Proposed terminology for differentiating between biomarker (nonadverse) versus adverse (population relevant) end points with respect to reproductive EDCs

<p><b>Copyright information:</b></p><p>Taken from "Screening and Testing for Endocrine Disruption in Fish—Biomarkers As “Signposts,” Not “Traffic Lights,” in Risk Assessment"</p><p></p><p>Environmental Health Perspectives 2005;114(S-1):106-114.</p><p>Published online 21 Oct 2005</p><p>PMCID:PMC1874181.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI</p

20 Years of fish immunotoxicology – what we know and where we are

<p>Despite frequent field observations of impaired immune response and increased disease incidence in contaminant-exposed wildlife populations, immunotoxic effects are rarely considered in ecotoxicological risk assessment. The aim of this study was to review the literature on immunotoxic effects of chemicals in fish to quantitatively evaluate (i) which experimental approaches were used to assess immunotoxic effects, (ii) whether immune markers exist to screen for potential immunotoxic activities of chemicals, and (iii) how predictive those parameters are for adverse alterations of fish immunocompetence and disease resistance. A total of 241 publications on fish immunotoxicity were quantitatively analyzed. The main conclusions included: (i) To date, fish immunotoxicology focused mainly on innate immune responses and immunosuppressive effects. (ii) In numerous studies, the experimental conditions are poorly documented, as for instance age or sex of the fish or the rationale for the selected exposure conditions is often missing. (iii) Although a broad variety of parameters were used to assess immunotoxicity, the rationale for the choice of measured parameters was often not given, remaining unclear how they link to the suspected immunotoxic mode of action of the chemicals. (iv) At the current state of knowledge, it is impossible to identify a set of immune parameters that could reliably screen for immunotoxic potentials of chemicals. (v) Similarly, in fish immunotoxicology there is insufficient understanding of how and when chemical-induced modulations of molecular/cellular immune changes relate to adverse alterations of fish immunocompetence, although this would be crucial to include immunotoxicity in ecotoxicological risk assessment.</p

FISH assays of pure cultures.

<p>DAPI staining (A, B, C); Pan-Flavo probe (D, E, F); <i>F. psychrophilum</i> probes (G, H, I) (100x). <i>F. psychrophilum</i> (DSM3660) (A, D, G); <i>Flavobacterium</i> spp. (B, E, H); <i>Chryseobacterium</i> spp. (C, F, I).</p

Fluorescent <em>In Situ</em> Hybridization: A New Tool for the Direct Identification and Detection of <em>F. psychrophilum</em>

<div><p><em>F. psychrophilum</em> is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent <em>in situ</em> hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect <em>F. psychrophilum</em> in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of <em>Flavobacterium</em> species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of <em>F. psychrophilum</em> infections in fish. We constructed a generic probe for the genus <em>Flavobacterium</em> (“Pan-Flavo”) and two specific probes targeting <em>F. psychrophilum</em> based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different <em>Flavobacterium</em> and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the <em>F. psychrophilum</em> probe, respectively). FISH was able to detect <em>F. psychrophilum</em> in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of <em>Flavobacterium</em> spp. and <em>F. psychrophilum</em>.</p> </div

ROC curves for cell suspension of pure strains; area under the curve (AUC) for FISH: 0.89, for culture method: 0.79.

<p>(A). ROC curves for spiked spleens; AUC for FISH: 0.84, for culture method: 0.6 (B).</p

Probes used, target microorganisms and DNA target regions. [Cyanine dye (CY3); Carboxyfluorescein (FAM)].

*<p><i>E.coli</i>, GenBank sequence HM371196.</p

Agreement between FISH and 16S rDNA sequencing (SEQ, used as gold standard) in the experiments carried out with the Pan-Flavo (Flavo285) probe and the combination of two <i>F. psychrophilum</i> (FlavoP77, FlavoP477) probes.

<p>SE: sensitivity; SP: specificity; PPV: positive predictive value, NPV: negative predictive value.</p

FISH assay on infected fish tissues.

<p>Pan-Flavo probe (A, B); <i>F. psychrophilum</i> probes (C, D). <i>F. psychrophilum</i> on skin (A, C) and <i>F. psychrophilum</i> in a spleen (B, D).</p

An example of biomarker interpretation for fish screening and testing of potential reproductive EDCs, applicable to single substances or to complex effluents (according to local or regional requirements)

<p><b>Copyright information:</b></p><p>Taken from "Screening and Testing for Endocrine Disruption in Fish—Biomarkers As “Signposts,” Not “Traffic Lights,” in Risk Assessment"</p><p></p><p>Environmental Health Perspectives 2005;114(S-1):106-114.</p><p>Published online 21 Oct 2005</p><p>PMCID:PMC1874181.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI</p> Abbreviations: ED, endocrine disruptor; GSI, gonadosomatic index; SSC, secondary sexual characteristics

DataSheet1_In vitro biotransformation assays using fish liver cells: Comparing rainbow trout and carp hepatocytes.pdf

Biotransformation assays using primary hepatocytes from rainbow trout, Oncorhynchus mykiss, were validated as a reliable in vitro tool to predict in vivo bioconcentration factors (BCF) of chemicals in fish. Given the pronounced interspecies differences of chemical biotransformation, the present study aimed to compare biotransformation rate values and BCF predictions obtained with hepatocytes from the cold-water species, rainbow trout, to data obtained with hepatocytes of the warm-water species, common carp (Cyprinus carpio). In a first step, we adapted the protocol for the trout hepatocyte assay, including the cryopreservation method, to carp hepatocytes. The successful adaptation serves as proof of principle that the in vitro hepatocyte biotransformation assays can be technically transferred across fish species. In a second step, we compared the in vitro intrinsic clearance rates (CLin vitro, int) of two model xenobiotics, benzo[a]pyrene (BaP) and methoxychlor (MXC), in trout and carp hepatocytes. The in vitro data were used to predict in vivo biotransformation rate constants (kB) and BCFs, which were then compared to measured in vivo kB and BCF values. The CLin vitro, int values of BaP and MXC did not differ significantly between trout and carp hepatocytes, but the predicted BCF values were significantly higher in trout than in carp. In contrast, the measured in vivo BCF values did not differ significantly between the two species. A possible explanation of this discrepancy is that the existing in vitro-in vivo prediction models are parameterized only for trout but not for carp. Therefore, future research needs to develop species-specific extrapolation models.</p