Surface Marker-Defined Head Kidney Granulocytes and B Lymphocytes of Rainbow Trout Express Benzo[a]pyrene-Inducible Cytochrome P4501A Protein

Abstract

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are immunotoxic to fish. Metabolism of PAHs in immune cells has been implicated in PAH immunotoxicity in mammals, but for fish the presence of metabolic enzymes in immune cells is less clear. The objective of this study was to examine localization and induction of the BaP-metabolizing biotransformation enzyme, cytochrome P4501A (CYP1A), in head kidney immune cells of rainbow trout (Oncorhynchus mykiss). In the first step, we measured induction of CYP1A-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity and CYP1A protein in head kidney of rainbow trout treated with a single intraperitoneal (ip) injection of 25 mg BaP/kg body weight. From days 3 to 10 postinjection, the BaP treatment led to a significant elevation of EROD and CYP1A protein in head kidney and liver, with CYP1A expression levels in the head kidney being much lower than in the liver. Next, we examined the cellular localization of CYP1A protein in the head kidney cell types: vascular endothelial, endocrine and lymphoid cells. CYP1A immunoreactivity was detectable only in BaP-treated trout, where it was localized in endothelial and lymphoid cells. Finally, we aimed to clarify which of the hematopoietic cell types possess CYP1A protein. Using double immunostaining for CYP1A and surface markers of rainbow trout immune cells, we identified B lymphocytes and granulocytes expressing inducible CYP1A protein and being the likely sites of BaP metabolism in the head kidney.

Expression of a highly differentiated phenotype and hepatic functionality markers in gilthead seabream (Sparus aurata L.) long-cultured hepatocytes: first morphological and functional in vitro characterization

“…However, the sustained CYP 1A production was further confirmed by the cellular localization both at 6 and 15d; a strong immunoreactivity was found in the cytoplasm of the hepatocytes, particularly along the perinuclear region. A similar pattern of CYP 1A immunostaining was previously found only in trout hepatocytes (Nakayama et al 2008).…”

“…As previously shown in lower vertebrates, including fish and lampreys, polyclonal antibody against mammalian proteins recognizes a broad range of cytoskeletal proteins, as well as mouse monoclonal antibodies recognize CKs (Markl and Franke 1988;Markl et al 1989;Bunton 1993Bunton , 1995Alarcón et al 1994Alarcón et al , 1997Zaccone et al 1995;Franke 2009). For cellular localization of CYP1A protein we chose a rabbit polyclonal antibody against fish CYP1A (CP 226), as previously shown by Nakayama et al (2008) to successfully locate CYP1A immunoreactivity in the cytoplasm of trout cells, thus suggesting a good sensitivity for the intracellular native protein.…”

Tissue-Specific Metabolism of Benzo[a]pyrene in Rainbow Trout (Oncorhynchus mykiss): A Comparison between the Liver and Immune Organs

“…For fish, the available database on tissue-specific expression of distinct cytochrome enzymes is limited. Best documented is the presence of CYP1A in the head kidney (e.g., Lorenzana et al, 1988;Pesonen et al, 1990), spleen (Taysse et al, 1998), or in distinct immune cell populations (Nakayama et al, 2008). Particularly the work in Buhler's group showed expression of a series of other CYP transcripts in the kidney of trout, mainly within the CYP1 and CYP3 families, both of which could be responsible for tissue differences in BaP metabolites (reviewed in Buhler and Wang-Buhler, 1998).…”

“…Microsomes from BaP-treated and control fish were analyzed. Enzyme kinetic analyses were performed in black 96-well plates with 250 ml of reaction volume per well using a protocol adapted from (Nakayama et al, 2008). As substrate, 7-ethoxyresorufin was used (16 mM).…”

Pathogen recognition and mechanisms in Atlantic cod (Gadus morhua) head kidney cells

“…However, the results of NFkb and AhR interactions seems to be cell type specific as Cyp1a is upregulated in other animal tissue like lung epithelial cells, rat astrocytes and human colon HT-29 cells [36] revealing a rather complicated picture. However, AhR is abundant in many cells of the mammalian immune system [39] and the immune system of fish is responsive to toxicants probably utilizing the same receptors [40]. The involvement of AhR and RXR in inflammation responses is far from being understood but the present observations may indicate an interesting connection between RXR, AhR and inflammation also in cod.…”