A guideline to frame stressor effects in freshwater ecosystems.

Environmental policies fall short in protecting freshwater ecosystems, which are heavily threatened by human pressures and their associated stressors. One reason is that stressor effects depend on the context in which they occur and it is difficult to extrapolate patterns to predict the effect of stressors without these being contextualized in a general frame. This study aims at improving existing decision-making frameworks such as the DPSIR approach (Driver-Pressure-State-Impact-Response) or ERA (Environmental Risk Assessment) in the context of stressors. Here, we delve into stressor-impact relationships in freshwater ecosystems and develop a guideline which includes key characteristics such as stressor type, stressor duration, location, the natural levels of environmental variables to which each ecosystem is used to, among others. This guideline is intended to be useful in a wide range of ecosystem conditions and stressors. Incorporating these guidelines may favor the comparability of scientific results and may lead to a substantial advancement in the efficacy of diagnosis and predictive approaches of impacts.

Are brown trout Salmo trutta fario and rainbow trout Oncorhynchus mykiss two of a kind? A comparative study of salmonids to temperature-influenced Tetracapsuloides bryosalmonae infection.

Proliferative kidney disease (PKD) of salmonids caused by Tetracapsuloides bryosalmonae causes high mortalities of wild brown trout (Salmo trutta fario) and farmed rainbow trout (Oncorhynchus mykiss) at elevated water temperatures. Here the aim was to compare the temperature-dependent modulation of T. bryosalmonae in the two salmonid host species, which display different temperature optima. We used a novel experimental set-up in which we exposed brown trout and rainbow trout to an identical quantified low concentration of T. bryosalmonae for a short time period (1 hr). We followed the development of the parasite in the fish hosts for 70 days. PKD prevalence and parasite kinetics were assessed using qPCR. Exposures were performed at temperatures (12°C and 15°C) that reflect an environmental scenario that may occur in the natural habitat of salmonids. T. bryosalmonae infection was confirmed earliest in brown trout kept at 15°C (day 7 post-exposure) while, in all other groups, T. bryosalmonae was not confirmed until day 15 post-exposure. Moreover, significantly greater infection prevalence and a faster increase of parasite intensity were observed in brown trout kept at 15°C than in all other groups. These results indicate that PKD is differentially modulated by water temperature in related host species.

What goes around comes around: an investigation of resistance to proliferative kidney disease in rainbow trout Oncorhynchus mykiss (Walbaum) following experimental re-exposure.

Rainbow trout Oncorhynchus mykiss surviving proliferative kidney disease (PKD) are reported not to develop the disease upon re-exposure. However, the mechanisms involved in the immune response to re-exposure are unknown. We examined disease susceptibility and the immune response of naive 1+ rainbow trout when first exposed to Tetracapsuloides bryosalmonae in comparison with that of 1+ rainbow trout re-exposed to T. bryosalmonae. PKD pathogenesis, parasite burden and transcriptional signatures of the host immune response were assessed at 10, 25 and 40 d.p.e (days post-exposure). In addition, we evaluated the presence of IgM+ B cells in the blood and the posterior kidney. The exposure of 1+ rainbow trout to T. bryosalmonae for the first time resulted in 100% infection prevalence, high parasite burdens and severe clinical PKD, while re-exposed fish were either able to avoid reinfection completely or mount an earlier and more efficient adaptive-type immune response. This response was characterized by a greater amount of IgM+ B cells in the blood and elevated mRNA levels of secretory IgM in the posterior kidney which minimized pathogen burden and kidney inflammation. Our findings suggest that rainbow trout is able to develop immune protection against T. bryosalmonae.

Aeromonas salmonicida type III secretion system-effectors-mediated immune suppression in rainbow trout (Oncorhynchus mykiss).

Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen in aquaculture. Together with other pathogens, it is characterized by the presence of a type 3 secretion system (T3SS). The T3SS is the main virulence mechanism of A. salmonicida. It is used by the bacterium to secrete and translocate several toxins and effector proteins into the host cell. Some of these factors have a detrimental impact on the integrity of the cell cytoskeleton, likely contributing to impair phagocytosis. Furthermore, it has been suggested that effectors of the T3SS are able to modulate the host's immune response. Here we present the first partial characterization of the immune response in rainbow trout (Oncorhynchus mykiss) infected with distinct strains of A. salmonicida either carrying (i) a fully functional T3SS or (ii) a functionally impaired T3SS or (iii) devoid of T3SS ("cured" strain). Infection with an A. salmonicida strain either carrying a fully functional or a secretion-impaired T3SS was associated with a strong and persistent immune suppression. However, the infection appeared to be fatal only in the presence of a fully functional T3SS. In contrast, the absence of T3SS was neither associated with immune suppression nor fish death. These findings suggest that the T3SS and T3SS-delivered effector molecules and toxins of A. salmonicida do not only impair the host cells' cytoskeleton thus damaging cell physiology and phagocytosis, but also heavily affect the transcription of critical immune mediators including the shut-down of important warning signals to recognize infection and induce immune defense.

Investigations into the temporal development of epitheliocystis infections in brown trout: a histological study.

Epitheliocystis in Swiss brown trout (Salmo trutta) is a chlamydial infection, mainly caused by Candidatus Piscichlamydia salmonis and Candidatus Clavichlamydia salmonicola. To gain a better understanding of the temporal development of infections in wild brown trout, we investigated epitheliocystis infections during the course of the summer and autumn months of a single year (2015), and compared this to sampling points over the span of the years 2012-2014. The survey focused on tributaries (Venoge and Boiron) of the Rhone flowing in to Lake Geneva. When evaluated histologically, epitheliocystis infections were found throughout the period of investigation with the exception of the month of June. Fifty to 86 animals per sampling were investigated. Highest prevalence and infection intensities were seen in September. A correlation between epitheliocystis infection and water temperatures was not evident. Interyear comparison revealed consistent levels of prevalence and infection intensities in late summer. The absence of infections in June, combined with the consistent interyear results, indicates seasonal fluctuation of epitheliocystis infections in brown trout with a reservoir persisting during winter months from which infections can re-initiate each year. This could either be at levels below detection limits within the brown trout population itself or in an alternative host.

National competence center for wildlife diseases in Switzerland: Mandate, development and current strategies.

The need for wildlife health surveillance has become increasingly recognized. However, comprehensive programs which cover a wide spectrum of species, pathogens and geographic areas are still lacking in most European countries and practical examples of systems in place remain scarce. This article provides an overview of the organization of wildlife health surveillance in Switzerland, with a focus on the development, current strategies and the activities of the national program carried out by the Centre for Fish and Wildlife Health (FIWI), University of Bern. This documentation may stimulate on-going discussions on the design and development of national wildlife health surveillance programs in other countries. Investigations into wildlife health in Switzerland date back to the 1950s. The FIWI acts as a national competence center for wildlife diseases on mandate of the Swiss federal authorities. The mandate includes four main activities: disease diagnostics, research, consulting and teaching. In line with this, the FIWI has made continuous efforts to strengthen a national network of field partners and implemented strategies to facilitate long-term and metastudies.

Assessing the impact of multiple stressors on aquatic biota: the receptor's side matters.

Aquatic ecosystems are confronted with multiple stress factors. Current approaches to assess the risk of anthropogenic stressors to aquatic ecosystems are developed for single stressors and determine stressor effects primarily as a function of stressor properties. The cumulative impact of several stressors, however, may differ markedly from the impact of the single stressors and can result in nonlinear effects and ecological surprises. To meet the challenge of diagnosing and predicting multiple stressor impacts, assessment strategies should focus on properties of the biological receptors rather than on stressor properties. This change of paradigm is required because (i) multiple stressors affect multiple biological targets at multiple organizational levels, (ii) biological receptors differ in their sensitivities, vulnerabilities, and response dynamics to the individual stressors, and (iii) biological receptors function as networks, so that actions of stressors at disparate sites within the network can lead via indirect or cascading effects, to unexpected outcomes.

Thyroid signaling in immune organs and cells of the teleost fish rainbow trout (Oncorhynchus mykiss).

Thyroid hormones are involved in modulating the immune system in mammals. In contrast, there is no information on the role played by these hormones in the immune system of teleost fish. Here we provide initial evidence for the presence of active thyroid signaling in immune organs and cells of teleosts. We demonstrate that immune organs (head kidney and spleen) and isolated leukocytes (from head kidney and peripheral blood) of the rainbow trout (Oncorhynchus mykiss) express both thyroid receptor α (THRA) and ß (THRB). Absolute mRNA levels of THRA were significantly higher than those of THRB. THRA showed higher expression in immune organs and isolated immune cells compared to the reference organ, liver, while THRB showed the opposite. In vivo exposure of trout to triiodothryronine (T3) or the anti-thyroid agent propylthiouracil (PTU) altered THR expression in immune organs and cells. Effect of T3 and PTU over the relative expression of selected marker genes of immune cell subpopulations was also studied. Treatments changed the relative expression of markers of cytotoxic, helper and total T cells (cd4, cd8a, trb), B lymphocytes (mIgM) and macrophages (csf1r). These findings suggest that the immune system of rainbow trout is responsive to thyroid hormones.

Emergence of canine distemper virus strains with modified molecular signature and enhanced neuronal tropism leading to high mortality in wild carnivores.

An ongoing canine distemper epidemic was first detected in Switzerland in the spring of 2009. Compared to previous local canine distemper outbreaks, it was characterized by unusually high morbidity and mortality, rapid spread over the country, and susceptibility of several wild carnivore species. Here, the authors describe the associated pathologic changes and phylogenetic and biological features of a multiple highly virulent canine distemper virus (CDV) strain detected in and/or isolated from red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), stone (Martes foina) and pine (Martes martes) martens, from a Eurasian lynx (Lynx lynx), and a domestic dog. The main lesions included interstitial to bronchointerstitial pneumonia and meningopolioencephalitis, whereas demyelination--the classic presentation of CDV infection--was observed in few cases only. In the brain lesions, viral inclusions were mainly in the nuclei of the neurons. Some significant differences in brain and lung lesions were observed between foxes and mustelids. Swiss CDV isolates shared together with a Hungarian CDV strain detected in 2004. In vitro analysis of the hemagglutinin protein from one of the Swiss CDV strains revealed functional and structural differences from that of the reference strain A75/17, with the Swiss strain showing increased surface expression and binding efficiency to the signaling lymphocyte activation molecule (SLAM). These features might be part of a novel molecular signature, which might have contributed to an increase in virus pathogenicity, partially explaining the high morbidity and mortality, the rapid spread, and the large host spectrum observed in this outbreak.

Developmental toxicity and endocrine disrupting potency of 4-azapyrene, benzo[b]fluorene and retene in the zebrafish Danio rerio.

This study examined the developmental toxicity of the polycyclic aromatic hydrocarbons (PAHs) 11H-benzo(b)fluorene (BBF) and 4-azapyrene (AP) in comparison to the known teratogen retene. Developmental toxicity assays were performed in zebrafish embryos exposed for 120 h. BBF and retene induced a similar dioxin-like phenotype, whereas AP showed distinct effects, particularly craniofacial malformations. Microarray analysis revealed that for BBF and retene, drug metabolism pathways were induced, which were confirmed by subsequent studies of cyp1a gene expression. For AP, microarray analysis revealed the regulation of genes involved in retinoid metabolism and hematological functions. Studies with a panel of CALUX(®) bioassays to screen for endocrine disrupting activity of the compounds also revealed novel antagonistic effects of BBF and retene on androgen and progesterone receptors. Classification analysis revealed distinct gene expression profiles for both individual and combined PAH exposure. This study highlights the potential health risk of non priority PAHs.

Brain CYP1A in seabream, Sparus aurata exposed to benzo(a)pyrene.

This study compares basal and induced expression of cytochrome P4501A-CYP1A in the brain of gilthead seabream, Sparus aurata. Larval or adult seabream were exposed to benzo(a)pyrene -B(a)P- and the CYP1A response was assessed by analyzing CYP1A mRNA (RT-PCR), CYP1A protein (expression levels: ELISA, western blotting; cellular localization: immunohistochemistry), and CYP1A catalytic activity (7-ethoxyresorufin-O-deethylase-EROD). In the brain of adult S. aurata, CYP1A immunostaining was generally detected in the vasculature. It was present in the neuronal fibers and glial cells of the olfactory bulbs and the ventral telencephalon. ELISA and RT-PCR analyses confirmed CYP1A expression in the brains of non-exposed seabream. B(a)P exposure led to increased CYP1A staining mainly in neuronal fibers and glial cells of the olfactory bulbs, but also in the vascular endothelia. EROD activity, however, could not be detected in the brain of adult seabream, neither in control nor in exposed fish. In the developing brain of S. aurata larvae, immunohistochemical staining detected CYP1A protein exclusively in endothelia of the olfactory placode and in retina. Staining intensity of CYP1A slightly increases with larval development, especially in vascular brain endothelia. Exposing the larvae to 0.3 or 0.5 microg B(a)P/L from hatching until 15 days post hatching (dph) did not result in enhanced CYP1A immunostaining in the brain. In samples of whole seabream larvae, both from controls and BaP treatments, neither CYP1A mRNA, protein nor catalytic activity were detectable. The results demonstrate that CYP1A is expressed already and inducible in the larval brain, but that the regional and cellular expression differs partly between larval and adult brain. This may have implications for the toxicity of CYP1A-inducing xenobiotics on early and mature life stages of seabream.

Long-term estrogen exposure of whitefish Coregonus lavaretus induces intersex but not Lake Thun-typical gonad malformations.

A high prevalence of gonad morphological variations has been observed in whitefish Coregonus lavaretus from Lake Thun (Switzerland). To clarify the role of endocrine disruption as a possible cause of the gonad alterations, whitefish were reared in a long-term laboratory experiment under exposure to 17 beta-estradiol (E2). Fish were fed from first-feeding until 3 yr of age at a daily rate of 0 (control), 0.5 or 50 microg E2 kg(-1) fish. E2 exposure resulted in a time- and concentration-dependent increase of prevalence and intensity of intersex gonads, i.e. gonads that macroscopically appeared as either testis or ovary but microscopically contained both male and female germ cells. Four types of intersex could be distinguished: Types 1 and 2 were composed of mainly male tissue, with Type 1 containing single oocytes and Type 2 displaying an ovary-like lamellar structure of the tissue. In Type 3, an increased percentage of the tissue was occupied by female germ cells, while in Type 4, the majority of the gonad tissue consisted of female germ cells. Chronic E2 exposure additionally resulted in a concentration-dependent shift of the sex ratio towards females, a reduced condition factor, retarded gonad growth together with delayed maturation of germ cells, and elevated levels of hepatic vitellogenin mRNA. However, Lake Thun-typical alterations of gonad morphology were not induced by chronic E2 exposure. The results provide evidence that estrogen-active compounds unlikely play a role in the etiology of gonad malformations in Lake Thun whitefish.

The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes.

Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.

Proliferative kidney disease (PKD) of rainbow trout: temperature- and time-related changes of Tetracapsuloides bryosalmonae DNA in the kidney.

Proliferative kidney disease (PKD) of salmonids, caused by Tetracapsuloides bryosalmonae, can lead to high mortalities at elevated water temperature. We evaluated the hypothesis that this mortality is caused by increasing parasite intensity. T. bryosalmonae-infected rainbow trout (Oncorhynchus mykiss) were reared at different water temperatures and changes in parasite concentrations in the kidney were compared to cumulative mortalities. Results of parasite quantification by a newly developed real-time PCR agreed with the number of parasites detected by immunohistochemistry, except for very low or very high parasite loads because of heterogenous distribution of the parasites in the kidney. Two experiments were performed, where fish were exposed to temperatures of 12, 14, 16, 18 or 19 degrees C after an initial exposure to an infectious environment at 12-16 degrees C resulting in 100% prevalence of infected fish after 5 to 14 days of exposure. While mortalities differed significantly between all investigated water temperatures, significant differences in final parasite loads were only found between fish kept at 12 degrees C and all other groups. Differences in parasite load between fish kept at 14 degrees C to 19 degrees C were not significant. These findings provide evidence that there is no direct link between parasite intensity and fish mortality.

How normal is abnormal? Discrimination between deformations and natural variation in gonad morphology of European whitefish Coregonus lavaretus.

The gonad morphology of whitefish Coregonus lavaretus collected in Lake Thun, Switzerland, and two neighbouring lakes was assessed in order to differentiate between 'normal' and 'abnormal' character states of gonad morphology, which had been previously described in C. lavaretus from Lake Thun (constrictions, asymmetries, aplasia, compartmentations, fusions and hermaphroditism). In total, 4668 fish were collected and analysed using two complementary sampling schemes: (1) monthly samples of catches by the commercial fishermen and (2) samples of ripe spawners of all known 33 spawning sites of the three lakes. Considerable variation in gonad morphology in C. lavaretus populations of all lakes was found. Notably, all deviation types were observed in fish of all three lakes. Asymmetries and constrictions were frequent in all three lakes and showed systematic differences in frequency between the two sampling strategies. This indicates that asymmetries and constrictions represent to a large extent natural variation in gonad morphology of C. lavaretus and are also prone to considerable measurement error. In contrast, aplasia, fusions, compartmentations and hermaphroditism occurred predominantly in one C. lavaretus form of Lake Thun and in particular in populations spawning at great depths. This suggests that these deviation types are probably reliable indicators for gonad deformations and supports the interpretation that Lake Thun harbours a unique case of deformed gonads in C. lavaretus of yet unknown origin.

Tissue-specific induction of EROD activity and CYP1A protein in Sparus aurata exposed to B(a)P and TCDD.

The purpose of this study was to compare xenobiotic CYP1A induction in liver, gills, and excretory kidney of gilthead seabream, Sparus aurata. Fishes were exposed via water for 20 days to different concentrations of benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). CYP1A was measured at the enzyme activity level as 7-ethoxyresorufin-O-deethylase (EROD) activity, and at the protein level by means of ELISA. The liver displayed the highest absolute levels of EROD activity, both under non-exposed and exposed conditions. Organ- or inducer-related differences in the time course of CYP1A induction were moderate; however, the magnitude of the induction response varied between the organs and between B(a)P and TCDD. In the case of TCDD, liver, and kidney yielded a comparable induction response, whereas in the case of B(a)P, the kidney showed a substantially higher maximum induction factor than the liver. In the gills, the two xenobiotics resulted in similar maximum induction factors. In B(a)P-exposed seabream, EROD activities and CYP1A protein levels showed a good correlation in all three organs, whereas with TCDD as inducer the correlation was poor, what was mainly due to a decrease of EROD activities at the higher concentrations of TCDD, while CYP1A protein levels showed no concomitant decline. Overall, the study revealed both similarities and differences in the time-, concentration-, and inducer-dependent CYP1A responses of the three target organs, liver, kidney, and gills. Although, the findings of this study principally confirm the notion of the liver as the major metabolic organ in fish, they also provide evidence for substantial metabolic potential in gills and particularly in the kidney.

Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and/or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive anti-VHSV-AB titres, but was negative in virus isolation. With IHNV, RT-PCR detected two positive samples not identified by virus isolation while in these fish the SPNT result had been questionable. One of the IHNV-positive samples represents the first detection of IHNV-RNA in wild brown trout in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus isolation. However, in a titration experiment under laboratory conditions, the sensitivity of RT-PCR was not significantly higher when compared with virus isolation.

Concentration- and time-dependent effects of the synthetic estrogen, 17alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio.

Partial or full life-cycle tests are needed to assess the potential of endocrine-disrupting compounds (EDCs) to adversely affect development and reproduction of fish. Small fish species such as zebrafish, Danio rerio, are under consideration as model organisms for appropriate test protocols. The present study examines how reproductive effects resulting from exposure of zebrafish to the synthetic estrogen 17alpha-ethinylestradiol (EE2) vary with concentration (0.05 to 10 ng EE2 L(-1), nominal), and with timing/duration of exposure (partial life-cycle, full life-cycle, and two-generation exposure). Partial life-cycle exposure of the parental (F1) generation until completion of gonad differentiation (0-75 d postfertilization, dpf) impaired juvenile growth, time to sexual maturity, adult fecundity (egg production/female/day), and adult fertilization success at 1.1 ng EE2 L(-1) and higher. Lifelong exposure of the F1 generation until 177 dpf resulted in lowest observed effect concentrations (LOECs) for time to sexual maturity, fecundity, and fertilization success identical to those of the developmental test (0-75 dpf), but the slope of the concentration-response curve was steeper. Reproduction of zebrafish was completely inhibited at 9.3 ng EE2 L(-1), and this was essentially irreversible as a 3-mo depuration restored fertilization success to only a very low rate. Accordingly, elevated endogenous vitellogenin (VTG) synthesis and degenerative changes in gonad morphology persisted in depurated zebrafish. Full life-cycle exposure of the filial (F2) generation until 162 dpf impaired growth, delayed onset of spawning and reduced fecundity and fertilization success at 2.0 ng EE2 L(-1). In conclusion, results show that the impact of estrogenic agents on zebrafish sexual development and reproductive functions as well as the reversibility of effects, varies with exposure concentration (reversibility at < or = 1.1 ng EE2 L(-1) and irreversibility at 9.3 ng EE2 L(-1)), and between partial and full life-cycle exposure (exposure to 10 ng EE2 L(-1) during critical period exerted no permanent effect on sexual differentiation, but life-cycle exposure did).

Induction of EROD activity by 1-phenylimidazole and beta-naphthoflavone in rainbow trout cultured hepatocytes: a comparative study.

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, beta-naphthoflavone (betaNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of betaNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist alpha-naphthoflavone (alphaNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. alphaNF and herbimicin provoked a decrease of EROD induction both by betaNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of betaNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.

Histopathological alterations, EROD activity, CYP1A protein and biliary metabolites in gilthead seabream Sparus aurata exposed to benzo(a)pyrene.

This study compared for seabream, Sparus aurata exposed to benzo(a)pyrene-B(a)P-, the response of molecular cytochrome P450 1A (CYP1A) and cellular histopathology biomarkers. Male gilthead seabream, Sparus aurata specimens were exposed for 20 days via water to a series of high B(a)P concentrations. CYP1A was assessed by measuring enzymatic activity (EROD) and CYP1A protein content, and cellular responses were evaluated by routine histopathological methods. In addition, biliary metabolites were measured in order to verify that B(a)P was absorbed and metabolised. Histological lesions, both in liver and gills, increased in parallel to B(a)P concentrations, with the majority of changes representing rather non-specific alterations. Hepatic EROD and CYP1A proteins data showed a concentration-dependent induction, while in the gills, EROD activity but not CYP1A proteins showed a non-monotonous dose response, with a maximum induction level at 200 microg B(a)P.L-1 and decreasing levels thereafter. The findings provide evidence that short-term, high dose exposure of fish can result in significant uptake and metabolism of the lipophilic B(a)P, and in pronounced pathological damage of absorptive epithelia and internal organs.