Isolated Rainbow Trout Hepatocytes Research Articles

An in vitro examination of selenium-cadmium antagonism using primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes.

The present study evaluated the ameliorative properties of selenium (Se) against cadmium (Cd)-induced oxidative stress, using isolated rainbow trout (Oncorhynchus mykiss) hepatocytes in primary culture as the model experimental system. Cadmium (Cd) is known to induce cytotoxic effects by disrupting cellular oxidative homeostasis. On the other hand, selenium (Se) is an essential component of biological antioxidative machinery, and thus may provide protection against the toxic insults of Cd by augmenting the cellular antioxidant response. However, Se, when present above the threshold concentration, can also induce reactive oxygen species (ROS) generation and cause oxidative damage. In this experiment, trout hepatocytes in primary culture were exposed to 100 µM Cd, alone or in combination with different concentrations (25-500 µM) of selenite (SeO3(2-)) or selenomethionine (SeMet) for 48 h. Our findings indicated that both chemical forms of Se, at the lowest concentration used (25 µM), significantly reduced Cd-induced cytotoxicity (measured as cell viability). In contrast, Se at higher concentrations (≥ 50 µM) did not offer any protection against a Cd induced decrease in cell viability. The reduced cytotoxicity of Cd in the presence of 25 µM selenite or SeMet was associated with reduced intracellular ROS production, recovery of the cellular thiol status (ratio of reduced and oxidized glutathione), and amelioration in the activities of major enzymatic antioxidants (superoxide dismutase, catalase, and glutathione peroxidase). Co-treatment of hepatocytes with Cd and pharmacological antioxidants (TEMPO and NAC) also reduced Cd-induced oxidative stress in trout hepatocytes. This provided further evidence that Se likely ameliorates Cd toxicity via different antioxidative mechanisms.

Application of XANES spectroscopy in understanding the metabolism of selenium in isolated rainbow trout hepatocytes: insights into selenium toxicity

Selenium (Se) is an essential element, but causes toxic effects in fish at a slightly elevated level beyond the threshold. However, the degree of Se toxicity differs depending on the chemical forms of Se (e.g., organic vs. inorganic) to which fish are exposed to. The mechanisms of Se metabolism and toxicity in fish, particularly at cellular level, are poorly understood. The present study was designed to examine the metabolic fate of different seleno-compounds, both inorganic and organic, in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) in primary culture using XANES spectroscopy. In cells exposed to 100 μM of selenate and selenite for 6-24 h, elemental Se was found to be the primary metabolite. Whereas, selenocystine appeared to be the major metabolite in cells exposed to 100 μM seleno-L-methionine for 6-24 h. Interestingly, we recorded L-methionine-γ-lyase activity in S9 fraction of cell lysate-an enzyme that directly catalyzes selenomethionine into methylselenol. We also found concurrent reduction of glutathione (GSH) concentration following reaction of seleno-L-methionine with cellular S9 fraction. Moreover, we observed a rapid increase in cellular reactive oxygen species (ROS) generation with increasing seleno-L-methionine exposure dose (100-1000 μM). These findings indicated the rapid cellular metabolism of seleno-L-methionine into methylselenol at higher exposure dose (≥100 μM), and the occurrence of GSH mediated redox cycling of methylselenol--a process that is known to produce reactive oxygen species (ROS). Overall, our results suggest that inorganic and organic selenium are metabolized through different metabolic pathways in rainbow trout hepatocytes. The findings of our study have important implications for understanding the chemical species-specific differences in Se toxicity to fish.

Assessment of cultured fish hepatocytes for studying cellular uptake and (eco)toxicity of nanoparticles

Environmental context. The production and application of engineered nanoparticles is rapidly increasing, and development of suitable models for screening nanoparticles for possible toxic effects is essential to protect aquatic organisms and support the sustainable development of the nanotechnology industry. Here, the suitability of isolated rainbow trout hepatocytes was assessed for high through-put toxicity screening of nanoparticles and for studying uptake of nanoparticles into cells. Abstract. Relatively little is known regarding the fate and possible toxic effects of engineered nanoparticles (ENPs) in the aquatic environment. We assessed the suitability of isolated trout hepatocytes for high throughput toxicity screening of ENPs, exposing them to a variety of metal and metal oxide nanoparticles and their bulk counterparts. We found no effects of the ENPs on cell viability, or on lipid peroxidation, with the exception of exposure to ZnO nanoparticles, or on glutathione-S-transferase (GST) levels, for exposure concentrations up to 500 μg mL–1. All ENPs, however, were internalised in the cultured hepatocytes, as shown by coherent anti-Stokes Raman scattering (CARS) as an imaging technique. Our findings suggest that fish hepatocyte cultures are suitable for studies investigating the cellular uptake of ENPs, but they do not appear to be sensitive to ENP exposure and thus not a good in vitro model for nanoparticle toxicity screening.

Profiling of benzophenone derivatives using fish and human estrogen receptor-specific in vitro bioassays

Benzophenone (BP) derivatives, BP1 (2,4-dihydroxybenzophenone), BP2 (2,2′,4,4′-tetrahydroxybenzophenone), BP3 (2-hydroxy-4-methoxybenzophenone), and THB (2,4,4′-trihydroxybenzophenone) are UV-absorbing chemicals widely used in pharmaceutical, cosmetics, and industrial applications, such as topical sunscreens in lotions and hair sprays to protect skin and hair from UV irradiation. Studies on their endocrine disrupting properties have mostly focused on their interaction with human estrogen receptor alpha (hERα), and there has been no comprehensive analysis of their potency in a system allowing comparison between hERα and hERβ activities. The objective of this study was to provide a comprehensive ER activation profile of BP derivatives using ER from human and fish origin in a battery of in vitro tests, i.e., competitive binding, reporter gene based assays, vitellogenin (Vtg) induction in isolated rainbow trout hepatocytes, and proliferation based assays. The ability to induce human androgen receptor (hAR)-mediated reporter gene expression was also examined. All BP derivatives tested except BP3 were full hERα and hERβ agonists (BP2 > THB > BP1) and displayed a stronger activation of hERβ compared with hERα, the opposite effect to that of estradiol (E 2). Unlike E 2, BPs were more active in rainbow trout ERα (rtERα) than in hERα assay. All four BP derivatives showed anti-androgenic activity (THB > BP2 > BP1 > BP3). Overall, the observed anti-androgenic potencies of BP derivatives, together with their proposed greater effect on ERβ versus ERα activation, support further investigation of their role as endocrine disrupters in humans and wildlife.

Endocrine Disruption of Water and Sediment Extracts in a Non-Radioactive Dot Blot/RNAse Protection-Assay Using Isolated Hepatocytes of Rainbow Trout (14 pp).Deficiencies between bioanalytical effectiveness and chemically determined concentrations and how to explain them

In order to evaluate the estrogenic activity of sediments and XAD water extracts of selected sites of the catchment area of the River Neckar, a river system in Southern Germany, an integrative assessment approach was used to assess the ecological hazard potential of endocrine-disrupting compounds in sediment and water. The approach is based on estrogen receptor-mediated vitellogenin synthesis induced in isolated hepatocytes of rainbow trout and quantified in a non-radioactive dot blot/RNAse protection-assay in parallel to comprehensive chemical analyses of estrogenic substances. Numerous investigated extracts revealed an estrogen activity comparable to that of the positive control (1 nM 17beta-estradiol corresponding to 270 ng/L in the test medium). Based on a concentration factor of 30 in the extracts and a recovery of XAD resins of approximately 80%, 17beta-estradiol equivalent concentrations between 20 and 26.7 ng/L could be calculated downstream of a sewage treatment plant (< 0.1 ng/L for a reference site). A comparison of the bioassay-derived Bio-TEQs (toxicity equivalents) and the Chem-TEQs revealed a high correlation with a Pearson coefficient of 0.85, indicating that the same ranking of the samples could be obtained with respect to the endocrine disrupting potential with both chemical and bioanalytical analysis. However, the TEQ concentrations computed from chemical analyses were significantly lower than the bioassay-derived TEQ concentrations. In fact, in none of the samples, more than 14% of the vitellogenin-inducing potency could be attributed to the substances (steroids, alkylphenols, bisphenol A, diethylstilbestrol) analyzed. A comparison of the endocrine disrupting potential of sediments extracted by the solvents acetone and methanol revealed lower biological effects for acetone-extracted samples. Possible reasons may be a masking of endocrine effects in acetone extracts by cytotoxicity, a low extraction efficiency of the solvent acetone, or anti- estrogen potencies of some extracted sediment compounds. Using a mass balance approach, the contribution of the compounds analyzed chemically (Chem-TEQs) to the total endocrine activity (Bio-TEQs) was calculated. Based on the very low detection limits, particularly of the steroids with their high TEF factors, results revealed that a calculation of the Chem-TEQs is associated with considerable scale inaccuracy: Whereas only 7-15% of the biological effectiveness (Bio-TEQs) could be explained by endocrine substances identified above the detection limits, the assumption of concentrations slightly below the given detection limits would result in a significant over estimation (137-197%) of the Bio-TEQs. Even the interassay variation of the dot blot assay with different fish donors for primary hepatocyte (factor 2-2.5) is relatively low, when compared to the large range of the Chem-TEQ concentrations (factor 20) obtained when applying different modes of calculation. Overall, only a minor portion of the endocrine activity detected by bioassays could be linked to compounds identified by chemical analysis. In vitro assays for assessment of endocrine activities are useful as sensitive integrating methods that provide quantitative estimates of the total activity of particular receptor-mediated responses. Although discrepancies may also result from different bioanalytical approaches, it is overall likely that bioanalytical and not chemical analytical approaches give the correct estimate of endocrine disrupting potencies in environmental samples. As a conclusion, assessment of endocrine disruption based on chemical analysis alone does not appear sufficient and further research into the spectrum of substances with potential endocrine activity as well as into additive or even synergistic effects in complex environmental samples is urgently needed.

Alterations in respiration rate of isolated rainbow trout hepatocytes exposed to the P-glycoprotein substrate rhodamine 123

Reducing intracellular xenobiotic concentration is an important defence strategy used by cells challenged with foreign chemicals. One mechanism used to achieve this goal is via the use of P-glycoproteins (P-gps), ATP-dependent transporters that mediate the removal of hydrophobic compounds from cells. The energetic costs of this mechanism are unknown, therefore, the activity and respiratory costs associated with the P-gp-mediated efflux of rhodamine 123 (R123) was measured in isolated rainbow trout hepatocytes. The accumulation of R123 was rapid and concentration-dependent. Initial accumulation rates were 1.79 ± 0.41, 7.29 ± 1.06 and 15.30 ± 1.74 ng R123/min/10 6 cells when exposed to 1, 5 and 10 μM R123, respectively. Efflux was measured in cells ‘pre-loaded’ with R123 at each concentration, resulting in initial efflux rates of 0.77 ± 0.12, 2.02 ± 0.35 and 3.51 ± 0.84 ng R123/min/10 6 cells, respectively. The baseline oxygen consumption rate of hepatocytes was 33.21 ± 1.09 ng O 2/min/10 6 cells. Respiration rates were significantly higher in cells exposed to 5 and 10 μM R123 (39.08 ± 0.80 and 41.72 ± 0.61 ng O 2/min/10 6 cells), representing increases over basal rates of 18.5 and 25.7%, respectively. Measurements of isolated mitochondrial respiration established that changes in hepatocyte oxygen consumption were not through the direct effects of R123 on mitochondria. The P-gp inhibitor, XR9576 significantly inhibited R123 efflux from cells with a concomitant return of respiration rates to baseline values. This study demonstrates that increased P-gp transport of xenobiotics can significantly raise cellular respiration rates and may result in higher energy costs for organisms living in P-gp-substrate contaminated environments.

Estrogenic effects of environmental chemicals: An interspecies comparison

The development of various in vitro screening methods has led to identification of novel estrogenic chemicals of natural and anthropogenic origin. In this study, the (anti)estrogenic potential of several environmental chemicals were compared in an array of in vitro test systems comprising: (i) competitive binding to estrogen receptors derived from the human breast cancer cell line MCF-7 (hER) and rainbow trout ( Oncorhynchus mykiss) (rtER), (ii) a proliferation assay with MCF-7 cells (E-SCREEN), and iii) induction of vitellogenin (rtVtg) in isolated rainbow trout hepatocytes. The results showed substantial differences in assay sensitivity for potent estrogens like 17β-estradiol, diethylstilbestrol and zearalenone (ranking order of sensitivity: E-SCREEN > hER ≈ rtER ≈ rtVtg). Chemicals like 4- n-nonylphenol and bisphenol A had higher relative binding affinity to the hER, whereas 4- t-butylphenol and 4- n-butylphenol showed highest affinity to the rtER. Zearalenone and the novel estrogen 4- t-butylhexanol displayed a considerable higher relative potency in the E-SCREEN than the rtVtg assay, whereas alkylphenols and the novel estrogen mimic 4- t-butyl-nitrobenzene were most potent in fish cells. Correlation analysis of data from the test systems suggest that interspecies differences is largely due to inter-assay variation of the ER-dependent cellular responses, whereas binding to the ER are fairly similar in the two species tested.

D-Glucose uptake in fish hepatocytes: mediated by transporter in rainbow trout ( Oncorhynchus mykiss), but only by diffusion in prespawning lamprey ( Lampetra fluviatilis) and in RTH-149 cell line

The transport of d-glucose into rainbow trout ( Oncorhynchus mykiss) and river lamprey ( Lampetra fluviatilis) hepatocytes, as well as into rainbow trout hepatoblastoma cell line RTH-149 was studied using tracer methods. The half-time for d-glucose equilibration was 15 s for rainbow trout. The half-times for the non-metabolizable d-glucose analog, 3- O-methyl- d-glucose equilibration were 8 s, 37 s and 38 s for rainbow trout, lamprey and RTH-149 cells, respectively. The 3- O-methyl- d-glucose was taken up by rainbow trout hepatocytes by facilitated diffusion in addition to simple diffusion. The uptake showed saturation kinetics with the K m of 37 mM and V max of 62 mmol kg −1 cells min −1. The uptake was sensitive to phloretin and cytochalasin B, but not affected by ouabain. The 3- O-methyl- d-glucose uptake by lamprey hepatocytes and RTH-149 cells showed no indication of saturation up to 160 mM, and was not affected by phloretin, cytochalasin B or ouabain, which suggests the mode of transport to be by passive diffusion. However, immunocytochemical stainings revealed the existence of mammalian type GLUT1 and GLUT2 transporters in all cells studied. The lack of a functioning carrier-mediated glucose uptake in lamprey hepatocytes might be due to its physiological state (prespawning starvation). The minor 3- O-methyl- d-glucose uptake into RTH-149 cells compared to freshly isolated rainbow trout hepatocytes might reflect low metabolic activity of the cell lines. Under the conditions applied the RTH-149 cell line is no suitable in vitro model for glucose transport in fish cells.

Intracellular pH regulation in rainbow trout (Oncorhynchus mykiss) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent.

We studied pH regulation in freshly isolated rainbow trout hepatocytes using microspectrofluorometry with the fluorescent dye BCECF. In accordance with earlier data on rainbow trout hepatocytes, ion substitution (N-methyl D-glucamine for sodium and gluconate for chloride) and transport inhibitor [10 microM M methyl isobutyl amiloride (MIA) to inhibit sodium/proton exchange and 100 microM DIDS to inhibit bicarbonate transport] studies in either Hepes-buffered or bicarbonate/carbon dioxide-buffered media (extracellular pH 7.6) indicated a role for sodium/proton exchange, sodium-dependent bicarbonate transport, and sodium-independent anion exchange in the regulation of hepatocyte pH. In Hepes-buffered medium, the activity of the sodium/proton exchanger (i.e. proton extrusion inhibited by MIA) was greater at 1% than at 21% oxygen. The oxygen dependency of the sodium/proton exchange is not caused by hydroxyl radicals, which appear to mediate the oxygen sensitivity of potassium-chloride cotransport in erythrocytes.

Cytological and Biochemical Effects of a Mixture of 20 Pollutants on Isolated Rainbow Trout ( Oncorhynchus mykiss) Hepatocytes

Isolated hepatocytes of rainbow trout ( Oncorhynchus mykiss) were exposed to different concentrations (1×, 10×, 50×) of a complex mixture of 20 environmentally relevant contaminants (PAHs, PCBs, pesticides) typical of the chemical burden of surface waters and sediments of small rivers in southwest Germany to investigate sublethal cytological and biochemical alterations. Results document that all concentrations clearly induced biochemical and morphological changes. The activities of enzymes such as lactate dehydrogenase and catalase as well as the rate of lipid peroxidation were significantly increased, whereas acid phosphatase activity decreased. Cytopathological effects included deformation of cellular shape, deformation and dilation of the nuclear envelope, increase in heterochromatin, nuclear inclusions, heterogeneity of mitochondria, vesiculation of RER cisternae, as well as increases in myelinated bodies, lysosomes, and peroxisomes. If compared with cytological alterations induced in isolated rainbow trout hepatocytes after exposure to sediment extracts from rivers of corresponding contamination, cytological effects of exposure to the complex chemical mixture displayed numerous similarities.

Rainbow trout ( Onchorhynchus mykiss) hepatic glucose transporter

We report identification of a rainbow trout hepatic glucose transporter sharing 58% and 52% amino acid identity with avian and mammalian GLUT2 sequences, respectively. The functionality of OnmyGLUT2 was assessed by expression in rainbow trout embryos. We also measured the transport of hexose in isolated rainbow trout hepatocytes. Inhibition of 3- O-methylglucose uptake by cytochalasin B, phloretin and 2-deoxy- d-glucose suggested the existence of a functional facilitative transporter in these cells. Expression of OnmyGLUT2 was found in the liver, kidney and intestine.

Glutathione S-transferases from rainbow trout liver and freshly isolated hepatocytes: purification and characterization

Glutathione S-transferases (GST) form an important family of biotransformation enzymes catalyzing the conjugation of glutathione to a great variety of xenobiotic compounds. The objective of this study was to compare the different characteristics of GST from freshly isolated rainbow trout hepatocytes with those corresponding to the total liver of the same fish, in order to establish the similarities. GST was purified by affinity chromatography and enzymatic activity was determined towards two substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETHA). The different isoenzymes were determined by HPLC associated with SDS-PAGE. Slight differences between the samples were obtained when the results corresponding to the enzyme activity were compared. HPLC results showed that all GST isoforms present in the total liver samples were represented in the isolated cells too, corresponding to isoforms with molecular masses of approximately 25.5 and 23.0 kDa.

Depletion of cellular protein thiols as an indicator of arylation in isolated trout hepatocytes exposed to 1,4-benzoquinone.

A method to measure protein thiols (PrSH), reduced and oxidized, was adapted to determine PrSH depletion in isolated rainbow trout hepatocytes exposed to arylating agent 1,4-benzoquinone (BQ). Toxicant analysis revealed rapid conversion of BQ to 1, 4-hydroquinone (HQ) upon addition to hepatocytes. Hepatocytes exposed to 200 microM BQ+HQ showed 80% decline in glutathione (GSH) (1 h), 30% loss of PrSH (6 h), and no loss of viability (24 h). Recoverable oxidized PrSH was detected only after 24 h (200 microM BQ+HQ). Exposure to 600 microM BQ+HQ caused rapid (10 min) loss of > 90% GSH and > 60% PrSH, with eventual cell death. Half of the PrSH depletion at 6 h observed in hepatocytes exposed to 600 microM BQ+HQ was recoverable by reduction with dithiothreitol. Following the loss of GSH in hepatocytes exposed to 600 microM BQ+HQ, cellular PrSH were susceptible to direct arylation and oxidation. Rainbow trout hepatocytes, which contained 10-fold less GSH than rat cells, had a GSH:PrSH ratio of 1:82 compared with rat ratios of 1:2 to 1:6. The methods reported are useful for further study and discrimination of reactive modes of action needed for prediction of aquatic organism susceptibility to these types of toxicants.

Accumulation and biotransformation of tri-N-butyltin by isolated rainbow trout hepatocytes

In the present study, metabolism of tributyltin (TBT) by isolated trout hepatocytes was investigated over a short exposure time. For this, the cells were exposed to TBT, and the determination of TBT and possible metabolites was performed. Cell viability and cytochrome P-450 activity were also measured. After 15 min of exposure to 1 μM TBT, 72 ± 23% of the total tin recovery was observed in the cellular fraction, and about 3 and 4% of the total amount of tin was recovered as dibutyltin (DBT) in the cells and incubation medium, respectively. The amount of DBT leveled off after 15 min, probably because of the inhibition of cytochrome P-450. Thus, TBT is metabolized by fish hepatocytes. However, as it also inhibits cytochrome P-450 activity, it has the potential to alter its own metabolism and that of other environmental pollutants.

A combination of fluorescent probes for evaluation of cytotoxicity and toxic mechanisms in isolated rainbow trout hepatocytes

The toxicity of 17 chemicals to freshly isolated rainbow trout hepatocytes was determined using four different fluorescent probes measuring different endpoints. Calcein was used to determine viability of the cells, rhodamine 123 and 5,5′,6,6′-tetrachloro-1,1′,3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) to monitor mitochondrial activity and 5-chloromethylfluorescein diacetate (CMFDA) to monitor intracellular glutathione (GSH) levels. An EC 50 value 10 times lower than that obtained with the calcein assay was taken to indicate a specific action. Two sets of test chemicals were used. The first set contained five chemicals with a known specific toxic action and two chemicals without a known main specific action at a cellular level, and this set was used to examine the specificity of the probes. The second set consisted of 10 chemicals from the MEIC (Multicenter Evaluation of In Vitro Cytotoxicity) chemical list and included only one chemical with a known main specific action at a cellular level. In the first set of chemicals the CMFDA assay detected all chemicals with known GSH-depleting action, and the results obtained with the second set of chemicals indicated that none of the chemicals act mainly by GSH depletion. However, the CMFDA assay showed one false positive (antimycin-A). The results obtained with the rhodamine 123 and the JC-1 assay were less clear. The rhodamine 123 assay recognized the three chemicals in set 1 interfering with mitochondrial function but gave one false positive (ethanol) in this set, whereas the JC-1 assay recognized only two of the chemicals and gave one false positive (menadione). Both assays failed to detect the only chemical in set 2 with a known action on mitochondria (KCN). We conclude that calcein and CMFDA are good candidates as fluorometric probes for detection of cell viability and depletion of GSH, respectively. However, rhodamine 123 and JC-1, on the other hand, do not appear to be good probes for screening of mitochondrial activity in rainbow trout hepatocytes.

The Use of Freshly Isolated Rainbow Trout Hepatocytes and Gill Epithelial Cells in Toxicity Testing

The Use of Freshly Isolated Rainbow Trout Hepatocytes and Gill Epithelial Cells in Toxicity Testing

The use of freshly isolated rainbow trout hepatocytes and gill epithelial cells in toxicity testing

The use of freshly isolated rainbow trout hepatocytes and gill epithelial cells in toxicity testing

The toxicity of the mutagen ‘MX’ and its analogue, mucochloric acid, to rainbow trout hepatocytes and gill epithelial cells and to Daphnia magna

The cytotoxicity of the, in Salmonella, potent mutagenic compound, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and its structural analogue 3,4-dichloro-5-hydroxy-2[5H]-furanone (mucochloric acid, MCA), was studied in freshly isolated rainbow trout hepatocytes and gill epithelial cells by determining 86Rb-leakage and decrease in fluorescence intensity in calcein AM-loaded cells. The acute toxicity of the compounds to Daphnia magna was studied by determining the concentration causing immobilization of the organism. MX proved to be more toxic than MCA both in the cellular assays and in the acute toxicity test with D. magna. MX was more toxic to hepatocytes than to gill epithelial cells. The uptake of [ 14C]MX was also much more efficient in hepatocytes than in gill epithelial cells. The uptake of [ 14C]MX in hepatocytes was not inhibited by taurocholic acid in excess, indicating that MX is not taken up by the carrier complex responsible for the uptake of taurocholate in the hepatocytes. Both the acute toxicity to D. magna and cytotoxicity of MX and MCA was rather low (EC 50 values > 0.1 mM) and we conclude that it is very unlikely that MX and MCA at concentrations occurring in recipients receiving chlorination effluents from pulp mills or chlorinated domestic sewage, as regards their acute toxicity, implies a risk for aquatic animals.

The use of freshly isolated gill epithelial cells in toxicity testing

The large number of chemicals in our environment, many of which have been insufficiently tested for toxicity, justifies the need for rapid and reliable in vitro testing to predict toxicity. In aquatic toxicology the use of cellular toxicity tests has been limited. In the present study, the usefulness of freshly isolated gill epithelial cells from rainbow trout in toxicity testing was evaluated by testing the cytotoxicity of 30 reference chemicals to the cells using the fluorescent cell viability probe Calcein-AM. The results were compared with previously obtained EC 50 values for the chemicals in freshly isolated rainbow trout hepatocytes, EC 50 values for immobilization of Daphnia magna and literature data on LC 50 values for fish. Regression analysis of the data obtained with gill epithelial cells and hepatocytes showed a correlation coefficient of 0.77 (r 2 = 0.59). The gill epithelial cells were slightly more sensitive than hepatocytes to the chemicals. The slope of the regression line was 0.79. Both cell types were less sensitive than D. magna and fish to the chemicals, but the EC 50 values for gill epithelial cells showed a better concordance with EC 50 values for immobilization in D. magna and with literature data for fish LC 50 values than the hepatocyte EC 50 values.

A comparison of the toxicity of 50 reference chemicals to freshly isolated rainbow trout hepatocytes and Daphnia magna

There is a need for rapid and cost-effective in vitro tests or test batteries in aquatic toxicology which could be used as tools in evaluating the toxicity of chemicals. In the present study the toxicity of 50 reference chemicals was evaluated by determining immobility in Daphnia magna (24 h incubation) and 86Rb-leakage in freshly isolated rainbow trout hepatocytes (3 h incubation). Regression analysis of the data on the EC 50 for Daphnia and EC 50 for the rubidium leakage in hepatocytes showed a correlation of 0.71 ( p < 0.0001). The slope of the regression line (0.68) differed markedly from a 1:1 correlation. Daphnia was more sensitive to the chemicals than hepatocytes and the discrepancy between the two tests increased with increasing toxicity of the chemicals. A comparison of the data from the hepatocyte test with published data on the toxicity of the reference chemicals to cultured mammalian hepatocytes (24 h incubation), indicated that the lower sensitivity of the fish hepatocytes compared to Daphnia is not due to the shorter incubation time used in the hepatocyte test. A comparison of the data from the Daphnia test with published data on the toxicity of the reference chemicals to various mammalian cells, showed a similar discrepancy between the Daphnia test and the cellular tests as in the case of the comparison between the Daphnia test and the rainbow trout hepatocyte test, indicating that the Daphnia test in general is more sensitive than conventional cellular cytotoxicity tests. It is concluded that further studies, using more sensitive endpoints than rubidium leakage, are needed before it can be decided whether or not freshly isolated rainbow trout hepatocytes are suitable in routine toxicity testing.