ABSTRACT: Atrazine (ATZ) and nonylphenol (NP) are commonly identified contaminants in aquatic habitats; however, relatively few studies have considered the impact of these endocrine disrupters on immune function. 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), 59 µg/L ATZ, 555 µg/L ATZ, 2.3 µg/L NP or 18 µg/L NP (measured concentrations) for 4 d. At the end of exposure, fish were assessed for general health indicators (liver somatic index (LSI), spleen somatic index (SSI), hematocrit), biochemical endpoints (plasma cortisol concentrations, lysozyme activity, and protein content), a cellular endpoint (blood leukocyte differential counts), and an integrated immune response at the organism level (host resistance challenge with Listonella anguillarum). Additionally, liver gene expression was assessed using a salmonid microarray (cGRASP, 32K version 1) for fish exposed to the (solvent) control, high ATZ (555 µg/L) and high NP (18 µg/L) treatments. Fish exposed to the high ATZ concentration exhibited 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 showed physiological (e.g. 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 alteration in immune system related pathways in the liver transcriptome. Fish exposed to high ATZ or NP concentrations suffered higher mortality than the control group following disease challenge with L. anguillarum. Microarray analysis of the liver transcriptome revealed a total of 211 unique, annotated differentially-regulated genes (DRGs) following high ATZ exposure and 294 DRGs following high NP exposure, with signatures that included alterations to a number of immune-system related processes and pathways. 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 and highlights the potential risk posed by these chemicals to wild fish populations. Total of 24 microarrays. 1 experimental sample and 1 pooled reference sample per microarray. Total of 8 individual samples (replicates) per treatment group. Three (3) treatment groups: control, atrazine (ATZ, 555 ug/L) and nonylphenol (NP, 18 ug/L).
