), and glycolysis (GAPDH). The results showed that the toxicogenomic properties of OSPW differed from those of OSLW and OSW. Gene transcripts that were influenced by OSW and OSLW, and strongly expressed in OSPW, were MT, CAT, GST (induction), CYP1A1, VTG, UNG/OGG, and PCNA. These genes are therefore considered not entirely specific to OSPW but to water in contact with OS. We also found gene transcripts that responded only with OSPW: SOD, GST (inhibition), MDR (inhibition), CYP3A4, GAPDH, GADD45, and APEX. Of these gene transcripts, the ones strongly associated with toxicity (loss of cell viability and RNA levels) were CYP3A4, GST, and GAPDH. Genes involved in DNA repair were also strongly related to the loss of cell viability but responded to both OSLW and OSPW. The observed changes in cell toxicity and gene expression therefore support the hypothesis that OSPW has a distinct toxic fingerprint from OSLW and OSW.

In vitro models of xenobiotic metabolism in trout for use in environmental bioaccumulation studies

1. In vitro screens are sought as informative, alternatives to the use of animals in vivo and to improve upon the current use of fish liver 9000 g supernatants (S9) in environmental risk assessment.2. The rates of ethoxyresorufin-O-deethylation (relative to S9 protein) measured under different conditions of culture of rainbow trout hepatocytes were significantly higher than those detected in S9, in the order of freshly isolated hepatocytes > 10-day spheroid cultures > primary hepatocytes in culture > S9. The percentage of conjugated metabolites was also similar between freshly isolated and spheroid cultured hepatocytes (9.9 and 13.5%).3. The rate of oxidation was enhanced (1.7 fold) when S9 was supplemented with cofactors for phase II conjugation but this was only approximately one tenth of the rate in freshly isolated hepatocytes (7.1 pmol/min/mg S9 protein equivalent).4. Hepatocytes also hydroxylated ibuprofen, producing two metabolites, in contrast to only one (identified as the 1-hydroxy derivative) using hepatic S9 fractions.5. Since the bioaccumulation potential of chemicals is often based on un-supplemented S9 in incubations ≥1 h when activity declines, it is recommended that predictability would be greatly improved through the use of hepatocyte spheroids, due to their maintenance of activity and longevity.