Cultures Of Rainbow Trout Hepatocytes Research Articles

The Combined Effect of Copper Nanoparticles and Microplastics on Transcripts Involved in Oxidative Stress Pathway in Rainbow Trout (Oncorhynchus Mykiss) Hepatocytes.

Copper nanoparticles (CuNPs) and microplastics (MPs) are two emerging contaminants of freshwater systems. Despite their co-occurrence in many water bodies, the combined effects of CuNPs and MPs on aquatic organisms are not well-investigated. In this study, primary cultures of rainbow trout hepatocytes were exposed to dissolved Cu, CuNPs, MPs, or a combination of MPs and CuNPs for 48h, and the transcript abundances of oxidative stress-related genes were investigated. Exposure to CuNPs or dissolved Cu resulted in a significant increase in the transcript abundances of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD). Exposure to CuNPs also led to an upregulation in the expression of Na+/K+ ATPase alpha 1 subunit (ATP1A1). Microplastics alone or in combination with CuNPs did not have a significant effect on abundances of the target gene transcripts. Overall, our findings suggested acute exposure to CuNPs or dissolved ions may induce oxidative stress in hepatocytes, and the Cu-induced effect on target gene transcripts was not associated with MPs.

Insights on the toxicity of selected rare earth elements in rainbow trout hepatocytes

The increasing extraction of rare earth elements (REEs) for technology applications raised concerns for contamination and toxicity in the environment. The purpose of this study was to examine the toxicity of the following REEs in primary cultures of rainbow trout hepatocytes: yttrium (Y), samarium (Sm), gadolinium (Gd), terbium (Tb) and lutetium (Lu). Hepatocytes were exposed to increasing concentrations of the above elements for 24 h at 15 °C and they were analyzed for viability, metallothioneins (MT), glutathione-S-transferase (GST) and arachidonate cyclooxygenase (COX) as markers of oxidative stress and inflammation. The results revealed that the cytoxicity of REEs were as follows in decreasing order: Y > Sm > Lu > Tb > Gd in concordance with published rainbow trout mortality data. While effects on GST and COX activities were marginal, MT levels were more strongly increased with the 2 most toxic REEs (Y and Sm) and Gd, while MT levels were decreased in the least toxic ones (Tb, Lu). While cell viability followed published trout mortality data, it also followed the redox potential and the glutathione affinity constant (log k). The capacity to induce/decrease MT levels was associated with ionic radius, log k (glutathione) and electronegativity. A proposed mechanism of toxicity for REEs is presented based on the chemical properties of REEs, namely the glutathione binding constant and ionic radius, in light of the observed effects in trout hepatocytes.

A Kinetic Study of Reactive Oxygen Species in Rainbow Trout Hepatocytes by Fluorometry.

The kinetics of reactive oxygen species (ROS) formation in a primary culture of rainbow trout hepatocytes was investigated using three fluorescent probes: 5-,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), dihydrorhodamine 123 (DHR 123), and dihydroethidium (DHE). The cell cultures were loaded with the three probes, separately. Hepatocytes were then exposed to Cu (0.15-10 mM) in serum-free Leibovitz's medium for 30 min before being quantified by a fluorescence plate reader during 30 min. Membrane integrity and glutathione (GSH) content were quantified using the fluorescent probes 5-carboxyfluorescein diacetate-acetoxymethyl ester (CFDA-AM) and monochlorobimane. Increasing ROS formation with increasing concentrations of Cu was shown using CM-H2DCFDA, whereas DHR 123 fluorescence decreased. Significant differences between control and treatment groups were observed at the highest concentrations (2.5 and 10 mM) for both probes. DHE fluorescence was lower than that of the other two probes and did not appear to be affected by any exposure. Additionally, a dose-dependent depletion of GSH and decreasing membrane integrity with increasing Cu concentrations were demonstrated, with significant effects observed at 2.5 and 10 mM for both endpoints. The results showed that both CMH2DCFDA and DHR 123 detected the development of their target Cu-induced ROS in trout hepatocytes but did so in opposite fashions. DHE was found to be unsuitable for detecting kinetics of ROS formation in this model system.

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.

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.

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 β-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: 4.6 μM) of the EROD activity. In addition, ICI also acted as a potent inhibitor of the EROD enzyme (IC 50: 0.6 μM). 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.

A stable fish reporter cell line to study estrogen receptor transactivation by environmental (xeno)estrogens

Cross-species differences between human and fish estrogen receptor (ER) binding by environmental chemicals have been reported. To study ER transactivation in a fish cellular context, we stably co-transfected the PLHC-1 fish hepatoma cell line with a rainbow trout estrogen receptor (rtER) and the luciferase reporter gene driven by an estrogen response element (ERE). This new cell model, called PELN-rtER (for PLHC-1- ERE- Luciferase- Neomycin), responded to 17β-estradiol (E2) in a both concentration- and temperature-dependent manner, as well as to environmental ER ligands from different chemical classes: natural and synthetic estrogens, zearalenone metabolites, genistein, alkyphenoles and benzophenone derivatives. The comparison with other in vitro models, i.e. human reporter cell lines (HELN-rtER, MELN) and vitellogenin induction in primary cultures of rainbow trout hepatocytes, showed an overall higher sensitivity of the human cells for a majority of ligands, except for benzophenone derivatives which were active at similar or lower concentrations in fish cells, suggesting species-specificity for these substances. Correlation analyses suggest that the fish cell line is closer to the trout hepatocyte than to the human cell context, and could serve as a relevant mechanistic tool to study ER activation in fish hepatic cellular context.

Cytotoxicity of aged cadmium-telluride quantum dots to rainbow trout hepatocytes

The purpose of this study was to examine the lethal and sublethal toxicity of cadmium telluride (CdTe) quantum dots (Q-dots) in primary cultures of rainbow trout hepatocytes. Hepatocytes were exposed to concentrations of positively coated and aged (2 months and 2 years) Q-dots for 48 h at 15°C. Post treatment, cellular analysis was done of cell viability, lipid peroxidation (LPO), DNA damage, metallothioneins (MT), labile zinc/cadmium and heat shock proteins (chaperones). Q-dots were found to be toxic to fish hepatocytes at a threshold concentration of 0.1 mg/l. These nanoparticles increased the levels of MT, labile zinc, DNA strand breaks and heat shock proteins of the 70 kDa family. The strongest response was observed with the molecular chaperone of the 70 kDa family, reaching a 7-fold induction in exposed cells. Overall, the assessment of multiple biomarkers in trout hepatocytes exposed to differently ‘aged’ Q-dots suggests that the cytotoxic responses to two-year-old positively coated CdTe Q-dots were largely due to the liberation of Cd2+.

Functional characterization of estrogen receptor subtypes, ERα and ERβ, mediating vitellogenin production in the liver of rainbow trout

The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ERα and ERβ, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ERα selective agonist, methyl-piperidino-pyrazole (MPP) an ERα selective antagonist, and diarylpropionitrile (DPN) an ERβ selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbow trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [ 3H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ERα could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ERβ. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ERα. On the other hand, once blocked ERα with MPP, the only manifestation of agonist activity of estradiol would be achieved via ERβ. In conclusion, the present results indicate that vitellogenin production is mainly mediated through ERβ, implying, furthermore that compounds which only exhibit ERα selectivity are not detected by vitellogenin bioassay.

Occurrence of pharmaceutical products in a municipal effluent and toxicity to rainbow trout ( Oncorhynchus mykiss) hepatocytes

Pharmaceutical and personal care products (PPCPs) are found in municipal effluents and represent the major sources of contamination for the aquatic environment. A preliminary chemical analysis of wastewater identified several compounds associated with PPCPs, including caffeine, ibuprofen, naproxen, oxytetracycline, novobiocin, carbamazepine, gemfibrozil, bezafibrate, trimethoprim, sulfamethoxazole, and sulfapyridine. The purpose of this study was to examine the cytotoxic and oxidative effects of these products and other wastewater-related products (i.e., coprostanol, cotinine, estradiol-17 β, nonylphenol, and cholesterol) in primary cultures of rainbow trout hepatocytes. The redox activity of various PPCPs in trout ( Oncorhynchus mykiss) liver microsomes was investigated in vitro by tracking the rate of oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the formation of lipid peroxidation (LPO) after a 60-min incubation period. In addition, primary cultures of rainbow trout hepatocytes were exposed to various drugs identified in the municipal effluent for 48 h at 15 °C. Our results show that most PPCPs (83%) accelerated the rate of NADPH oxidation in the presence of microsomes and 72% of them increased LPO in microsomal membranes. LPO levels were significantly correlated ( R = 0.5 ; P < 0.05 ) with the number of functional groups on the molecule's backbone (i.e., number of O, S, N, P/number of C and H) and negatively so ( R = - 0.44 ; P < 0.05 ) with the octanol/water partition coefficient, suggesting that nucleophilicity and hydrophobicity are related to oxidative activity for these compounds. Exposure of trout hepatocytes to these products leads in many cases to decreased cell viability, increased CYP3A-related monooxygenase activity (benzylether resorufin dealkylase), and LPO. No induction of CYP1A1-related activity (7-ethoxyresorufin O-deethylase) was observed. Moreover, municipal effluent extracts (ethanol) were able to increase all the above responses in a dose-dependent manner. These results suggest that the basic redox properties of PPCPs could influence oxidative metabolism in liver cells and lead to oxidative damage. These products have the potential to produce a toxic response in aquatic organisms and the above biomarkers were shown to respond readily to PPCPs in aquatic organisms.

Modulation of Ah receptor and CYP1A1 expression by α-naphthoflavone in rainbow trout hepatocytes

The objective of this study was to evaluate whether α-naphthoflavone (ANF) modulates aryl hydrocarbon receptor (AhR) signaling in rainbow trout ( Oncorhynchus mykiss). AhR and cytochrome P450 1A1 (CYP1A1) protein and mRNA content were used as indictors of AhR signaling. Primary culture of rainbow trout hepatocytes were exposed to different concentrations of ANF (10 − 9 –10 − 5 M), while β-naphthoflavone (BNF 10 − 10 –10 − 6 M) and a combination of ANF and BNF were used to elucidate the impact of ANF on AhR signaling. ANF increased AhR and CYP1A1 protein expression in a concentration-related manner; the maximal induction was about 50% that of BNF. Despite the differences in protein content between ANF and BNF stimulation, the maximal AhR and CYP1A1 mRNA abundance seen with the high concentrations of ANF and BNF were similar. ANF significantly decreased (∼50%) BNF-induced AhR protein expression (only at 10 − 9 M), but not CYP1A1 protein and gene expression. In addition, ANF at a sub-maximal concentration (10 − 7 M) did not affect BNF-induced AhR protein content, but increased the sensitivity of hepatocytes to BNF-mediated CYP1A1 protein expression. Taken together, the mode of action of ANF appears similar to BNF, including modulation of AhR expression and activation of AhR-mediated signaling in rainbow trout hepatocytes. Overall, ANF is not only a partial AhR agonist, but may also modify BNF-mediated AhR signaling in trout hepatocytes.

Study of potential toxic effects on rainbow trout hepatocytes of surface water treated with chlorine or alternative disinfectants

This study evaluates the effects of disinfection on the formation of toxic compounds in lake water treated with sodium hypochlorite, chlorine dioxide and peracetic acid (a disinfectant not previously used in drinking water processes). Chlorine reacts with the natural organic matter or contaminants in surface waters and produces a complex mixture of disinfection by-products (DBPs), some of which have been shown to be carcinogenic, mutagenic and/or teratogenic in animal studies. To define the potential toxicity on aquatic animals, disinfected drinking waters obtained from a pilot plant fed with water coming from Lake Trasimeno (Perugia) were collected, adsorbed by using silica C 18 cartridges, and then eluted in sequence with ethylacetate, dichloromethane and methanol. The eluates were concentrated under vacuum and resuspended in dimethylsulfoxide (DMSO). Primary cultures of rainbow trout hepatocytes were exposed to three amounts of water concentrate (equivalent to 0.5, 0.25 and 0.125 l of disinfected water per ml of culture medium) for 24 h at 20 °C, after which their viability, glutathione content (GSH), free radical production (ROS) and cytochrome P4501A3 expression were determined. The disinfected water samples were collected during different seasons in order to evaluate a possible correlation between seasonal water variations and formation of toxic compounds. None of the water concentrates showed any cytotoxic effect or variations in GSH content, but significant increases in ROS production were detected in the autumn water concentrates from the treatments with sodium hypochlorite and chlorine dioxide. Cytochrome P4501A3 expression was not modified in the hepatocytes exposed to the water concentrates irrespective to the sampling season.

Effects of human pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes.

Pharmaceuticals are found in the aquatic environment but their potential effects on non-target species like fish remain unknown. This in vitro study is a first approach in the toxicity assessment of human drugs on fish. Nine pharmaceuticals were tested on two fish hepatocyte models: primary cultures of rainbow trout hepatocytes (PRTH) and PLHC-1 fish cell line. Cell viability, interaction with cytochrome P450 1A (CYP1A) enzyme and oxidative stress were assessed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium bromide tetrazolium (MTT), 7-ethoxyresorufin-o-deethylase (EROD) and dichlorofluorescein (DCFH-DA) assays, respectively. The tested drugs were clofibrate (CF), fenofibrate (FF), carbamazepine (CBZ), fluoxetine (FX), diclofenac (DiCF), propranolol (POH), sulfamethoxazole (SFX), amoxicillin (AMX) and gadolinium chloride (GdCl(3)). All substances were cytotoxic, except AMX at concentration up to 500 microM. The calculated MTT EC(50) values ranged from 2 microM (CF) to 651 microM (CBZ) in PLHC-1, and from 53 microM (FF) to 962 microM (GdCl(3)) in PRTH. CF, FF, and FX were the most cytotoxic drugs and induced oxidative stress before being cytotoxic. Compared to hepatocytes from human and dog, fish hepatocytes seemed to be more susceptible to the peroxisome proliferators (PPs) CF and FF. In PLHC-1 cells none of the tested drugs induced the EROD activity whereas POH appeared as a weak EROD inducer in PRTH. Moreover, in PRTH, SFX, DiCF, CBZ and to a lesser extend, FF and CF inhibited the basal EROD activity at clearly sublethal concentrations which may be of concern at the biological and chemical levels in a multipollution context.

Copper impact on heat shock protein 70 expression and apoptosis in rainbow trout hepatocytes

The mechanism underlying copper hepatotoxicity was investigated in primary cultures of rainbow trout hepatocytes maintained in Leibovitz-15 media. CuSO 4 treatment (0, 25, 50, 100 and 200 μM) resulted in a dose-dependent elevation in heat shock protein 70 (hsp70) expression at 24 and 48 h post-exposure. There was no effect of copper (200 μM CuSO 4) on hepatotoxicity at 24 h, whereas longer exposures (48 h) resulted in increased lactate dehydrogenase (LDH) leakage and apoptosis, demonstrated by fluorescence nuclear staining and DNA fragmentation. Vitamin C (1 mM), a free radical scavenger, inhibited this copper-induced apoptosis implying a role for reactive oxygen species in copper toxicity. However, no parallel inhibition of either LDH leakage or hsp70 protein expression was observed with vitamin C suggesting that at least two independent mechanisms are involved in the cellular response to copper. Also, copper exposed (24 h) cells were unable to mount an hsp70 response to a standardized heat shock (+15 °C for 1 h), even in the presence of vitamin C. Together, these results suggest that hepatotoxicity of copper includes impairment of hsp70 response to subsequent stressors and/or signals, which is crucial for protecting cells from proteotoxicity.

Toxic effects of wastewaters collected at upstream and downstream sites of a purification station in cultures of rainbow trout hepatocytes.

The toxic effects of wastewater samples, collected in December 1998, from upstream (U) and downstream (D) sites of the purification station of the town of Nice (South-East France on the coast of the Mediterranean Sea) were assessed undiluted and at various dilutions (75%, 50%, and 25% of collected water sample), on trout hepatocyte cultures treated for 48 or 72 h. Chemical contamination (PCBs, PAHs, Cd, Cu, Pb, and Zn) was also evaluated by chemical analysis. The water samples from the upstream site were more cytotoxic than those from the downstream site. The induction of CYP1A enzyme and metallothioneins (MTs) were selected as specific indicators of exposure to organic contaminants and metals, respectively. CYP1A-related EROD activity as well as protein expression were found to be greatly induced after 72 h exposure of the hepatocytes to the undiluted water samples (U(100%) and D(100%)), but CYP1A1 mRNA was significantly overexpressed only by samples from the upstream site. Maximal MT levels were reached after 48 h of treatment with the least concentrated water samples (U(25%) and D(25%)). Glutathione S-transferase (GST) activities were similarly increased under the same conditions. On the other hand, there was no significant glutathione peroxidase (GPx) activity response. Induction of apoptosis was analyzed by using as markers both the fragmentation of the nuclear DNA into oligonucleosomal-length fragments recognized as a "DNA ladder" and the activation of DEVD (Asp-Glu-Val-Asp)-dependent protease considered as the central mediator of programmed cell death. Significant DNA cleavage was only detectable after 72-h exposure to the most concentrated water samples from upstream sites (U(75%) and U(100%)). DEVD-dependent protease activities were significantly increased, mainly in cells exposed to U(75%) and D(25%) for 72 h. In addition, pollution-related DNA damage assessed by using the Comet assay was approximatively 1.5 times greater than that of the control level with the undiluted water samples U(100%) and D(100%), after 72-h and 48-h exposure, respectively. The present study shows that such a multibiomarker-based approach could provide complementary information, for aquatic pollution monitoring, about the early biochemical effects in cells exposed to complex chemical pollution and could be considered as early warning systems to aquatic pollution.

Polycyclic aromatic hydrocarbons as inducers of cytochrome P4501A enzyme activity in the rainbow trout liver cell line, RTL‐W1, and in primary cultures of rainbow trout hepatocytes

In order to investigate cell-specific differences in the response of in vitro models to environmental toxicants, we compared the capacity of nine polycyclic aromatic hydrocarbons (PAHs) to induce cytochrome P4501A (CYPIA) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes and a rainbow trout liver cell line, RTL-W1. Induction of CYPIA was estimated from the catalytic activity of 7-ethoxyresorufin-O-deethylase (EROD) and compared by median effective concentration (EC50) values, induction spans, and benzo[a]pyrene induction equivalency factors for inducing PAHs. The influence of culture conditions was investigated with respect to the presence or absence of serum and varying exposure times. Both in vitro systems lead to an identical classification of the PAHs in noninducing (anthracene, fluoranthene, phenanthrene, and pyrene) and inducing compounds with a similar ranking of inducing PAHs. Mean EC50 values in RTL-W1 cells were, respectively, 343 and 266 nM for benzo[a]anthracene, 57 and 92 nM for BaP, 134 and 283 nM for benzo[b]fluoranthene, 455 and 270 nM for chrysene, and 98 and 116 nM for 3-methylcholanthrene. Compared to primary hepatocytes, the RTL-W1 cell line was more sensitive in its EROD response to the presence or absence of serum and to the increase in exposure time, which led to higher EC50 values.

Regulation of cytochrome P450 in a primary culture of rainbow trout hepatocytes.

Primary cultures of fish hepatocytes have been used as a convenient model for studies on cytochrome expression. Here we have further examined the regulation of CYP enzymes in this model. A transient increase in CYP1A1 messenger ribonucleic acid (mRNA) and 7-ethoxyresorufin-O-deethylase (EROD) activity occurred within h after medium change. This event implies that either an exogenous, quickly metabolized CYP1A1 inducer was introduced to the hepatocytes with the fresh medium, or that the mechanical act of changing the medium disrupts the cell homeostasis, which in turn activates CYP1A1 transcription or alternatively stabilizes CYP1A1 mRNA. CYP1A1 has been shown to be highly inducible in primary cultures of rainbow trout hepatocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via an aryl hydrocarbon (Ah) receptor-mediated activation of gene transcription. In the present study, CYP1A1 was strongly induced by TCDD, whereas CYP2K1, a constitutively expressed cytochrome P450 (CYP), was refractory to the same treatment. Cycloheximide efficiently blocked protein synthesis in the cell culture, and thus the apparent half-life of CYP1A1 (measured as EROD activity) could be estimated. In cells treated with TCDD for 24 h the CYP1A1 apparent half-life was estimated to be 15.9 h. When ethoxycoumarin-O-deethylase activity was used as an indicator of CYP levels, a considerably longer half-life of 27.1 h was estimated. The level of CYP2K1 remained constant throughout the study and was not sensitive to cycloheximide exposure (30 h), indicating a considerably longer half-life of this protein in cell culture.

2,3,7,8-Tetrachlorodibenzo- p-dioxin alters melatonin metabolism in fish hepatocytes

Pineal hormone melatonin is an important regulator of endocrine and circadian rhythms in vertebrates. Since liver is assumed to be the major organ in the metabolism of this indole hormone, we investigated the effect of the known Ah-receptor agonist, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on melatonin metabolism in fish hepatocytes as well as the in vitro effect of melatonin on trout hepatic microsomal cytochrome P4501A (CYP1A) catalyst. Primary cell cultures of rainbow trout hepatocytes were exposed to [ 3H]melatonin (1 nM to 1 μM) alone and in combination with TCDD (50 pM) at 15°C for 24 or 48 h. Analysis of melatonin and its metabolites in the culture medium and hepatocytes by HPLC revealed that about 96% of the added [ 3H]melatonin was metabolised after 24 h in both control and TCDD treated cultures. 3H-radioactivity was found mainly in the culture medium and less than 5% of the total 3H-radioactivity retained inside hepatocytes. Of the HPLC separated metabolites, one coeluted with 6-hydroxymelatonin and one unknown metabolite eluted after 6-hydroxymelatonin. In addition, two other metabolites were more water-soluble than 6-hydroxymelatonin and were considered to be conjugated products. Treatment of the hepatocytes with TCDD increased the amount of the major oxidated product, 6-hydroxymelatonin, about 2.5-fold after 24 h and 1.2-fold after 48 h exposure, respectively when compared with the control cultures. Whereas the amount of the unknown metabolite eluting after 6-hydroxymelatonin decreased about 1.3-fold after 24 h and 1.2-fold after 48 h exposure, respectively. Melatonin alone did not affect P4501A associated EROD-activity or CYP1AmRNA levels in the primary hepatocyte cultures. TCDD-treatment increased EROD-activity 3 to 5-fold and respective CYP1AmRNA content 6 to 14-fold, when compared with the control or melatonin-treated cultures. Furthermore, melatonin competitively inhibited EROD-activity in liver microsomes with a Ki value of 62.06±3.78 μM. The results show that TCDD alters metabolic degradation of melatonin in hepatocytes and suggest that P4501A may be an important P450 isoenzyme involved in oxidative metabolism of melatonin in fish liver.

Heat Shock Proteins and Physiological Stress in Fish

SYNOPSIS. This paper reviews the generalized stress response in fish at the cellular and neuroendocrine levels. The focus of this review is to examine the possible relationships between the stress responses at these two levels in fish. It focuses primarily on the heat shock protein 70 (hsp70). Thus, the descriptions of the endocrine and the cellular stress responses are followed by a discussion of how hsps may be related to the stress hormones adrenaline and cortisol. Preliminary evidence shows that adrenaline causes an increase in hsp70 in primary cultures of rainbow trout hepatocytes. Cortisol does not directly affect hsp70 levels in fish tissues; however, in primary cultures of trout hepatocytes, cortisol decreased the stressor-induced increase in hsp 70. A wide range of abiotic and biological stressors have been shown to induce hsp induction in many types of fish cells, including cell lines, primary cell cultures, and in tissues from whole animals. Heat shock proteins has been implicated in the protection of sulphate transport in the renal epithelium of the flounder against the damaging effects of heat stress. Heat shock proteins likely confer thermotolerance in fish, as well as tolerance to cytotoxic effects of environmental contaminants and other non-thermal stressors.

Assessment of PCBs and hydroxylated PCBs as potential xenoestrogens: In vitro studies based on MCF-7 cell proliferation and induction of vitellogenin in primary culture of rainbow trout hepatocytes.

In the present study, four structurally diverse polychlorinated biphenyls (PCBs) were chosen from a set of 20 PCBs selected to represent the 154 tetra- through hepta-chlorinated biphenyls. The purpose was to determine estrogenic activities of the chosen PCBs and five of their hydroxylated derivatives (OH-PCBs). A human breast cancer cell line (MCF-7) and primary cultures of rainbow trout (Oncorhyncus mykiss) hepatocytes were used to determine estrogenic effects. The PCBs 2,2',4,6,6'-pentachlorobiphenyl (104) and 2,2',3, 4', 5,6,6'-heptachlorobiphenyl (188), and the hydroxylated PCBs 2,2', 4',6'-tetrachloro-4-biphenylol (4'-50), 2',4', 6'-trichloro-4-biphenylol (4'-30), 2',3,5, 5'-tetrachloro-4-biphenylol (4'-72), 2',3,3',5', 6'-pentachloro-4-biphenylol (4'-112), and 2',3,4',5, 6'-pentachloro-4-biphenylol (4'-121) significantly increased MCF-7 cell proliferation. The coaddition of hydroxytamoxifen, an estrogen antagonist, inhibited increased cell proliferation. The activity of the hydroxylated PCBs 4'-50 and 4'-30 was significantly higher at all nominal concentrations tested as compared to the corresponding PCB, viz., PCB 104. The hydroxylated PCBs 4'-50, 4'-30, 4'-72 and 4'-112 induced vitellogenin synthesis in rainbow trout hepatocytes. Significant differences were found in the MCF-7 system between the parent PCB and its hydroxylated derivative, viz., for 4'-50/4'-30 and 104, and in the rainbow trout hepatocyte assay between 4'-112 and 112, respectively. No activity was observed for PCB 58 in any of the two assays in the present study. Even though cells from two different species (human and fish) are used in the present study, the results obtained by the two methods agree fairly well. In both studies the hydroxylated PCBs were more active than the PCBs, and 4'-30 was the most active compound second only to 17beta-estradiol. http://link.springer-ny. com/link/service/journals/00244/bibs/37n2p145.html