Estrogenic response of bisphenol A in rainbow trout (Oncorhynchus mykiss)

Bisphenol A (BPA) previously shown to possess xenoestrogenic activities was administered to rainbow trout (Oncorhynchus mykiss) through a continuos flow system. The estrogenic response expressed as the induction of vitellogenin (VTG) synthesis was measured during 12 days of exposure, using a direct sandwich ELISA. Quantification of internal liver and muscle concentrations of non-metabolised BPA was performed by LC-MS at the end of the exposure period. A significant induction of the VTG synthesis was obtained at 500 μg BPA/l exposure, although an increase in the ratio of responding animals was observed already between 40 and 70 μg BPA/l. An increase in VTG levels was observed for the 500 μg BPA/l group over the study period, whereas constant or decreasing levels could be detected in the low exposure groups between days 6 and 12. Average internal liver concentrations of BPA increased from 0.22 to 4.36 μg/g for the 10-500 μg BPA/l groups. However, BPA could not be detected in muscle tissue below an exposure level of 70 μg BPA/l. A dose response relationship was established between the internal liver concentrations of BPA and the corresponding VTG responses, with a P<0.001 and a correlation coefficient of 0.66.     

Cytotoxicity of atorvastatin and simvastatin on primary rainbow trout (Oncorhynchus mykiss) hepatocytes

Statins are cholesterol-lowering pharmaceuticals and commonly prescribed drugs in European countries. Their discharge into the aquatic environment has increased in the last few years and they are present at detectable levels in most sewage effluents. The aim of the present study was to quantify the cytotoxic effects of acid and lactone forms of two statins, atorvastatin and simvastatin, as well as selected metabolites (ortho- and para-hydroxy atorvastatin acid, ortho-hydroxy atorvastatin lactone, simvastatin hydroxyl carboxylic acid, and 3′′hydroxy simvastatin lactone) to hepatocytes from rainbow trout (Oncorhynchus mykiss). Hepatocytes were exposed for 24, 48, and 72 h to different concentrations of each test substance (0.4–400 μM). Cytotoxicity was measured as metabolic inhibition and loss of membrane integrity with the fluorescent probes alamar blue (AB) and 5-carboxyfluorescein diacetate, acetoxymethyl ester (CFDA–AM), respectively. Atorvastatin, simvastatin, and ortho-hydroxy atorvastatin lactone had dose-dependent cytotoxic effects on hepatocytes. Simvastatin was more toxic than atorvastatin and the lactone form more toxic than the acid form. Exposure time affected atorvastatin and ortho-hydroxy atorvastatin lactone but not simvastatin toxicity.     

Estrogenic effect of dietary 4-tert-octylphenol in rainbow trout (Oncorhynchus mykiss)

The estrogenic effect of dietary 4-tert-octylphenol (octylphenol) in rainbow trout Oncorhynchus mykiss was investigated. Octylphenol was administered orally to sexually immature rainbow trout every second day for 11 days in doses between 0.4 and 50 mgkg(-1)2 d(-1). Plasma vitellogenin was measured at day 0, 6 and 11 and at the end of the experiments, the amounts of octylphenol retained in liver and muscle were determined. Increases in average plasma vitellogenin levels were seen at exposure to 40 mg octylphenol kg(-1) every second day; the most sensitive fish responded to 30 mgkg(-1). Doses below 20 mg octylphenol kg(-1)2 d(-1) had no effect. The ED(50) value for induction of vitellogenin synthesis was 35 mg octylphenol kg(-1)2 d(-1). Only 1 to 2 per thousand of the total amount of octylphenol administered orally over the 11 days experimental period was retained in muscle and liver at the end of the experiment. A clear dose-related increase was observed for concentrations of octylphenol in both liver and muscle of fish exposed to doses between 0.4 and 50 mgkg(-1)2 d(-1). A significant correlation was found between the concentrations of octylphenol in the liver and vitellogenin level in plasma.     

Effect of iprodione, a dicarboximide fungicide, on primary cultured rainbow trout (Oncorhynchus mykiss) hepatocytes.

As is known from literature, iprodione, a dicarboximide fungicide, has a highly specific action, with a capacity to cause oxidative damage through production of free oxygen radicals (ROS), but it does not appear to be species selective. Since this substance is able to diffuse in water, evaluation of its capacity to induce oxidative damage in an aquatic organism such as the rainbow trout (Oncorhynchus mykiss) was considered of particular interest. A study was, therefore, undertaken to investigate the effect of iprodione on free radicals (ROS) and malondialdehyde (MDA) production, reduced glutathione (GSH) content and catalase activity (CAT), in primary cultured trout hepatocytes, following treatment with 0.2, 0.3 and 0.4 mM concentrations for a 24-h period. The iprodione 0.3 and 0.4 mM concentrations increased both ROS and MDA production and decreased GSH content and CAT activity. These results suggest that iprodione is able to produce oxidative damage in primary cultured fish hepatocytes, thus confirming that its action is specific, but not species selective. It is also well known that ROS production in fungi is due to interaction with the flavin enzyme NADPH cytochrome c reductase to the extent that the normal electron flow from NADPH to cytochrome c is blocked. In contrast, we observed that, in primary cultured trout hepatocytes, iprodione appears to have no effect on NADPH cytochrome c reductase activity. It is, therefore, possible to presume that the mechanism of oxidative damage in trout hepatocytes differs from that observed in fungi. Moreover, our experiments also demonstrate that iprodione is able to induce "in vitro" CYP1A1, leading to the conclusion that the production of ROS is due to this phenomenon.     

The partial pressure of oxygen affects biomarkers of oxidative stress in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes

Oxidative stress, the imbalance between production of reactive oxygen species and the cellular detoxification of these reactive compounds, is believed to be involved in the pathology of various diseases. Several biomarkers for oxidative stress have been proposed to serve as tools in toxicological and ecotoxicological research. Not only may exposure to various pro-oxidants create conditions of cellular oxidative stress, but hyperoxic conditions may also increase the production of reactive oxygen species. The objective of the current study was to determine the extent to which differences in oxygen partial pressure would affect biomarkers of oxidative stress in a primary culture of hepatocytes from rainbow trout (Oncorhynchus mykiss). Membrane integrity, metabolic activity, levels of total and oxidized glutathione (tGSH/GSSG) was determined, as well as mRNA expression levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), gamma-glutamyl-cystein synthetase (GCS) and thioredoxin (TRX). The results show that different biomarkers of oxidative stress are affected when the cell culture is exposed to atmospheric oxygen, and that changes such as increased GSSG content and induction of GSSG-R and GSH-Px can be reduced by culturing the cells under lower oxygen tension. Oxygen tension may thus influence results of in vitro based cell research and is particularly important when assessing parameters in the antioxidant defence system. Further research is needed to establish the magnitude of this effect in different cellular systems.     

Biotransformation of the xenoestrogen 4-tert-octylphenol in hepatocytes of rainbow trout (Oncorhynchus mykiss)

1. The biotransformation of a 4-tert-alkylphenol in rainbow trout (Oncorhynchus mykiss) liver was studied to determine the possible fate and activity of these xenoestrogens in fish. 2. Primary trout hepatocytes were incubated with 30 μM 4-(1′, 1′,3′, 3′-tetramethylbutyl)[U-¹⁴C]phenol (4-tert-octylphenol; 4-t-OP) for up to 3 h. Radiolabelled metabolites were detected by radio-HPLC and the structures were determined by GC-MS analysis of the conjugated or aglycone products. 3. During the first 15 min, 4-t-OP was metabolized at 1.06 pmol·min^?1·10^?6 cells. The amount of parent compound metabolized was maximum after a 1-h incubation, when 86% of 4-t-OP was transformed to five other products. 4. The major metabolite comprised 61% of the total recovered radioactivity and was identified as 4-t-OP-β-glucuronide. 5. The remaining metabolites were formed from the hydroxylation of 4-t-OP on either the C2 (w-3) or C4 (omega) positions of the alkyl chain, or ortho on the aromatic ring to form a catechol. These oxidized products were also metabolized to glucuronide derivatives conjugated on the phenol ring. 6. The results suggest that oestrogenic alkylphenols could be rapidly transformed in fish liver by both phase I and II metabolic pathways to a number of conjugated products which are unlikely to be active at the oestrogen receptor.     

Assessing combined toxicity of estrogen receptor agonists in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes

The presence of highly complex mixtures of chemicals in the environment challenges our ability to assess single chemical effects and the interaction that occurs with cellular receptor targets and regulation of endocrine processes. In this study concentration addition (CA) and independent action (IA) prediction models were used to assess the combined toxicity of mixtures of environmental relevant estrogen receptor (ER) agonists (hormones and anthropogenic pollutants) in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes using the ER-mediated production of vitellogenin (Vtg) as a biological marker (biomarker) for estrogenicity. Nine of the eleven tested chemicals induced the production of Vtg and the parameters from the fitted concentration-response curves were used to model four mixtures containing four (17β-estradiol, estrone, estriol and diethylstilbestrol), five (musk ketone, 4-tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene), seven (17β-estradiol, estrone, estriol, diethylstilbestrol, 4-tert-octylphenol, bisphenol A and o,p′-DDT) and nine compounds (17β-estradiol, estrone, estriol, diethylstilbestrol, musk ketone, 4-tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene). The CA and IA prediction model proved to be a good estimation for the combined effect of mixtures of ER agonists at low relative mixture concentration (e.g. relative to the maximum mixture concentrations used), but a deviation from the prediction models was observed when exposing hepatocytes to high relative mixture concentrations. The CA and IA prediction models’ ability to predict the combined estrogenic effect of complex mixtures, especially in the low concentration-response range, is of ecological relevance since organisms in the environment generally encounter low concentrations of chemicals from a wide array of chemical groups that may not elicit estrogenic effects on their own.     

Biotransformation of the xenoestrogen 4-tert-octylphenol in hepatocytes of rainbow trout (Oncorhynchus mykiss)

1. The biotransformation of a 4-tert-alkylphenol in rainbow trout (Oncorhynchus mykiss) liver was studied to determine the possible fate and activity of these xenoestrogens in fish. 2. Primary trout hepatocytes were incubated with 30 microM 4-(1',1',3',3'-tetramethyl-butyl)[U-14C]phenol (4-tert-octylphenol; 4-t-OP) for up to 3 h. Radiolabelled metabolites were detected by radio-HPLC and the structures were determined by GC-MS analysis of the conjugated or aglycone products. 3. During the first 15 min, 4-t-OP was metabolized at 1.06 pmol x min(-1) x 10(-6) cells. The amount of parent compound metabolized was maximum after a 1-h incubation, when 86% of 4-t-OP was transformed to five other products. 4. The major metabolite comprised 61% of the total recovered radioactivity and was identified as 4-t-OP-beta-glucuronide. 5. The remaining metabolites were formed from the hydroxylation of 4-t-OP on either the C2 (omega-3) or C4 (omega) positions of the alkyl chain, or ortho on the aromatic ring to form a catechol. These oxidized products were also metabolized to glucuronide derivatives conjugated on the phenol ring. 6. The results suggest that oestrogenic alkylphenols could be rapidly transformed in fish liver by both phase I and II metabolic pathways to a number of conjugated products which are unlikely to be active at the oestrogen receptor.     

Abamectin effects on rainbow trout (Oncorhynchus mykiss)

The effect of abamectin (ABM) on rainbow trout (Oncorhynchus mykiss) was studied. The acute toxicity of ABM on rainbow trout was established, following the target 58-h water bath exposure of ABM concentrations from 0.6 to 4.5 μg/l, on the basis of which LD₇₅ (4.0 μg/l) was calculated. The histological changes in organs showed a direct toxicity of ABM for rainbow trout since degenerative changes in brain and kidney and—to a minor extent—in liver were established. The values of the ABM residues in fish muscle tissue with skin were proportional to the exposed concentrations of ABM.     

Induction of oxidative stress by selenomethionine in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss)

Fish are exposed to environmental selenium predominantly in the form of dietary selenomethionine (SeMet). The present study was designed to investigate the role of oxidative stress in the toxicity of SeMet using isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) as the model experimental system. Cells were exposed to an increasing range of SeMet (0–1000 μM) over 24 h, and the time-dependent effects on cell viability, response of enzymatic antioxidants, thiol redox, intracellular calcium balance and caspase-mediated apoptosis were evaluated. SeMet was found to be toxic only at the highest exposure dose (1000 μM), with ～15% decrease in cell viability. Although modest increases in the activities of antioxidant enzymes were recorded following SeMet exposure, the ratio of reduced to oxidized glutathione decreased in a dose-dependent manner, suggesting a gradual progression towards an oxidative intracellular environment. The peroxidation of membrane lipids also increased with increasing SeMet exposure dose. In addition, a rapid increase in intracellular calcium level and the activation of caspase 3/7 enzymes were recorded at the highest exposure dose, indicating that SeMet at a high exposure dose causes cell death probably via apoptosis. Overall, our study demonstrated that oxidative stress plays a key role in the cytotoxicity of SeMet in fish.     

Inhibition of oxygen consumption by pentachlorophenol and tetrachloroguaiacol in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) were exposed for 24 h to concentrations representing 100, 50 and 25% of the 96 h-LC50 of pentachlorophenol (PCP) or tetrachloroguaiacol (TCG), and their oxygen consumption, cardiac output, heart rate and stroke volume were measured at regular intervals. Oxygen consumption either remained stable at basal levels (PCP), or increased to 130% of basal levels (TCG) when fish were exposed to the 96 h-LC50 of each chemical. However, oxygen consumption decreased to about 50-60% of basal levels when fish were exposed to concentrations of PCP or TCG representing 50 and 25% of the 96 h-LC50. This decrease in oxygen consumption did not appear to affect cardiac function since cardiac output, heart rate and stroke volume remained stable. PCP is best known for its capacity to uncouple oxidative phosphorylation and increase oxygen consumption. However, this study showed that it can also decrease oxygen consumption, and that the effects of PCP and TCG on fish metabolism are similar.     

Extrahepatic disposition of 3H-aflatoxin B1 in the rainbow trout (Oncorhynchus mykiss).

Whole-body autoradiography in rainbow trout (Oncorhynchus mykiss) after oral and intravenous administration of 3H-labelled aflatoxin B1 showed labelling of several extrahepatic tissues, such as the uveal melanin and the vitreous humour of the eyes, the trunk and head kidney, the olfactory rosettes and the pyloric caecae. Liquid chromatography of extracts of the vitreous humour showed that unmetabolized 3H-AFB1 was the main labelled material present at this site. Liquid chromatography of extracts of the uveal melanin showed presence of aflatoxicol and aflatoxin B1 in proportions of about 3:1. The binding to the pigment is probably due to a hydrophobic type of interaction with the melanin. Microautoradiography showed that melanin-containing cells in the trunk and head kidney and in the olfactory rosettes also accumulated high amounts of radioactivity. In the trunk kidney there was, in addition, a labelling of the second segment of the proximal tubules and of the distal tubules and the collecting ducts. Studies in vitro with microsomal and 12,000 x g supernatant preparations of the trunk kidney showed formation of DNA- and protein-bound metabolites from the aflatoxin B1. It is probable that the bioactivation of the aflatoxin B1 is confined to the cytoplasm of the cells, may be related to excretion and/or absorption processes. Microautoradiography of the olfactory rosettes, showed labelling of the sensory epithelium, but not the indifferent epithelium. A low formation of protein-bound aflatoxin B1-metabolites was found in incubations with microsomal preparations of this tissue. The same observation was made in incubations with microsomal preparations of the head kidney. In the pyloric caeca bound metabolites were observed in vivo at a level comparable to that found in the trunk kidney. Our results suggest that retention and metabolism in some extrahepatic tissues might be of importance as concerns the toxicologic potential of aflatoxin B1 in the rainbow trout.     

Energetic costs of pyrene metabolism in isolated hepatocytes of rainbow trout, Oncorhynchus mykiss

The respiratory costs of pyrene exposure and biotransformation were examined in isolated hepatocytes of adult rainbow trout, Oncorhynchus mykiss. Baseline oxygen consumption rates measured at an acclimation temperature of 7.5 degrees C and during an acute temperature increase to 15 degrees C were 10.1 +/- 0.1 and 22.6 +/- 0.4 ng O(2)/min/mg cells, respectively. Hepatocytes exposed to pyrene at 1, 5 and 10 microg/ml exhibited concentration-dependent increases in oxygen consumption. Respiration rates of cells exposed to these concentrations at their acclimation temperature were 12.5 +/- 0.1, 14.7 +/- 0.1 and 17.1 +/- 0.2 ng O(2)/min/mg cells, respectively. Exposure of cells to pyrene at 15 degrees C also elevated oxygen consumption to a maximum of 34.4 +/- 0.3 ng O(2)/min/mg cells, however, the relationship with pyrene concentration was biphasic. The major metabolite identified through a series of solvent extractions, acid hydrolysis, and synchronous fluorometric spectroscopy was conjugated 1-hydroxypyrene. At 7.5 degrees C, increased pyrene metabolism correlated with increased hepatocyte respiration rates. At 15 degrees C, however, pyrene metabolism reached a maximum at 5 microg/ml, suggesting saturation of detoxification enzymes, which correlated with maximum respiration rates at this concentration. Measures of respiration by isolated mitochondria indicated that changes in hepatocyte oxygen consumption were not through direct effects of pyrene on mitochondria. This study indicates that significant respiratory costs may be accrued by teleost hepatocytes actively metabolizing and secreting xenobiotic compounds.     

Effects of deltamethrin on rainbow trout (Oncorhynchus mykiss)

The aim of this study was to assess the effect of deltamethrin on rainbow trout (Oncorhynchus mykiss). Control and experimental group of fish were exposed to Decis EW 50 pesticide preparation (active substance 50 g/l of deltamethrin). The acute semistatical toxicity test lasting 96 h was performed on rainbow trout juveniles. The 96hLC₅₀ value of Decis EW 50 was 0.02 mg/l. Examination of haematological and biochemical profile and histological tissue examination was performed on 1–2-year-old rainbow trout after 96 h of exposure to Decis EW 50 in a concentration of 0.02 mg/l. The experimental group showed significantly lower values (p < 0.05) of plasma glucose, alanine aminotransferase, cholinesterase and significantly higher (p < 0.05) values of erythrocyte count, haemoglobin content, haematocrit and plasma total protein, albumins, ammonia, aspartate aminotransferase, creatinekinase and calcium compared to the control group. The deltamethrin-based Decis EW 50 pesticide preparation was classified among substances strongly toxic for fish.     

Comparative pharmacokinetics and bioavailability of oxolinic acid and oxytetracycline in rainbow trout (Oncorhynchus mykiss).

1. The pharmacokinetics and bioavailability of oxolinic acid and oxytetracycline were studied in rainbow trout at a water temperature of 16 degrees C after intravascular (10 and 20 mg/kg, respectively) and oral (75 mg/kg) dosing. 2. The pharmacokinetics were best described by a two-compartment open model giving distribution half-lives of 0.31 h and 1.53 h, and elimination half-lives of 69.7 h and 60.3 h for oxolinic acid and oxytetracycline, respectively. The respective volumes of distribution (Vdarea) were 1.94 l/kg and 1.34 l/kg. 3. The apparent oral bioavailability for oxolinic acid and oxytetracycline was 13.6% and 5.6%. 4. The plasma protein binding was 27% for oxolinic acid and 55% for oxytetracycline. 5. Both drugs were well tolerated, the acute oral toxicities (LD50) exceeding 4000 mg/kg.     

Temperature-dependent vitellogenin-mRNA expression in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes at 14 and 18 degrees C.

In order to study the influence of temperature on vitellogenin gene and estrogen receptor gene expression in primary hepatocytes from rainbow trout (Oncorhynchus mykiss), cells were exposed to 17β-estradiol, bisphenol-A and nonylphenol for 48 and 96 hr. Induction of vitellogenin-mRNA expression was detected in a non-radioactive dot blot/RNAse protection assay and by RT-PCR. In the dot blot/RNAse protection assay, the estrogenic potentials of bisphenol-A and nonylphenol were about 10⁴- to 10⁵-fold and 10⁵-fold lower than that of 17β-estradiol, respectively. The relative estrogenic potential did not show any difference between 14 and 18°C. In contrast, at 18°C, RT-PCR analysis revealed increased amounts of vitellogenin- and estrogen receptor-mRNA after 12 and 24 hr of exposure to 17β-estradiol, if compared to 14°C. Owing to increased vitellogenin gene expression at 18°C, the sensitivity of primary hepatocytes to 17β-estradiol and bisphenol-A could be increased.     

Effects of nonylphenol on estrogen receptor conformation, transcriptional activity and sexual reversion in rainbow trout (Oncorhynchus mykiss).

Estrogenic potency of 4-n-nonylphenol diethoxylate, 4-n-nonylphenol (NP) and metabolites were tested using two bioassays: rainbow trout hepatocyte culture and recombinant yeast stably expressing rainbow trout estrogen receptor (rtER) and containing estrogen-dependent reporter genes. Since NP was the only compound active in both systems, its interaction with rtER was studied in more detail. Qualitative and quantitative differences were observed in the presence of 17beta-estradiol (E2) or NP when estrogen-dependent promoters containing one to three estrogen-responsive elements were used in yeast. Moreover, limited proteolysis of rtER after E2 or NP binding presented different patterns after SDS-PAGE analysis suggesting that NP induces a differential conformation of rtER compare to E2. This finding may have important implications with respect to the biological activity of NP. Thus, the effects of NP on the activation of an E2-dependent gene and on sexual differentiation were assessed on all-male trout embryos exposed to NP for 1 h per day for 10 days. Although in situ hybridization demonstrated that E2, and to a lesser extend NP, were able to increase rtER mRNA level in the liver of embryos, no indication of total or partial sexual reversion was observed (even in E2 treated fishes) when the gonads were examined 8 months after hatching.     

Role of flavin-containing monooxygenase in the in vitro biotransformation of aldicarb in rainbow trout (Oncorhynchus mykiss).

1. The in vitro biotransformation of 14C-aldicarb was examined in liver, kidney, and gill microsomes from the rainbow trout (Oncorhynchus mykiss). 2. In all tissues the major metabolite was aldicarb sulphoxide. Addition of the cytochrome P-450 inhibitor, N-benzylimidazole, failed to alter significantly aldicarb sulphoxide levels, while co-incubation with the flavin-containing monooxygenase substrates, N,N-dimethylaniline or methimazole, caused significant decreases in sulphoxide formation in liver and gill microsomes. 3. Aldicarb sulphoxide formation was optimal at pH 8.0, and had Michaelis-Menten kinetics with an apparent Km of 46.7 microM and a Vmax of 0.216 nmol/min per mg. 4. Aldicarb sulphoxide formation was competitively inhibited by co-incubation with N,N-dimethylaniline in liver microsomes. These data indicate that flavin-containing monooxygenase plays an important role in the in vitro biotransformation of aldicarb in rainbow trout.