Page 375 - The Toxicology of Fishes

Liver Toxicity 355

Pregnane X Receptor (PXR) in Fish
While the NR1I family has been extensively studied in mammalian species, much fewer functional data
for these members of the NR family are available for teleosts and for cartilaginous fishes. PXR has been
identified in pufferfish, zebrafish, and medaka, and mining their genomes suggests the presence of a
single PXR/CAR-like gene in each species. These teleost receptors retain similarity to mammalian PXR,
having a 50- to 60-amino-acid insert between helices 1 and 3 (Moore et al., 2002), and may be a
functional precursor of mammalian PXR. Interestingly, transactivation studies with a chimeric
Gal4–zfLBD system from zebrafish PXR demonstrated limited diversity of functional ligands. While
responsive to prototypic PXR ligands (nifedipine, phenobarbital, clotrimazole, and 5β-pregnane-3,20-
dione), transactivation was low in comparison to mammalian PXR. These differences, however, may
reflect the high degree of sequence variation in the ligand-binding domain. Comparative cross-species
analysis of PXR suggests a high degree of variation at this site (Reschly and Krasowski, 2006). Analysis
of endogenous biliary ligands demonstrated that zebrafish PXR activation was restricted to the C27 bile
alcohol cyprinol sulfate, the major zebrafish bile salt. By comparison, C24 bile acids (found in birds
and mammals) were ineffective (Krasowski et al., 2005a). Fish bile acids are highly variable, with some
species containing C27 bile alcohols and acids and others C24 bile alcohols and acids. Some, such as
the medaka, contain both C27 and C24 bile acid components (Lee Hagey, pers. commun.). The role of
PXR as an endogenous sensor for bile acids may be highly species specific and subject to selective
adaptations depending on the metabolic requirements.
From a toxicological perspective, it is important to know whether PXR is associated with induction
of teleost P450 enzymes, specifically CYP3A. In one study, typical mammalian PXR receptor agonists,
including dexamethasone (DEX), the macrocyclic antibiotic rifampicin (RIF), and the synthetic steroid
pregnenolone-16α-carbonitrile (PCN), were seemingly ineffective at altering teleost CYP gene transcrip-
tion (Celander et al., 1989, 1996). In tilapia (Oreochromis niloticus), however, PCN treatment resulted
in a twofold induction of CYP3A proteins (Pathiratne and George, 1996). Similarly, exposure of zebrafish
to PCN resulted in a slight increase in PXR, CYPA, and MDR1 gene transcription. No change was
observed with other prototypic PXR agonists, including the antimycotic clotrimazole (CTZ) or the
antianginal drug nifedipine (NIF) (Bresolin et al., 2005). 4-Nonylphenol, the major byproduct of alkyl-
phenol ethoxylates, induces CYP3A in several fish (Hasselberg et al., 2005; Kullman et al., 2004). In a
recent study, nonylphenol-induced expression of CYP3A correlated with an increased expression of
salmon PXR, suggesting possible coregulation (Meucci and Arukwe, 2006). A similar finding was
observed with DDE, illustrating the broad substrate affinity of Atlantic salmon PXR to environmental
contaminants (Mortensen and Arukwe, 2006). In a recent review by Goksøyr (2006) on endocrine
disruptors in the marine environment, the nature of the effect of such compounds was considered to be
due to dose-dependent routing and cross-talk between different classes of nuclear receptors. Zebrafish
CYP3A65 transcription is enhanced by administration of DEX and RIF (Tseng et al., 2005). Interestingly,
CYP3A65 is additionally responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), indicating possible
cross-talk between PXR and AhR. To date, however, there exists only indirect evidence that teleost PXR
is associated with CYP expression based on similarity in response to mammalian PXR ligands. Although
gene sequences for PXR have been identified, few functional data exist regarding the role of PXR in
the transcriptional activation of hepatic metabolic genes and their physiological significance. In vitro
transactivation studies and identification of cognate hormone response elements upstream of teleost
CYP3A genes may provide some evidence that nuclear receptors are involved in transcriptional regula-
tion; however, this has not been determined to date.

Constitutive Androstane Receptor (CAR) in Fish
Genomic data have revealed a wide diversity of CYP2 genes in several fish species (Nelson, 2003).
Findings suggest that this CYP family has greatly diversified in fishes as compared to mammals; however,
both structure and function have been conserved. To date, the functional properties of few teleost CYP2
genes have been thoroughly investigated. Heterologus expression of CYP2M1 and CYP2P demonstrated
a diversified role in xenobiotic and endobiotic metabolism (Oleksiak et al., 2003; Yang et al., 1998).
Unlike mammals, however, teleosts exhibit an apparent lack of CYP2B and CYP2 gene induction