Alteration of immune function endpoints and differential expression of estrogen receptor isoforms in leukocytes from 17ß-estradiol exposed rainbow trout (Oncorhynchus mykiss).

While the endocrine system is known to modulate immune function in vertebrates, the role of 17ß-estradiol (E2) in cellular immune function of teleosts is poorly understood. The cellular and molecular responses of juvenile rainbow trout (Oncorhynchus mykiss) to E2 treatment were evaluated by exposing fish to 0.47±0.02µg/L E2 (mean±SEM) for either 2 or 7d, with a subsequent 14d recovery period. After 2 and 7d of exposure to E2, hematocrit was significantly lower than in control fish. Lipopolysaccharide-induced lymphocyte proliferation was elevated on day 2 and concanavalin A-induced lymphocyte proliferation was reduced following 7d of E2 exposure. Four estrogen receptor (ER) transcripts were identified in purified trout head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL). While the mRNA abundance of ERß1 and ERß2 was unaffected by treatment, ERα1 was up-regulated in HKL and PBL following 7d of E2 exposure. ERα2 was up-regulated in HKL after 7d of E2 exposure, but down-regulated in PBL after 2 and 7d of treatment. All parameters that were altered during the E2 exposure period returned to baseline levels following the recovery period. This study reports the presence of the full repertoire of ERs in purified HKL for the first time, and demonstrates that ERα transcript abundance in leukocytes can be regulated by waterborne E2 exposure. It also demonstrated that physiologically-relevant concentrations of E2 can modulate several immune functions in salmonids, which may have widespread implications for xenoestrogen-associated immunotoxicity in feral fish populations inhabiting contaminated aquatic environments.

Immunotoxic and cytotoxic effects of atrazine, permethrin and piperonyl butoxide to rainbow trout following in vitro exposure.

For many current use pesticides, limited information exists on their cytotoxicity and immunotoxicity in non-target organisms such as fish. We examined the effects of atrazine, permethrin and piperonyl butoxide (PBO) exposure, in vitro, on rainbow trout (Oncorhynchus mykiss) lymphocyte viability and proliferation. Purified rainbow trout peripheral blood leukocytes (PBLs) were exposed in vitro to the test chemicals (0, 0.01, 0.1, 1 and 10 µM) for 96 h, with and without the mitogen lipopolysaccharide. All three chemicals caused a decrease in both lymphocyte viability and proliferation at 10 µM, while atrazine also suppressed proliferation of PBLs at 1 µM. The in vitro toxicity of these chemicals to this salmonid underscores the need for further investigation using in vivo studies and host resistance models.

Reduction in swimming performance in juvenile rainbow trout (Oncorhynchus mykiss) following sublethal exposure to pyrethroid insecticides.

While the lethal toxicity of pyrethroid insecticides to fish is well documented, their sublethal physio-behavioral effects remain poorly characterized. Known pyrethroid-associated changes to insect neuromuscular function may translate into similar effects in fish, thereby altering swimming ability and affecting foraging, predator avoidance, and migration. Three experiments were conducted using critical (Ucrit) and burst (Umax) swimming speeds to assess the sublethal effects of the pyrethroids permethrin and deltamethrin in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to deltamethrin (100, 200, or 300 ng/L) or permethrin (1, 2, or 3 µg/L) in water for 4 d, and assessed for swimming performance. Deltamethrin (200 and 300 ng/L) reduced Ucrit, but not Umax, while both swim performance measurements were unaffected by permethrin. Subsequent experiments used only Ucrit to assess deltamethrin exposure. In a time course experiment, deltamethrin (300 ng/L) reduced Ucrit after 1 and 4 d of exposure, but after 7 d of exposure Ucrit was fully recovered. Finally, deltamethrin (1, 2, or 3 µg/L) reduced Ucrit after 1h bath exposures similar to recommended protocols for deltamethrin based sea-lice treatment in aquaculture. The real-world implications of the revealed pyrethroid-associated swimming ability reductions in salmon may be important in areas close to aquaculture facilities.

The effects of an in vitro exposure to 17ß-estradiol and nonylphenol on rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes.

While xenoestrogens are routinely detected in the aquatic environment, there is little understanding of the immunotoxicological consequences of exposure to these chemicals in fish, or of the mechanisms through which these effects are mediated. This study was conducted to determine if estrogen receptors (ERs) are present in fish leukocytes and to characterize the effects of 17ß-estradiol (E2) and the xenoestrogen nonylphenol (NP) on immune system endpoints in rainbow trout (Oncorhynchus mykiss). Q-PCR was used to confirm that freshly isolated peripheral blood leukocytes (PBLs) express ERs. Following 96-h incubations with E2 or NP (1 nM to 10 µM), PBL ER transcription was again examined using Q-PCR and lipopolysaccharide (LPS)-stimulated proliferation was assessed using flow cytometry. While the transcription of all four forms of rainbow trout ER was unaffected by treatment with E2 or NP, transcription of ERα1 and ERα2 was down-regulated following LPS stimulation. Both E2 and NP, at concentrations of ≥100 nM and 10nM respectively, suppressed leukocyte proliferation. This first report of ERs in rainbow trout PBLs suggests a mechanism through with E2 and other xenoestrogens can modulate immune function. These results highlight the potential for xenoestrogens to impact host resistance to pathogens in wild fish populations.

Toxicity of atrazine and nonylphenol in juvenile rainbow trout (Oncorhynchus mykiss): effects on general health, disease susceptibility and gene expression.

Atrazine (ATZ) and nonylphenol (NP) are commonly identified contaminants in aquatic habitats; however, few studies have considered the impact of these endocrine disrupters on immune function and resistance to disease. This study examined the immunotoxicological effects of ATZ and NP at multiple levels of biological organization. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to a solvent control (0.00625%, v/v anhydrous ethanol), or sub-lethal concentrations of ATZ (59 µg/L and 555 µg/L) or NP (2.3 µg/L or 18 µg/L) for 4d. At the end of exposure, fish were assessed for a number of physiological endpoints, including a host resistance challenge, and liver gene expression was assessed using a salmonid microarray (cGRASP, 32K version 1). While the low ATZ and low NP treatments had no measurable effects on the physiological endpoints measured, fish exposed to the high ATZ concentration (555 µg/L) exhibited significantly elevated plasma cortisol, a decrease in SSI, and decreased lymphocytes and increased monocytes in peripheral blood, with suppression of early immune system processes apparent at the molecular level. In contrast, fish exposed to the high NP concentration (18 µg/L) showed physiological (e.g. significantly elevated LSI) and gene expression changes (e.g. induction of vitellogenin) consistent with estrogenic effects, as well as decreased lymphocytes in the peripheral blood and more limited alterations in immune system related pathways in the liver transcriptome. Fish exposed to high ATZ or NP concentrations incurred higher mortality than control fish following a disease challenge with Listonella anguillarum, while fish exposed to the lower concentrations were unaffected. Microarray analysis of the liver transcriptome revealed a total of 211 unique, annotated differentially regulated genes (DRGs) following high ATZ exposure and 299 DRGs following high NP exposure. Functional (enrichment) analysis revealed effects on immune system function, metabolism, oxygen homeostasis, cell cycle, DNA damage, and other processes affected by ATZ or NP exposure. Overall, this study provides evidence at multiple levels of biological organization that both ATZ and NP are immunotoxic at sub-lethal concentrations and highlights the potential risk posed by these chemicals to wild fish populations.

Immunotoxicological effects of a sub-chronic exposure to selected current-use pesticides in rainbow trout (Oncorhynchus mykiss).

Many current-use pesticides (CUPs) are found at increasing concentrations in aquatic environments, yet relatively little is known about their effects on the immune system of fish. We examined the in vivo effects of three pesticides (chlorothalonil, cypermethrin and pentachlorophenol) on the immune system of juvenile rainbow trout (Oncorhynchus mykiss) by assessing a suite of innate immune function tests, as well as a host resistance test using Listonella anguillarum. Increased activity of phagocytic leukocytes, as evidenced using flow cytometry, was observed following 28-day exposures to pentachlorophenol (1 microg/L and 2 microg/L), but not for cypermethrin or chlorothalonil, although a trend of increasing activity was noted for chlorothalonil. No recovery was observed during a 14-day post-exposure chlorothalonil experiment, as evidenced by continued elevation of respiratory burst and percent phagocytic cells at the lowest exposure concentrations (100 ng/L and 200 ng/L), suggesting a prolonged CUP-induced impact on the immune system. No effects of any pesticide on body weights, direct lethality, serum lysozyme or relative leukocyte differential were observed, suggesting that modulation of the cellular responses of the innate immune system represents a sensitive sub-lethal endpoint for these three pesticides. However, a lack of detectable effects of these CUPs on host resistance to L. anguillarum in our study may reflect a dose-response range that did not elicit an effect on those immune responses responsible for control and clearance of this particular pathogen. Additional research may provide more insight into the immunomodulatory effects of these and other CUPs, and the implications for host resistance against a variety of bacterial, viral and macroparasitic pathogens.