Elsevier

Aquatic Toxicology

Volume 147, February 2014, Pages 121-128
Aquatic Toxicology

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

https://doi.org/10.1016/j.aquatox.2013.12.017Get rights and content

Highlights

  • Injection of benzo[a]pyrene results in sustained excretion for up to 42 days.

  • Differential effects between cell types are distinguishable using monoclonal antibodies.

  • Decreases in certain leukocytes occurred most strongly 21 days after exposure.

  • Pre-exposure followed by antigen challenge caused a significant reduction in antibody.

  • Different time profiles of effects suggest a complex mechanism of action.

Abstract

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 100 mg/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 100 mg/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.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) represent one of the most ubiquitous groups of toxicants in the environment; they are formed through incomplete combustion of organic materials, namely from petrochemical sources. It has been known for nearly a century that PAHs are carcinogenic (Waldron, 1983) and more recent evidence suggests some of these compounds can be immunomodulators in fishes (reviewed by Reynaud and Deschaux, 2006). Understanding the mechanism of these immune effects is vital in being able to predict and mitigate potential population level effects in the wild.

Exposure to PAHs has demonstrated a number of effects on the immune function of fishes in laboratory studies. Decreased lymphocyte proliferation was observed in Japanese medaka (Oryzias latipes) dosed intraperitoneally (i.p.) with benzo[a]pyrene (BaP) (Carlson et al., 2002) as well as in bluegill sunfish (Lepomis macrochirus) following dietary exposure to three different PAHs (Connelly and Means, 2010). Altered in vitro proliferation of leukocytes from common carp was observed after i.p. injection of 3-methylcholanthrene (3-MC); the nature of this effect depended on presence or absence of a mitogen (Reynaud and Deschaux, 2005). A number of studies with fishes have shown that exposure to PAHs affects phagocyte function demonstrated by decreased oxidative burst or lysozyme activity (reviewed by Reynaud and Deschaux, 2006). A study done with rainbow trout exposed in situ to oil sands contaminated water were observed to have decreased leucocyte counts in blood, and decreased circulating antibody in response a pathogen associated molecular pattern; PAHs were concluded to be a potential causative agent for this effect (McNeill et al., 2012). More detailed studies are needed to understand the mechanism of effect on specific immune cell types in fishes.

The study of immune toxicity at the cellular level in fishes has been limited by availability of antibodies specific for cell surface markers, a key tool in mammalian toxicant models. It is now feasible to characterize the majority of leucocyte populations in rainbow trout using monoclonal antibodies (mAbs) (MacDonald et al., 2012). Recently, a model involving rainbow trout (Oncorhynchus mykiss) and immune challenge with inactivated Aeromonas salmonicida (iA.s.) has been developed (Hoeger et al., 2004a, Hoeger et al., 2004b, Hoeger et al., 2005, Hogan et al., 2010, McNeill et al., 2012). We have designed a staggered toxicant exposure-immune challenge design, reducing biosafety and animal welfare concerns associated with use of live pathogens. Combined with the mAbs, this allows for examination of how pre-exposure to a toxicant affects the immune system at the cellular distribution level, in vivo, in multiple tissues. With an interest in PAHs, we have decided to use BaP as a model compound as it is accepted as being among the most biologically potent of the PAHs, and is used in much of the supporting literature on this topic.

There are few in vivo studies investigating the effects of BaP on the rainbow trout immune system. With this study, we aim to add information at a different biological level of organization than many studies in this field have provided. The focus of this study is to elucidate the time course of effects of i.p. injected BaP on the immune system of the model fish species rainbow trout (O. mykiss). Our hypothesis is that BaP will selectively reduce the number of certain leucocyte populations in a tissue-specific manner. Secondly, it is hypothesized that this toxicity will lead to a reduction in antibody production because adaptive immune response requires the coordination of a number of leucocyte types. Finally, we seek to further develop the rainbow trout immune toxicity model for use in environmental toxicology studies. In the present study, we use tissue specific absolute leucocyte counts obtained by flow cytometry, to determine what dose and duration of intraperitoneally injected BaP could cause a detectable change in B-cell, T-cell, myeloid cell, and thrombocyte populations from blood, spleen and head kidney. Subsequently, antibody production in response to an immune challenge, iA.s., was evaluated after a pre-exposure to the most effective dose and duration of BaP exposure.

Section snippets

Experimental design

Included in this study are three distinct experiments. In Experiment 1 rainbow trout were exposed to one of 3 doses of BaP (Sigma Aldrich, St. Louis, USA; 0, 25, 100 mg/kg) and lethal sampling was conducted at three time-points (7, 14, and 21 d); the goal of this study was to determine a dose and duration of BaP exposure that would cause a detectable change in the immune cell profile of blood, spleen, and head kidney. Experiment 2 exposed rainbow trout to one of 6 concentrations of iA.s.; the

Statistical analysis

The ELISA data was expressed as the negative logarithm of the antibody titre. The titre value was determined by the lowest dilution of serum at which antibody could be detected. The threshold for detection was defined as the next highest dilution that had an absorbance greater than twice the value of the most dilute in the dilution series (all antibody titres were diluted to background levels). Those data points that didn’t have detectable antibody within the range of dilutions tested were

Experiment 1 – BaP effect of dose and time

No mortalities were observed in any of the BaP trials. Statistical analysis revealed a significant increase in liver size at 7, 14, and 21 d in fish exposed to high dose (100 mg/kg). A significant decrease in spleen size at low (25 mg/kg) and high dose (100 mg/kg) was observed in the 21 d experiment (Table 2). Bile metabolites showed that a single injection of BaP in corn oil resulted in a sustained exposure to BaP for the 21 d of the experiment (Fig. 1). At the high dose (100 mg/kg), approximately

Discussion

Bile metabolite analysis indicated that a single i.p. injection of BaP resulted in sustained excretion of BaP and its metabolites for 21 d and substantial excretion was occurring even at 42 d. Rainbow trout leukocytes including myeloid cells, B cells, and T cells were reduced in absolute numbers in a time dependent manner in blood, spleen, and head kidney after this BaP exposure. Reductions were not seen in erythrocytes or thrombocytes, indicating that BaP has a selective effect on particular

Funding

This work was supported by Syncrude Canada, Suncor Energy, Shell Albian Sands, Total E&P Canada, and Canadian Natural Resources Limited under the auspices of Canadian Oil Sands Network for Research and Development, by NSERC CRD grants and a Canada Research Chair held by Michael R. van den Heuvel. Personal support for Laura J. Phalen was through an NSERC Alexander Graham Bell Canadian Graduate Scholarship, a PEI Innovation Scholarship, and a Dr. Regis Duffy Graduate Scholarship.

Supplementary data

A table including the absolute cell counts (complete and differentiated) with standard error for experiments 1 and 2 are included in a single table as supplementary data. This data is presented in part as percentages of appropriate controls in Fig. 2.

Acknowledgements

The authors wish to thank Gillian MacDonald, Karen Thorpe, Scott Roloson, Collin Arens, Gailene Tobin, Michael Coffin, Kyle Knysh, Travis James, and Brad Scott for their help during sampling. Additional thanks to Michael Coffin for reviewing the manuscript.

References (30)

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