Transcriptional response in rainbow trout (Oncorhynchus mykiss) B cells and thrombocytes following in vivo exposure to benzo[a]pyrene.

Immune toxicity of polycyclic aromatic hydrocarbons (PAHs) in fishes has been frequently reported but the reasons for differential cell toxicity remains unclear. Rainbow trout were exposed in vivo with a single intraperitoneal injection of corn oil or 100mg/kg of the immunotoxic PAH benzo[a]pyrene (B[a]P) in corn oil. Leukocytes were harvested from head kidney, spleen and blood after 14days, the optimal time for B cell depletion found in a previous study. The mRNA expression of five cytochrome P450 (CYP) enzymes, the aryl hydrocarbon receptor (AhR), and an intrinsic pathway apoptosis checkpoint (p53) in B cells and thrombocytes were examined. Transcript levels were measured in immunomagnetically-isolated B cells and thrombocytes from those tissues as well as in liver as B cells had been previously shown to be responsive the BaP whereas thrombocytes were not. There was induction of CYP1A1 in liver, blood B cells, and blood and spleen thrombocytes; CYP1B1 in blood B cells, blood and spleen thrombocytes; CYP1A3 in liver, blood and spleen B cells, and blood thrombocytes; CYP1C1 in liver; and AhR in liver and spleen thrombocytes. There was no change in CYP1C2, or p53 mRNA levels across tissues or cell type. Induction in mRNA was observed 14 d after exposure, indicating a prolonged physiological effect of a single B[a]P injection. CYP1A1 and CYP1A3 were the most abundantly expressed CYP genes and CYP1B1 was generally least abundant. B[a]P-induced thrombocytes had a significantly different pattern of CYP expression than either liver or B cells. Given the importance of metabolites in the toxicity of PAHs, differences in CYP expression between tissues may explain differences in toxicity previously observed between B cells and thrombocytes.

The effects of benzo[a]pyrene on leucocyte distribution and antibody response in rainbow trout (Oncorhynchus mykiss).

Polycyclic aromatic hydrocarbons (PAHs) are a group of compounds with immunotoxic and carcinogenic potential that may pose a threat to fish populations. This study aims to utilize a newly developed fish immunotoxicology model to determine the immune tissue/cell population level effects of PAHs on rainbow trout, using benzo[a]pyrene (BaP) as a representative immunotoxic PAH. Intraperitoneal injection of 25 or 100mg/kg BaP resulted in sustained exposure as indicated by biliary fluorescence at BaP wavelengths for up to 42 days. A new flow cytometry method for absolute counts of differential leucocyte distributions in spleen, blood, and head kidney was developed by combining absolute quantitative counts of total leukocytes in the tissue (3,3'-dihexyloxacarbocyanine iodide (DiOC6) dye) with relative differential counts using monoclonal antibodies for B cells, T cells, myeloid cells, and thrombocytes. Experiments indicated dose- and time-dependent decreases in the absolute number of B cells, myeloid cells, or T cells in blood, spleen, or head kidney after 7, 14 or 21 d of exposure. There was no change in the absolute numbers of erythrocytes or thrombocytes in any tissue. When rainbow trout were exposed to inactivated Aeromonas salmonicida after a 21 d exposure to 100mg/kg BaP, circulating antibody concentrations were decreased by 56%. It was concluded that BaP has a cell lineage-specific toxic effect on some immune cells of rainbow trout, and causes a decrease in circulating antibody levels.

Immunotoxic effects of oil sands-derived naphthenic acids to rainbow trout.

Naphthenic acids are the major organic constituents in waters impacted by oil sands. To investigate their immunotoxicity, rainbow trout (Oncorhynchus mykiss) were injected with naphthenic acids extracted from aged oil sands tailings water. In two experiments, rainbow trout were injected intraperitoneally with 0, 10, or 100 mg/kg of naphthenic acids, and sampled after 5 or 21 d. Half of the fish from the 21 d exposure were co-exposed to inactivated Aeromonas salmonicida (A.s.) to induce an immune response. A positive control experiment was conducted using an intraperitoneal injection of 100 mg/kg of benzo[a]pyrene, a known immune suppressing compound. T-lymphocytes, B-lymphocytes, thrombocytes, and myeloid cells were counted in blood and lymphatic tissue using flow cytometry. In the 5d exposure, there was a reduction in blood leucocytes and spleen thrombocytes at the 100 mg/kg dose. However, at 21 d, leucocyte populations showed no effects of exposure with the exception that spleen thrombocyte populations increase at the 100 mg/kg dose. In the 21 d exposure, B- and T-lymphocytes in blood showed a significant Dose × A.s. interaction, indicating stimulated blood cell proliferation due to naphthenic acids alone as well as due to A.s. Naphthenic acid injections did not result in elevated bile fluorescent metabolites or elevated hepatic EROD activity. In contrast to naphthenic acids exposures, as similar dose of benzo[a]pyrene caused a significant decrease in B- and T-lymphocyte absolute counts in blood and relative B-lymphocyte counts in spleen. Results suggest that the naphthenic acids may act via a generally toxic mechanism rather than by specific toxic effects on immune cells.

The immunological effects of oil sands surface waters and naphthenic acids on rainbow trout (Oncorhynchus mykiss).

There is concern surrounding the immunotoxic potential of naphthenic acids (NAs), a major organic constituent in waters influenced by oil sands contamination. To assess the immunological response to NAs, rainbow trout (Oncorhynchus mykiss) waterborne exposures were conducted with oil sands-influenced waters, NAs extracted and purified from oil sands tailings waters, and benzo[a]pyrene (BaP) as a positive control. After a 7d exposure, blood, spleen, head kidney, and gill samples were removed from a subset of fish in order to evaluate the distribution of thrombocytes, B-lymphocytes, myeloid cells, and T-lymphocytes using fluorescent antibodies specific for those cell types coupled with flow cytometry. The remaining trout in each experimental tank were injected with inactivated Aeromonas salmonicida and held in laboratory water for 21 d and subjected to similar lymphatic cell evaluation in addition to evaluation of antibody production. Fluorescent metabolites in bile as well as liver CYP1A induction were also determined after the 7 and 21 d exposure. Oil sands waters and extracted NAs exposures resulted in an increase in bile fluorescence at phenanthrene wavelengths, though liver CYP1A was not induced in those treatments as it was with the BaP positive control. Trout in the oil sands-influenced water exposure showed a decrease in B- and T-lymphocytes in blood as well as B-lymphocytes and myeloid cells in spleen and an increase in B-lymphocytes in head kidney. The extracted NAs exposure showed a decrease in thrombocytes in spleen at 8 mg/L and an increase in T-lymphocytes at 1mg/L in head kidney after 7d. There was a significant decrease in antibody production against A. salmonicida in both oil sands-influenced water exposures. Because oil sands-influenced waters affected multiple immune parameters, while extracted NAs impacts were limited, the NAs tested here are likely not the cause of immunotoxicity found in the oil sands-influenced water.