Influence of o'p-DDD on the physiological response to stress in Arctic charr (Salvelinus alpinus)

Various toxicants have previously been held responsible for an impaired capacity of fish from polluted environments to elevate their cortisol levels in response to stress. In the present study we investigated the responses to stress in o'p-DDD [2-(chlorophenyl)-2-(4-chlorphenyl)-1,1-dichloroethane] exposed (given a single, oral dose of 75 mg o'p-DDD/kg fish) and unexposed Arctic charr. After o'p-DDD administration fish were left undisturbed and without being fed for 28 days, when they were subjected to an acute handling stress. At 1, 3, 7 and 23 h following stress, primary (ACTH and cortisol secretion) and secondary (plasma Cl levels and energy mobilisation) components of the stress response were monitored. As the nutritional state of wild fish may influence this potential biomarker response, the fish had been subjected to a restricted feed ration prior to o'p-DDD administration in order to obtain marked within-group variations in condition factor. No effects of o'p-DDD were observed on post-stress hormone secretion (i.e. peak post-stress plasma ACTH and cortisol levels), nor on plasma chloride levels. However, other results obtained provided evidence for a metabolic depression by o'p-DDD, witnessed by consistently lower plasma glucose levels before and after stress in these contaminated fish. This may be related to the finding that during the 30-day period between o'p-DDD administration and stress treatment, toxicant treated fish lost less weight in comparison to their sham-treated counterparts. Nutritional state did not appear to influence the performance of the charr in the present experiment, as correlations between the parameters measured and condition factor or lipid contents on an individual basis in all cases turned out non significant. Overall, the results contrast with those of previous in vivo and in vitro studies on fish, which concluded that comparable headkidney o'p-DDD levels impaired interrenal steroidogenesis. Although we conclude that the effects of o'p-DDD on Arctic charr metabolism were not associated with the stress response, we propose that they may well interfere with the animals' ability to cope with stress in the long term, or may compromise other physiological processes, such as smoltification. Finally, the high level of integration of components involved in the stress response complicates the identification of single stress-sensitive indices as biomarkers applicable in environmental monitoring programmes.     

Hematological,biochemical and histopathological changes in Caspian brown trout (Salmo trutta caspius Kessler, 1877) following exposure to sublethal concentrations of chlorpyrifos

The main objective of this study was to survey the acute and sublethal effect of chlorpyrifos (CPF) on Caspian brown trout (Salmo trutta caspius). Toxicity tests were performed for the determination of 96?h median lethal concentration (96hLC50). For this purpose, nominal concentrations of CPF including 0.0125, 0.025, 0.05, 0.1, 0.2 and 0.3?mgL^??¹. The 96hLC50 value of CPF was 0.026?mgL^?1. The sublethal effect was determined on the basis of acute toxicity, hematological effects, biochemical plasma profile, behavioral and histopathological changes. The experimental group exposed to CPF at nominal concentration of 0.0026?mgL^?1 (10% 96hLC50) for 30 days showed significant changes in hematological and biochemical profiles and caused histopathological changes in liver, gill and kidney. Based on these data, CPF is acute toxic for Caspian trout.     

Toxicokinetics of tetrabromoethylcyclohexane (TBECH) in juvenile brown trout (Salmo trutta) and effects on plasma sex hormones

Technical 1,2-dibromo-4-(1,2 dibromoethyl)cyclohexane or tetrabromoethylcyclohexane (TBECH) used primarily as an additive flame retardant in polystyrene foams, contains two diastereoisomers, α- and β- present in equimolar amounts. At temperatures in excess of 125 °C, isomerization to two other isoforms, δ- and γ- is possible. The recent detection of TBECH in the environment and studies suggesting that isomers are androgenic prompted us to examine the toxicokinetics and biochemical effects of one of the isomers, β-, in a controlled laboratory environment. Juvenile brown trout (Salmo trutta) were exposed to three different amounts of the β-isomer (low, medium and high) via the food followed by a period in which they were exposed to unfortified food. A fourth group of fish was exposed to unfortified food for the duration of the experiment. On days 0, 7, 14, 21, 35, 49, 56, 63, 77, 91, 105, and 133, eight fish from each treatment group were euthanized and liver, plasma, lower jaw (i.e., thyroid tissue) and gonad were collected and the remaining tissue (‘whole-fish’) was retained. β-Isomer content was measured in whole-fish and in liver while estradiol (E2), 11-ketotestosterone (11-KT) and testosterone (T) were measured in plasma. Based on liver and gonad somatic indices, no apparent effects on liver or gonad development in fish from any of the treatment groups were observed. The bioaccumulation of β-isomer was similar in fish from all treatment groups with steady-state occurring before the end of the uptake phase. Depuration of the β-isomer from fish obeyed first order kinetics and there were no statistically significant differences in the depuration half life (t_1/2) among the treatment groups: 22.5 ± 10.4 (low), 13.5 ± 5.9 (med) and 13.8 ± 2.2 (high) days. Steady-state biomagnification factors were much smaller than 1 for fish in all treatment groups. Debrominated metabolites were not detected in composite liver or whole-fish extracts and there was no evidence of isomerization of the β-isomer to other isoforms in vivo. While there were occasional differences among treatment groups in circulating plasma E2, T and 11-KT levels there was no clear, temporal trend or dose-response.     

Gonad histology and vitellogenin concentrations in brown trout (Salmo trutta) from Danish streams impacted by sewage effluent

Brown trout (Salmo trutta) collected from a number of Danish streams impacted by sewage effluent were examined for alterations to gonadal development and induction of vitellogenin synthesis. Among fish collected in June/July 2000/2001 and November 2002 higher levels of plasma vitellogenin were found in males from six streams impacted by sewage effluent compared to males from their respective reference sites. A direct non-competitive ELISA was developed for brown trout vitellogenin in order to perform the vitellogenin measurements. Intersex in females with no apparent relation to sewage effluent exposure was observed at all sites. In one stream, male brown trout with a very high level of vitellogenin were concomitantly found to have a high degree of vacuolation of the testes and a presence of only the early spermatogenic stage, spermatogonia. The cause of these alterations to the testis structure is unclear. However, as a high level of plasma vitellogenin in these males indicated estrogenic exposure, the vacuolation might also be a result of endocrine disruption causing delayed or disrupted spermatogenesis.     

Social interactions, predation behaviour and fast start performance are affected by ammonia exposure in brown trout (Salmo trutta L.)

In fish, fast starts are brief, sudden accelerations during predator-prey encounters. They serve for escape and predation and are therefore ecologically important movements. Fast starts are generated by glycolytic muscle performance and are influenced by many internal and external factors. It is known that ammonia pollution has a major effect on the glycolytic muscle action, thus creating conditions in which fast start performance might be reduced and predation rates altered. Therefore, escape response and predation strikes were investigated in brown trout (Salmo trutta) of 10 and 20 cm body length exposed to an elevated (1 mg l(-1)) ammonia concentration for 24 and 96 h. Various locomotor and behavioural variables were measured. In C-starts, i.e. an escape start where the fish bends into a C-shaped position, ammonia exposure had no effect on response latency. After 96 h of exposure, cumulative distance, maximum swimming speed and turning radius of the prey were all significantly reduced and the escape went in no definite direction. The effect of ammonia exposure was more pronounced in large fish than in small fish. Predation strikes were also affected. Distance, speed and turning radius were significantly lower in exposed fish. Agonistic behaviour of dominant fish was significantly reduced and fish spent more time resting. Predator behaviour was also altered and the number of prey captured was reduced. This study shows that ammonia exposure affects brown trout escape response mainly through a reduction in fast start velocity and through an impairment of directionality. Thus, in addition to a reduced strength of the response, ammonia exposure could also reduce the fish's elusiveness facing a predator. Predation rate and social interactions are disrupted and predator-prey relationships could be altered.     

Water-borne diclofenac affects kidney and gill integrity and selected immune parameters in brown trout (Salmo trutta f. fario)

The detection of residues of various pharmaceuticals in surface waters during the last two decades has prompted concerns about possible adverse effects of this kind of pollution on aquatic organisms. The objective of the present study was to investigate effects of the non-steroidal anti-inflammatory drug diclofenac, one of the pharmaceuticals most prevalent in surface waters, on brown trout (Salmo trutta f. fario), a salmonid species native to German rivers. Brown trout were exposed to 0.5, 5 and 50 microg/L diclofenac for 7, 14 and 21 days, whereby the lowest exposure concentration is comparable with concentrations commonly found in the aquatic environment. Fish exposed to diclofenac displayed significantly reduced haematocrit levels after 7 and 14 days of exposure. After 21 days, trout were examined for histopathological alterations, whereby diclofenac exposure resulted in increased monocyte infiltration in the liver, telangiectasis in gills, and the occurrence of interstitial hyaline droplets, interstitial proteinaceous fluid and mild tubular necrosis in trunk kidney. Concurrent immunohistological analysis revealed an increase of granulocyte numbers in primary gill filaments, as well as granulocyte accumulation and increased major histocompatibility complex (MHC) II expression in kidney, suggestive of an inflammatory process in these organs. Moreover, the ability of diclofenac to hinder the stimulation of prostaglandin E2 synthesis was shown in head kidney macrophages of brown trout in vitro. These findings support the hypothesis that environmental exposure of fish to diclofenac provokes the same mechanism of action in these non-target organisms as previously described for mammalian species and can thus lead to similar (possibly adverse) effects. In general, the present study suggests that exposure of brown trout to diclofenac in concentration ranges commonly found in the environment can result in adverse effects in various organs and possibly compromise the health of affected fish populations.     

Molecular and cellular effects of chemicals disrupting steroidogenesis during early ovarian development of brown trout (Salmo trutta fario)

A range of chemicals found in the aquatic environment have the potential to influence endocrine function and affect sexual development by mimicking or antagonizing the effects of hormones, or by altering the synthesis and metabolism of hormones. The aim of this study was to evaluate whether the effects of chemicals interfering with sex hormone synthesis may affect the regulation of early ovarian development via the modulation of sex steroid and insulin‐like growth factor (IGF) systems. To this end, ex vivo ovary cultures of juvenile brown trout (Salmo trutta fario) were exposed for 2 days to either 1,4,6‐androstatriene‐3,17‐dione (ATD, a specific aromatase inhibitor), prochloraz (an imidazole fungicide), or tributyltin (TBT, a persistent organic pollutant). Further, juvenile female brown trout were exposed in vivo for 2 days to prochloraz or TBT. The ex vivo and in vivo ovarian gene expression of the aromatase (CYP19), responsible for estrogen production, and of IGF1 and 2 were compared. Moreover, 17β‐estradiol (E2) and testosterone (T) production from ex vivo ovary cultures was assessed. Ex vivo exposure to ATD inhibited ovarian E2 synthesis, while T levels accumulated. However, ATD did not affect ex vivo expression of cyp19, igf1, or igf2. Ex vivo exposure to prochloraz inhibited ovarian E2 production, but did not affect T levels. Further prochloraz up‐regulated igf1 expression in both ex vivo and in vivo exposures. TBT exposure did not modify ex vivo synthesis of either E2 or T. However, in vivo exposure to TBT down‐regulated igf2 expression. The results indicate that ovarian inhibition of E2 production in juvenile brown trout might not directly affect cyp19 and igf gene expression. Thus, we suggest that the test chemicals may interfere with both sex steroid and IGF systems in an independent manner, and based on published literature, potentially lead to endocrine dysfunction and altered sexual development. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 199–206, 2014.     

Effect of potassium ethylxanthate and sodium diethyldithiocarbamate on the accumulation and disposition of nickel in the brown trout (Salmo trutta)

Brown trouts, Salmo trutta, were exposed to water containing 0.1 or 10 micrograms/l of 63Ni2+, alone or with potassium ethylxanthate or sodium diethyldithiocarbamate. After one and three weeks the accumulation and disposition of the 63Ni2+ in the fish were examined by liquid scintillation spectrometry and whole-body autoradiography. The sodium diethyldithiocarbamate was found to greatly enhance the uptake of 63Ni2+ in several tissues of the trouts. Potassium ethylxanthate was without effect. Diethyldithiocarbamate is known to form lipophilic complexes with metals, including nickel, and a facilitated penetration of the complexed nickel over the cellular membranes of the gills and other tissues is a likely mechanism underlying our results. The ethylxanthate is also able to form a lipophilic nickel-chelate, although of a lower lipophilicity than the nickel-diethyldithiocarbamate-complex. This variance in lipophilicity may explain why the disposition of the 63Ni2+ was affected by the diethyldithiocarbamate, but not by the ethylxanthate.     

The Hypothalamic-Pituitary-Thyroid (HPT) Axis in Fish and Its Role in Fish Development and Reproduction

Bony fishes represent the largest vertebrate class and are a very diverse animal group. This chapter provides a thorough review of the available scientific literature on the thyroid system in these important vertebrate animals. The molecular components of the hypothalamic-pituitary-thyroid (HPT) axis in this group correspond closely to those of mammals. The thyroid tissue in the fishes is organized as diffuse follicles, with a few exceptions, rather than as an encapsulated gland as is found in most other vertebrate species. The features of this diffuse tissue in fishes are reviewed with an emphasis on feedback relationships within the HPT axis, the molecular biology of the thyroid system in fishes, and comparisons versus the thyroid systems of other vertebrate taxa. A review of the role of thyroid hormone in fish development and reproduction is included. Available information about the HPT axis in fishes is quite detailed for some species and rather limited or absent in others. This review focuses on species that have been intensively studied for their value as laboratory models in assays to investigate disruption in normal function of the thyroid system. In addition, in vitro and in vivo assay methods for screening chemicals for their potential to interfere with the thyroid system are reviewed. It is concluded that there are currently no in vitro or in vivo assays in fish species that are sufficiently developed to warrant recommendation for use to efficiently screen chemicals for thyroid disruption. Methods are available that can be used to measure thyroid hormones, although our ability to interpret the causes and implications of potential alterations in T₄ or T₃ levels in fishes is nonetheless limited without further research.     

Effect of sodium isopropylxanthate,potassium amylxanthate and sodium diethyldithiocarbamate on the uptake and distribution of cadmium in the brown trout (Salmo trutta)

Brown trout, Salmo trutta, were exposed to water containing 1 μg/l of ¹⁰⁹Cd²⁺, alone or with sodium isopropylxanthate, potassium amylxanthate or sodium diethyldithiocarbamate, respectively. After one week the uptake and distribution of the ¹⁰⁹Cd²⁺ in the fish were examined by whole-body autoradiography and gamma spectrometry. Sodium diethyldithiocarbamate was found to enhance the uptake of the ¹⁰⁹Cd²⁺ in several tissues of the fish and this effect increased with increasing concentration of the carbamate. Potassium amylxanthate induced increase in the levels of ¹⁰⁹Cd²⁺ in several tissues, whereas the brain was the only tissue with increased concentration of ¹⁰⁹Cd²⁺ in the presence of sodium isopropyl-xanthate. A likely mechanism for the enhanced uptake of the ¹⁰⁹Cd²⁺ may be a facilitated penetration over the gill membranes of the lipophilic complexes formed between the studied compounds and the cadmium. A facilitated passage through cellular membranes may also be important for the increased uptake of the metal in other tissues. An elevated uptake of cadmium by the xanthates or the diethyldithiocarbamate may constitute an increased risk for noxious effects of the metal.     

Reproductive impairment and induction of alkaline-labile phosphate, a biomarker of estrogen exposure, in fathead minnows (Pimephales promelas) exposed to waterborne 17β-estradiol

The objectives of this research were: (1) to assess the effects of waterborne 17β-estradiol [E2; (17β)-estra-1,3,5(10)-triene-3,17-diol; CAS RN 50-28-2] on the reproduction of fathead minnows (Pimephales promelas) as a benchmark to which xeno-estrogens can be compared, and (2) to correlate the effects on reproductive function with plasma vitellogenin expression, measured as alkaline-labile phosphorous. Histopathological changes were also noted but are reported elsewhere. Duplicate groups of six fish (3 male and 3 female) were exposed to waterborne E2 at nominal concentrations of 10, 1, and 0.1 nM (2724, 272.4, and 27.24 ng l⁻¹) administered via a flow-through proportional diluter apparatus for 19 days. An ethanol carrier solvent was used at a final tank concentration of 1 ppm v/v in the treated tanks and in the solvent control tanks; the latter did not receive E2. Duplicate control tanks received neither ethanol nor E2. Dissolved E2 concentrations, measured throughout the exposure period using an ELISA, averaged 79% of nominal concentrations in the treated tanks. ELISA-detectable concentrations of E2 were found in all tanks (ranging from 3.5 to 15 ng E2 l⁻¹), including the control and solvent control tanks, which indicated that fish in the untreated tanks may have been the source of some E2. The EC50 (concentration expected to cause 50% effect), based on measured E2 concentrations, for inhibition of egg production was 120 ng E2 l⁻¹ (log₁₀ EC50=2.08±1.22, ±S.E.). The EC50 for induction of vitellogenin (measured as plasma alkaline-labile phosphate) in males was 251 ng E2 l⁻¹ (log₁₀ EC50=2.40±0.33, ±S.E.). No vitellogenin induction plateau was observed in females, therefore no EC50 could be calculated. Egg production, expressed as eggs laid per female, was significantly correlated with plasma vitellogenin in both males (linear r²=0.46, P<0.03) and females (linear r²=0.81, P<0.0004), though the relationship was stronger with female plasma vitellogenin expression than with males. The primary effect of E2 exposure on female fathead minnows appeared to be alteration of the timing of recrudescence including vitellogenin production. Spawning was inhibited in a way that indicated that exposure to waterborne E2 may have ‘reset' the cycle of recrudescence toward the beginning of the oogenic cycle. Vitellogenin induction in male fathead minnows was strongly correlated with E2 exposure, but less so with egg production. The results of this experiment link a biochemical indicator of waterborne estrogen exposure, vitellogenin, with a reproductive performance indicator, egg production, an important parameter affecting fish populations in the environment.     

Species-specific sensitivity to selenium-induced impairment of cortisol secretion in adrenocortical cells of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis)

Species differences in physiological and biochemical attributes exist even among closely related species and may underlie species-specific sensitivity to toxicants. Rainbow trout (RT) are more sensitive than brook trout (BT) to the teratogenic effects of selenium (Se), but it is not known whether all tissues exhibit this pattern of vulnerability. In this study, primary cultures of RT and BT adrenocortical cells were exposed to selenite (Na₂SO₃) and selenomethionine (Se-Met) to compare cell viability and ACTH-stimulated cortisol secretion in the two fish species. Cortisol, the primary stress hormone in fish, facilitates maintenance of homeostasis when fish are exposed to stressors, including toxicants. Cell viability was not affected by Se, but selenite impaired cortisol secretion, while Se-Met did not (RT and BT EC₅₀ > 2000 mg/L). RT cells were more sensitive (EC₅₀ = 8.7 mg/L) to selenite than BT cells (EC₅₀ = 90.4 mg/L). To identify the targets where Se disrupts cortisol synthesis, selenite-impaired RT and BT cells were stimulated with ACTH, dbcAMP, OH-cholesterol, and pregnenolone. Selenite acted at different steps in the cortisol biosynthesis pathway in RT and BT cells, confirming a species-specific toxicity mechanism. To test the hypothesis that oxidative stress mediates Se-induced toxicity, selenite-impaired RT cells were exposed to NAC, BSO and antioxidants (DETCA, ATA, Vit A, and Vit E). Inhibition of SOD by DETCA enhanced selenite-induced cortisol impairment, indicating that oxidative stress plays a role in Se toxicity; however, modifying GSH content of the cells did not have an effect. The results of this study, with two closely related salmonids, provided additional evidence for species-specific differences in sensitivity to Se which should be considered when setting thresholds and water quality guidelines.     

Developmental and subcellular effects of chronic exposure to sub-lethal concentrations of ammonia,PAH and PCP mixtures in brown trout (Salmo trutta f. fario L.) early life stages

Brown trout (Salmo trutta f. fario L.) early life stages were studied for physiological effects caused by chronic exposure to sub-acute levels of unionised ammonia, a mixture of PCP and PAHs, and a combination of ammonia and the mixture of organics during the entire embryonic development. Nominal concentrations of tested compounds were based on field data. Accumulation data for PAHs and PCP in trout tissue reflected respective water concentrations of PCP and PAHs. Physiological responses were studied by early life stage tests (ELST) and by the analysis of the 70 kDa stress protein (hsp70). Endpoint responses in the ELST were: accelerated development, pre-hatching, and increased heart rates. For these endpoints, response levels were highest in the ammonia treatment, followed by the exposure to the PCP/PAH mixture. Weight was reduced in embryos treated with the PCP/PAH mixture, but not in the group treated with this mixture combined with ammonia. Induction of hsp70 by the test agents was found to be stage-specific with increased response levels at advanced developmental stages. In both the ELST and hsp70 analysis, response levels were lower in the combined ammonia/PCP/PAH treatment than in groups treated with either ammonia or the PCP/PAH mixture alone.     

In vivo assessment of genotoxic potential of brown shammah (smokeless tobacco) in bone marrow cells of mice

This study was aimed to assess the genotoxicity of brown shammah (BS), a local form of smokeless tobacco, popular in Middle East countries including Yemen, Saudi Arabia and Sudan. The genotoxicity was explored using in vivo chromosomal aberration (CA), micronucleus (MN) and sperm abnormality (SA) assays. In addition, oxidative stress was also determined using various hepatic markers. Swiss albino mice were selected for the study, which were divided in to 5 groups of six animals each. They include, negative control (NC, received only vehicle) as well as positive control group (PC, received vehicle for 2 weeks followed by administration of cyclophosphamide, CP). Depending upon their dose, three BS treated animal groups were BS-100, 300 and 900 mg/kg. Doses of BS were obtained by suspending BS in 0.5% CMC (carboxy methyl cellulose) and orally administered once a day for 2 weeks. Significant augmentation of the average percentage of aberrant metaphase (AM), CA per cells and suppressed mitotic activity was observed on post administration of BS. In addition, BS increased the occurrence of MNPCEs (micronucleated polychromatic erythrocytes) formation, induced cytotoxicity and increased percentage of abnormal sperms as compared to NC. Moreover, BS also induced oxidative stress as the activities of hepatic superoxide dismutase (SOD) and glutathione (GSH) were reduced and malondialdehyde (MDA) content were increased by BS. Cyclophosphamide was utilized as clastogen, showed anticipated positive results and confirmed the sensitivity of test system. Therefore, it may be deduced from the study that the BS possesses genotoxic effects on mice bone marrow and germ cells in vivo.     

Effects of a pyrethroid pesticide on endocrine responses to female odours and reproductive behaviour in male parr of brown trout (Salmo trutta L.)

Reproductive behaviour of brown trout (Salmo trutta L.) from an anadromous stock was studied in a large stream water aquarium. Four adult males and two ovulated females were placed in the aquarium together with eight mature male parr. Four of the parr were exposed during the previous 4 days to two concentrations (0.1 or 1.0 microgl(-1)) of the pyrethroid pesticide cypermethrin (a disrupter of olfactory receptor function) and four of the parr to the solvent ethanol. The behaviour of all fish was followed for 24h and then blood and milt was collected. Exposure to the higher concentration of cypermethrin disturbed the reproductive behaviour of the parr. They displayed fewer courting events, spent less time near the nesting females and had lower volumes of strippable milt. They also had significantly lower amounts of 11-ketotestosterone (11-KT) in the blood plasma than the control group. The higher cypermethrin group also had significantly lower levels of all these variables than the lower cypermethrin group, apart from strippable milt that showed no significant differences between two groups. No significant differences in non-reproductive behaviours were observed between any of the groups. In the control fish, there were significant positive correlations between (a) the number of courting events and the amount of time spent near the female, (b) blood plasma levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and time spent near the female and (c) plasma levels of 17,20beta-P and the number of courting events. Further, in control fish, higher plasma levels of 17,20beta-P were observed in parr interacting with a female compared to those with no female contacts. A priming experiment confirmed a previous study that cypermethrin damages olfactory reception. Parr exposed to cypermethrin had significantly lower blood plasma levels of 17,20beta-P and 11-KT than control males after exposure to ovarian fluid and urine (known to contain reproductive priming pheromones). When ethanol-exposed males were exposed to ovarian fluid and urine they had significantly higher plasma levels of 17,20beta-P compared to those exposed to water only.     

Induction and characterization of hemoprotein(s) P-450 and monooxygenation in rainbow trout (Salmo gairdneri).

Hepatic microsomes obtained from control rainbow trout showed relatively low monooxygenase activities toward benzo[a]pyrene, ethoxycoumarin, ethoxyresorufin, and ethylmorphine as substrates. However, benzo[a]pyrene hydroxylation, ethoxycoumarin-O-deethylation, and ethoxyresorufin-O-deethylation were greatly induced by pretreatment of trout with β-naphthoflavone and Aroclor 1242. No increase in ethylmorphine-N-demethylation, liver/body ratio, or yield of microsomal protein were observed. Phenobarbital pretreatment of trout failed to affect any of the parameters studied. The λ_max of carboxyferrocytochrome P-450 was at 449 nm in control microsomes, and although a small (40%) increase in total hemoprotein(s) P-450 was seen after β-naphthoflavone pretreatment no hypsochromic shift of the λ_max was observed. No significant changes in the $\text{430}\text{455}$ ratio of the EtNC ligand spectra were noted after induction. Type I and Type II binding spectra were observed in all microsomal preparations examined. The use of specific inhibitors of monooxygenation (α-naphthoflavone and metyrapone) indicated that control and induced trout hepatic hemoprotein(s) P-450 were similar to rat cytochrome P₁-450. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence of a novel hemoprotein in hepatic microsomes after pretreatment of trout with β-naphthoflavone or Aroclor 1242. This induced protein had a molecular weight of approximately 57,000.     

Metal accumulation and metallothionein in two populations of brown trout, Salmo trutta, exposed to different natural water environments during a run-off episode

Cd, Cu and Zn concentrations were measured in ambient water as well as in gills, liver and kidney tissues of two natural populations of brown trout (Salmo trutta) during a run-off episode in two rivers with different metal compositions due to mining pollution. Metallothionein (MT) was also measured in these tissues. The two rivers, Rugla (Cu contaminated) and Naustebekken (Cd and Zn contaminated), are located in two neighboring drainage basins separated by the topographic divide near the city of Røros in the County of Sør-Trøndelag, Norway. In Rugla, the Cu concentration increased from 15 μg/l at the low water level to 41 μg/l during the run-off episode. In Naustebekken, corresponding values for Cd were 90–170 ng/l and those for Zn were 49–91 μg/l. Gill concentrations of Cu and Cd/Zn MT in both populations of native trout clearly reflect the presence of these metals in the rivers during the run-off, in accordance with the hypothesis of protection caused by MT induction. When Rugla trout were transferred to Naustebekken and vice versa, both the amounts of MT itself and the Cu contents reflected the concentration of this metal in the new environment, indicating that MT induction also protects against acutely increased metal levels. The measured levels of MT in both native and transferred trout can account for all the Cd present in the tissues, but not for all of the Cu and Zn. The capacity of MT to regulate Cd and Cu in the trout populations in their natural habitat therefore seems clearly present. Our data also indicate that the MT I and II isoforms may bind metals selectively.     

Assessment of DNA damage and molecular responses in Labeo rohita (Hamilton, 1822) following short-term exposure to silver nanoparticles

The increasing application of silver nanoparticles (Ag-NPs) both in industries and in agricultural fields has led to its accumulation in the aquatic ecosystem through water run-off. In the present study, the effects of Ag-NPs in the liver of Labeo rohita, were investigated at genomic and cellular level for seven days at the concentrations of 100, 200, 400 and 800 μg l⁻¹ by using 18 and 29 nm sizes of Ag-NPs. The Ag-NPs sizes of 18 and 29 nm were synthesized by a chemical method using atomic force microscopy with the zeta potential of −55 mV and−31.4 mV respectively. They were found to be spherical with smooth surfaces. Assessment of genotoxic effects of the particles in the fish using single-cell gel electrophoresis showed DNA damage on exposure to concentrations of 400 and 800 μg l⁻¹. Histopathological examination of the liver revealed vacuolar degeneration, hepatocytes have undergone total degeneration and high accumulation of Ag-NPs that depicted both time and dose-dependent relationships. Furthermore, the expression study of stress-related genes showed down-regulation, due to the production of free radicals and reactive oxygen species. Ag-NPs can cause both DNA damage and affect the cellular responses of L. rohita.     

The use of primary hepatocytes from brown trout (Salmo trutta lacustris) and the fish cell lines RTH-149 and ZF-L for in vitro screening of (anti)estrogenic activity of wood extractives.

Wood extractives are constituents of wood present in pulp and paper mill effluents, which may cause reproductive disturbances in fish. In the present study, we examined three cellular in vitro bioassays in order to assess (anti)estrogenic potencies of the wood extractives dehydroabietic acid (DHAA), isopimaric acid (IPA), betulinol (BET), hydroxymatairesinol (HMR), a phytosterol preparation (ULT), an oxidized phytosterol preparation (OX) and the model estrogen 17beta-estradiol (E2). The test systems used were primary hepatocyte cultures from brown trout and two piscine liver cell lines, RTH-149 and ZF-L. Estrogenicity was measured as vitellogenin (Vtg) secretion in cell culture medium. The primary hepatocytes cultures responded to E2 in a dose-dependent way. Vtg induction was inhibited with a simultaneous exposure to 4-hydroxytamoxifen (4-HT) indicating an estrogen receptor mediated response. DHAA and ULT induced a weak statistically non-significant Vtg production, and weak additive effects were found in some combination treatments of wood extractives and E2. Additionally, a pulp mill effluent tested on primary hepatocytes induced Vtg production when exposed at a 1% dilution. The cell lines secreted negligible amounts of Vtg upon E2 stimulation, which was neither dose-dependent nor inhibited by 4-HT. In conclusion, trout primary hepatocytes could be useful for assessing (anti)estrogenic potencies of compounds, and the wood extractives and a pulp mill effluent showed only weak or no estrogenic activity in this model system.