The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio): Expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers

Abstract

The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map into CYP2AA1 near the substrate access channels, suggesting differing substrate specificity. Zebrafish CYP2AA1 transcript was expressed predominantly in intestine, while CYP2AA2 was most highly expressed in kidney, suggesting differing roles in physiology. In liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1, but not CYP2AA2 in liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish.

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

“…QPCR were run on a MyIQ single-color real-time thermocycler with IQ Sybr green Supermix from Bio-Rad. The E -ΔΔCt method was used for relative quantification (Livak and Schmittgen, 2001), using the geometric mean of aryl hydrocarbon receptor nuclear translocator 2 ( arnt2 ) and elongation factor 1 alpha ( ef1α ) Ct values for normalization (Kubota et al, 2013; Morrison et al, 2014). Primers were: 5’-TACAGGAGGTGGAAGGAAAGGTG-3’ (F) and 5’-GACGACCACCAAGGGCATAGATAAC-3’ (R) for CYP20A1 .…”

“…Functions can be inferred for many CYPs in gene families 5-51, based on activities of known mammalian orthologs, yet predicted function has been confirmed in few cases (e.g., the lanosterol 14α-demethylase CYP51) (Morrison et al, 2014). In gene families 1-3, there has been much lineage specific expansion, resulting in co-orthologs, most with unknown function (Kubota et al, 2013). Thus, many zebrafish CYPs still are properly considered orphans, including CYP20A1.…”

BDE-99, but not BDE-47, is a transient aryl hydrocarbon receptor agonist in zebrafish liver cells

“…The induction of biotransformation enzymes in mammals is mediated by three main transcription factors: the AHR, the PXR and the constitutive androstane receptor (CAR) (Köhle and Bock, 2009). Like the AHR, the PXR is a xenobiotic sensor responsible for a wide range of ligand-mediated activities, and induces genes such as CYP2s and CYP3s in both rodents and fish (Waxman, 1999;Bresolin et al, 2005;Bainy et al, 2013;Kubota et al, 2013). Phylogenetic analysis indicates that unlike mammalian models, zebrafish models do not contain Car , which highlights the potential key function of Pxr in zebrafsh.…”

Cyp2aa9 regulates haematopoietic stem cell development in zebrafish

“…However, this genomic locus share poor synteny with mammalian CYP2 genes3435, and the different Cyp2aa members may have distinct substrate specificities32. Previous studies have demonstrated that the Cyp2aas transform a broad range of xenobiotic substrates3536, whereas little is known regarding their physiological functions. Our study has identified physiological roles of Cyp2aa9 in HSCs formation during embryonic development.…”