Natural factors to consider when using acetylcholinesterase activity as neurotoxicity biomarker in Young-Of-Year striped bass (Morone saxatilis).

Acetylcholinesterase (AChE) activity is one of the most common biomarkers of neurotoxicity used in aquatic organisms. However, compared to its extensive use as biomarker, the effects of natural factors on AChE activity remain unclear especially in estuarine fishes. The aim of this study was to evaluate the effects of natural factors on AChE activity of striped bass (Morone saxatilis) juveniles. Brain AChE activity was measured in YOY (Young-Of-Year) individuals collected monthly from August 2007 to January 2008 at 12 different sites in the San Francisco Estuary system. The spatio-temporal variability of AChE was analyzed relative to water temperature and salinity as well as fish size. AChE activity was highly positively correlated with water temperature and to a lesser extent negatively with fish size while no relationship was detected with salinity. Taking into account these natural factors when using AChE as a biomarker will help to determine and understand the effects of neurotoxic contaminants on fish in estuarine systems.

Effects of neurotoxic insecticides on heat-shock proteins and cytokine transcription in Chinook salmon (Oncorhynchus tshawytscha).

This study investigated sublethal, molecular effects of two current-use insecticides, chlorpyrifos (CP) and esfenvalerate (EV) in juvenile Chinook salmon. Heat-shock protein (hsp60, hsp70, hsp90) expression was quantified by Western blotting in muscle, liver and gill, and transcription of four cytokines (TGF-beta, IL-1beta, IGF-1, Mx-protein) was measured by real-time TaqMan PCR in anterior kidney and spleen. Expression of hsp was increased in muscle and liver at 1.2 and 7.2 microg/L CP, and at 0.01 and 0.1 microg/L EV, respectively. Transcription of IL-1beta and TGF-beta was elevated in kidney at 1.2 microg/L CP, while EV had no effect. No changes in cytokine transcription were observed in the spleen. Our results show that these insecticides cause cellular effects at environmental concentrations, and that hsps are sensitive indicators of sublethal exposure to CP and EV. In addition, CP may exert immunotoxic effects by altering the transcription of important mediators of the fish immune system.

Maternal transfer of xenobiotics and effects on larval striped bass in the San Francisco Estuary.

Aquatic ecosystems around the world face serious threats from anthropogenic contaminants. Results from 8 years of field and laboratory investigations indicate that sublethal contaminant exposure is occurring in the early life stages of striped bass in the San Francisco Estuary, a population in continual decline since its initial collapse during the 1970s. Biologically significant levels of polychlorinated biphenyls, polybrominated diphenyl ethers, and current-use/legacy pesticides were found in all egg samples from river-collected fish. Developmental changes previously unseen with standard methods were detected with a technique using the principles of unbiased stereology. Abnormal yolk utilization, brain and liver development, and overall growth were observed in larvae from river-collected fish. Histopathological analyses confirmed and identified developmental alterations. Using this methodology enabled us to present a conclusive line of evidence for the maternal transfer of xenobiotics and their adverse effects on larval striped bass in this estuary.

Expression of immune-regulatory genes in juvenile Chinook salmon following exposure to pesticides and infectious hematopoietic necrosis virus (IHNV).

Impairment of fish immune function as a consequence of polluted aquatic environments can result in changes in susceptibility to disease. In this study, we investigated the effects of two widely used insecticides, chlorpyrifos (CP) and esfenvalerate (EV), and a pathogen, infectious hematopoietic necrosis virus (IHNV), singly and in combination, on survival and cytokine (Mx protein, IL-1beta, TGF-beta and IGF-1) expression in juvenile Chinook salmon. Fish were exposed for 96 h to sublethal concentrations of CP (3.7 microg L(-1)) or EV (0.08 microg L(-1)), allowed to recover in clean water for seven days, then exposed to IHNV (6.4x10(5)TCID(50)mL(-1)) for 1.5h. Mortality was recorded daily, and spleen and anterior kidney samples were collected on day 4 (after CP or EV treatment), day 20 and day 60 (after CP or EV treatment and subsequent IHNV exposure) of the experiment. Significant mortality after 60 days was observed following exposure to EV (17%) or IHNV (20%), and prior insecticide exposure did not synergize the acute effects of pathogen treatment. By day 4, exposure to CP as well as EV led to a significant decrease of Mx protein and IL-1beta expression; by day 20, EV-exposed fish significantly overexpressed IL-1beta. Mx protein transcription was up-regulated in spleen and kidney of all IHNV-exposed fish groups by day 20. All but one treatment (EV) led to significantly decreased IGF-1 transcription in spleen on days 20 and 60, whereas a short-term increase was seen after CP exposure (day 4). In kidney, decreases of IGF-1 transcription were less pronounced. TGF-beta transcription was up-regulated in CP/IHNV and EV/IHNV exposure groups. Our results indicate that CP and EV alter the expression of cytokines, but this did not negatively affect the ability of fish to survive a subsequent exposure to IHNV. Induced TGF-beta transcription indicated that the combined stressors affected fish in a synergistic manner, but the consequences are unknown. Increased transcription of Mx protein was a reliable indicator of virus exposure.

Comparisons of tissue-specific transcription of stress response genes with whole animal endpoints of adverse effect in striped bass (Morone saxatilis) following treatment with copper and esfenvalerate.

Changes in the gene transcription of stress response genes in resident fish can be powerful biomarkers for the identification of sublethal impacts of environmental stressors on aquatic ecosystems. In this study, we tested the effects of two reference toxicants, copper (Cu) and the pyrethroid insecticide esfenvalerate [(S)-alpha-cyano-3-phenoxybenzyl-(S)-2-(4-chlorophenyl)-3-methylbutyrate], on lethal (mortality) and sublethal endpoints (growth, swimming behavior, transcription levels of stress response genes) in juvenile (81-90-day-old) striped bass (Morone saxatilis). We established cellular stress response markers for proteotoxicity (HSP70, HSP90), phase I detoxification mechanism (CYP1A1), metal-binding (metallothionein), as well as immune-function and pathogen-defense (TGF-beta, Mx-protein, nRAMP). Quantitative real-time TaqMan PCR was used to examine tissue-specific changes in the transcriptome of liver, spleen, white muscle, anterior kidney and gills after 7-day Cu exposures and 24-h esfenvalerate exposures. On the transcriptome level, exposure to Cu showed strongest effects on the transcription of metallothionein in spleen tissue, causing a 4-fold increase of mRNA at 42ppb total Cu and a 10-fold increase at 160 ppb Cu. Exposure to Cu also caused significant tissue-specific changes in gene transcription for immune-system related genes. Esfenvalerate exposure had tissue-specific effects on the transcription of HSP70, HSP90 and CYP1A1. The most significant effects were detected in liver tissue after exposure to 0.64 microg/L esfenvalerate. Our results show that the stress response at the transcriptome level is a more sensitive indicator for Cu and esfenvalerate exposures at low concentrations than swimming behavior, growth or mortality. The accuracy of studies on quantitative changes in the transcriptome can benefit from an initial evaluation or the inclusion of several different tissues and the use of multiple housekeeping genes.

Pesticide and pathogen: heat shock protein expression and acetylcholinesterase inhibition in juvenile Chinook salmon in response to multiple stressors.

Rapid expression of heat shock protein (hsp) families in response to a variety of stressors has been demonstrated in many organisms, including fish. The present 60-d challenge study was designed to compare hsp induction in juvenile Chinook salmon following exposure to individual pesticides, virus, and both stressors combined. Heat shock protein expression patterns over time were monitored and related to the extent of virus infection and mortality. Acetylcholinesterase (AChE) inhibition and recovery in response to applied stressors were measured in brain. High enzyme inhibition levels have been correlated with imminent mortality, and other sublethal physiological effects have been observed in fish concurrent with depressed AChE activity. Mortality was elevated considerably in fish exposed to 0.08 microg/L of the pyrethroid esfenvalerate (EV). Mortality due to infectious hematopoietic necrosis virus (IHNV) was lower in groups previously treated with pesticides; however, these fish died sooner than individuals exposed to virus only. Both pesticides, EV and the organophosphate (OP) chlorpyrifos (CP), as well as virus exposure, induced hsp expression, but highest hsp levels were observed after the combined treatments, suggesting an additive effect between virus and pesticides. Highest virus titers were accompanied by strongest hsp induction, indicating a connection between virus concentration and hsp expression. In conclusion, the measurement of hsp expression appears to be a very sensitive, integrative indicator of stress. Esfenvalerate and IHNV did not affect AChE activity, and exposure to 3.7 microg/L CP led to significantly inhibited AChE for at least 20 d. The time required for complete recovery of AChE activity raises concern about deleterious behavioral effects.

Synergistic effects of esfenvalerate and infectious hematopoietic necrosis virus on juvenile chinook salmon mortality.

Sublethal concentrations of pollutants may compromise fish, resulting in increased susceptibility to endemic pathogens. To test this hypothesis, juvenile chinook salmon (Oncorhynchus tshawytscha) were exposed to sublethal levels of esfenvalerate or chlorpyrifos either alone or concurrently with infectious hematopoietic necrosis virus (IHNV). Three trials were performed with fish exposed to concentrations of IHNV between 0.8 x 10(2) and 2.7 x 10(6) plaque-forming units/ml and to 5.0 microg/L of chlorpyrifos or 0.1 microg/L of esfenvalerate. The presence and concentration of IHNV in dead fish were assayed by virus isolation and plaque assay techniques, respectively. Among groups exposed to both esfenvalerate and IHNV, 83% experienced highly significant (p < 0.001) mortality, ranging from 20 to 90% at 3 d post-virus exposure, and cumulatively died from 2.4 to 7.7 d sooner than fish exposed to IHNV alone. This trend was not seen in any other treatment group. Virus assays of dead fish indicate a lethal synergism of esfenvalerate and IHNV. Chlorpyrifos had no observed effect on total mortality or IHNV susceptibility. The present results suggest that accepted levels of pollutants may be seemingly nonlethal to fish but, in fact, be acting synergistically with endemic pathogens to compromise survivorship of wild fish populations through immunologic or physiologic disruption.

Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos.

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 microg/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 microg/l), but not a low dose (1.2 microg/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were approximately 30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was undetectable. Together these data suggest that (1) carboxylesterase activity inhibition may be a more sensitive biomarker for OP exposure than AChE activity, (2) neither AChE nor carboxylesterase activity are biomarkers for pyrethroid exposure, (3) CYP1A protein is not a sensitive marker for these agrochemicals and (4) slow hydrolysis rates may be partly responsible for acute pyrethroid toxicity in fish.

Molecular and cellular biomarker responses to pesticide exposure in juvenile chinook salmon (Oncorhynchus tshawytscha).

We determined the effects of two pesticides, chlorpyrifos and esfenvalerate in juvenile Chinook salmon. Four to five month old salmon were exposed to a range of pesticide concentrations, and tissue samples of surviving fish were analyzed for stress protein expression, cytokine transcription, and acetylcholinesterase (AChE) activity. At the highest concentrations, both pesticides led to complete mortality, whereas medium and low concentrations resulted in high survival rates. Significant differences in stress protein expression, cytokine transcription and AChE activity were found between control and surviving chlorpyrifos-exposed fish. Elevated stress protein expression was the only detectable response to esfenvalerate.