Aryl Hydrocarbon Receptor Signaling Is Functional in Immune Cells of Rainbow Trout (Oncorhynchus mykiss).

The arylhydrocarbon receptor (AhR) is an important signaling pathway in the immune system of mammals. In addition to its physiological functions, the receptor mediates the immunotoxic actions of a diverse range of environmental contaminants that bind to and activate the AhR, including planar halogenated aromatic hydrocarbons (PHAHs or dioxin-like compounds) and polynuclear aromatic hydrocarbons (PAHs). AhR-binding xenobiotics are immunotoxic not only to mammals but to teleost fish as well. To date, however, it is unknown if the AhR pathway is active in the immune system of fish and thus may act as molecular initiating event in the immunotoxicity of AhR-binding xenobiotics to fish. The present study aims to examine the presence of functional AhR signaling in immune cells of rainbow trout (Oncorhynchus mykiss). Focus is given to the toxicologically relevant AhR2 clade. By means of RT-qPCR and in situ hybdridization, we show that immune cells of rainbow trout express ahr 2α and ahr 2ß mRNA; this applies for immune cells isolated from the head kidney and from the peripheral blood. Furthermore, we show that in vivo as well as in vitro exposure to the AhR ligand, benzo(a)pyrene (BaP), causes upregulation of the AhR-regulated gene, cytochrome p4501a, in rainbow trout immune cells, and that this induction is inhibited by co-treatment with an AhR antagonist. Taken together, these findings provide evidence that functional AhR signaling exists in the immune cells of the teleost species, rainbow trout.

Evaluation of endocytosis of silica particles used in biodegradable implants in the brain.

Silica nanoparticles embedded in a biodegradable scaffold have been proposed to offer several advantages when used in laser-tissue-soldering of blood vessels in the brain. During degradation, these nanoparticles are likely to be released into the surrounding brain tissue. The aim of this study was to investigate possible cellular uptake mechanism(s) of the two silica nanoparticle types in microglial cells as well as their effect on autophagy and inflammatory cytokines. The nanoparticle uptake was analysed quantitatively using high-content analysis. Nanoparticle incubation did not modulate cytokine secretion and autophagy at any time point investigated. The nanoparticles were taken up by the microglia cells in a time- and particle-dependent manner. The maximal uptake was reached after 4hours and the nanoparticles were found in the endoplasmic reticulum and lysosomes. Macropinocytosis and phagocytosis were predominantly responsible for the uptake, whereas clathrin- and caveolin-independent endocytosis were involved to a minor extent.

The teleostean liver as an immunological organ: Intrahepatic immune cells (IHICs) in healthy and benzo[a]pyrene challenged rainbow trout (Oncorhynchus mykiss).

The existence of a resident population of intrahepatic immune cells (IHICs) is well documented for mammalian vertebrates, however, it is uncertain whether IHICs are present in the liver of teleostean fish. In the present study we investigated whether trout liver contains an IHIC population, and if so, what the relative cellular composition of this population is. The results provide clear evidence for the existence of an IHIC population in trout liver, which constitutes 15-29% of the non-hepatocytes in the liver, and with a cellular composition different to that of the blood leukocyte population. We also analyzed the response of IHICs to a non-infectious liver challenge with the hepatotoxic and immunotoxic chemical, benzo[a]pyrene (BaP). Juvenile trout were treated with BaP (25 or 100mg/kgbw) at levels sufficient to induce the molecular pathway of BaP metabolism while not causing pathological and inflammatory liver changes. The IHIC population responded to the BaP treatments in a way that differed from the responses of the leukocyte populations in trout blood and spleen, suggesting that IHICs are an independently regulated immune cell population.

Tissue-specific metabolism of benzo[a]pyrene in rainbow trout (Oncorhynchus mykiss): a comparison between the liver and immune organs.

Polycyclic aromatic hydrocarbons (PAHs) are immunotoxicants in fish. In mammals, phase I metabolites are believed to be critically involved in the immunotoxicity of PAHs. This mechanism has been suggested for fish as well. The present study investigates the capacity of immune organs (head kidney, spleen) of rainbow trout, Oncorhynchus mykiss, to metabolize the prototypic PAH, benzo[a]pyrene (BaP). To this end, we analyzed 1) the induction of enzymatic capacity measured as 7-ethoxyresorufin-O-deethylase (EROD) activity in immune organs compared with liver, 2) the organ profiles of BaP metabolites generated in vivo, and 3) rates of microsomal BaP metabolite production in vitro. All measurements were done for control fish and for fish treated with an intraperitoneal injection of 15 mg BaP/kg body weight. In exposed trout, the liver, head kidney, and spleen contained similar levels of BaP, whereas EROD induction differed significantly between the organs, with liver showing the highest induction factor (132.8×), followed by head kidney (38.4×) and spleen (1.4×). Likewise, rates of microsomal metabolite formation experienced the highest induction in the liver of BaP-exposed trout, followed by the head kidney and spleen. Microsomes from control fish displayed tissue-specific differences in metabolite production. In contrast, in BaP-exposed trout, microsomes of all organs produced the potentially immunotoxic BaP-7,8-dihydrodiol as the main metabolite. The findings from this study show that PAHs, like BaP, are distributed into immune organs of fish and provide the first evidence that immune organs possess inducible PAH metabolism leading to in situ production of potentially immunotoxic PAH metabolites.

Immunotoxic effects of environmental toxicants in fish - how to assess them?

Numerous environmental chemicals, both long-known toxicants such as persistent organic pollutants as well as emerging contaminants such as pharmaceuticals, are known to modulate immune parameters of wildlife species, what can have adverse consequences for the fitness of individuals including their capability to resist pathogen infections. Despite frequent field observations of impaired immunocompetence and increased disease incidence in contaminant-exposed wildlife populations, the potential relevance of immunotoxic effects for the ecological impact of chemicals is rarely considered in ecotoxicological risk assessment. A limiting factor in the assessment of immunotoxic effects might be the complexity of the immune system what makes it difficult (1) to select appropriate exposure and effect parameters out of the many immune parameters which could be measured, and (2) to evaluate the significance of the selected parameters for the overall fitness and immunocompetence of the organism. Here, we present - on the example of teleost fishes - a brief discussion of how to assess chemical impact on the immune system using parameters at different levels of complexity and integration: immune mediators, humoral immune effectors, cellular immune defenses, macroscopical and microscopical responses of lymphoid tissues and organs, and host resistance to pathogens. Importantly, adverse effects of chemicals on immunocompetence may be detectable only after immune system activation, e.g., after pathogen challenge, but not in the resting immune system of non-infected fish. Current limitations to further development and implementation of immunotoxicity assays and parameters in ecotoxicological risk assessment are not primarily due to technological constraints, but are related from insufficient knowledge of (1) possible modes of action in the immune system, (2) the importance of intra- and inter-species immune system variability for the response against chemical stressors, and (3) deficits in conceptual and mechanistic assessment of combination effects of chemicals and pathogens.