The liver transcriptome of suckermouth armoured catfish (Pterygoplichthys anisitsi, Loricariidae): Identification of expansions in defensome gene families.

Pterygoplichthys is a genus of related suckermouth armoured catfishes native to South America, which have invaded tropical and subtropical regions worldwide. Physiological features, including an augmented resistance to organic xenobiotics, may have aided their settlement in foreign habitats. The liver transcriptome of Pterygoplichthys anisitsi was sequenced and used to characterize the diversity of mRNAs potentially involved in the responses to natural and anthropogenic chemicals. In total, 66,642 transcripts were assembled. Among the identified defensome genes, cytochromes P450 (CYP) were the most abundant, followed by sulfotransferases (SULT), nuclear receptors (NR) and ATP binding cassette transporters (ABC). A novel expansion in the CYP2Y subfamily was identified, as well as an independent expansion of the CYP2AAs. Two expansions were also observed among SULT1. Thirty-two transcripts were classified into twelve subfamilies of NR, while 21 encoded ABC transporters. The diversity of defensome transcripts sequenced herein could contribute to this species' resistance to organic xenobiotics.

An Aryl Hydrocarbon Receptor from the Salamander Ambystoma mexicanum Exhibits Low Sensitivity to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Structural features of the aryl hydrocarbon receptor (AHR) can underlie species- and population-specific differences in its affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These differences often explain variations in TCDD toxicity. Frogs are relatively insensitive to dioxin, and Xenopus AHRs bind TCDD with low affinity. Weak TCDD binding results from the combination of three residues in the ligand-binding domain: A354 and A370, and N325. Here we sought to determine whether this mechanism of weak TCDD binding is shared by other amphibian AHRs. We isolated an AHR cDNA from the Mexican axolotl (Ambystoma mexicanum). The encoded polypeptide contains identical residues at positions that confer low TCDD affinity to X. laevis AHRs (A364, A380, and N335), and homology modeling predicts they protrude into the binding cavity. Axolotl AHR bound one-tenth the TCDD of mouse AHR in velocity sedimentation analysis, and in transactivation assays, the EC50 for TCDD was 23 nM, similar to X. laevis AHR1ß (27 nM) and greater than AHR containing the mouse ligand-binding domain (0.08 nM). Sequence, modeled structure, and function indicate that axolotl AHR binds TCDD weakly, predicting that A. mexicanum lacks sensitivity toTCDD toxicity. We hypothesize that this characteristic of axolotl and Xenopus AHRs arose in a common ancestor of the Caudata and Anura.

Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in zebrafish (Danio rerio).

The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain (DBD) was highly conserved, with 74% identity to human PXR-DBD, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for mammalian PXR or constitutive androstane receptor (NR1I3). The results establish a foundation for PXR studies in this vertebrate model. PXR allelic variation and the differences between the full-length PXR and the LBD in reporter assays have implications for assessing the action of PXR ligands in zebrafish.

Cytochrome P450 diversity and induction by gorgonian allelochemicals in the marine gastropod Cyphoma gibbosum.

BACKGROUND: Intense consumer pressure strongly affects the structural organization and function of marine ecosystems, while also having a profound effect on the phenotype of both predator and prey. Allelochemicals produced by prey often render their tissues unpalatable or toxic to a majority of potential consumers, yet some marine consumers have evolved resistance to host chemical defenses. A key challenge facing marine ecologists seeking to explain the vast differences in consumer tolerance of dietary allelochemicals is understanding the biochemical and molecular mechanisms underlying diet choice. The ability of marine consumers to tolerate toxin-laden prey may involve the cooperative action of biotransformation enzymes, including the inducible cytochrome P450s (CYPs), which have received little attention in marine invertebrates despite the importance of allelochemicals in their evolution. RESULTS: Here, we investigated the diversity, transcriptional response, and enzymatic activity of CYPs possibly involved in allelochemical detoxification in the generalist gastropod Cyphoma gibbosum, which feeds exclusively on chemically defended gorgonians. Twelve new genes in CYP family 4 were identified from the digestive gland of C. gibbosum. Laboratory-based feeding studies demonstrated a 2.7- to 5.1-fold induction of Cyphoma CYP4BK and CYP4BL transcripts following dietary exposure to the gorgonian Plexaura homomalla, which contains high concentrations of anti-predatory prostaglandins. Phylogenetic analysis revealed that C. gibbosum CYP4BK and CYP4BL were most closely related to vertebrate CYP4A and CYP4F, which metabolize pathophysiologically important fatty acids, including prostaglandins. Experiments involving heterologous expression of selected allelochemically-responsive C. gibbosum CYP4s indicated a possible role of one or more CYP4BL forms in eicosanoid metabolism. Sequence analysis further demonstrated that Cyphoma CYP4BK/4BL and vertebrate CYP4A/4F forms share identical amino acid residues at key positions within fatty acid substrate recognition sites. CONCLUSIONS: These results demonstrate differential regulation of CYP transcripts in a marine consumer feeding on an allelochemical-rich diet, and significantly advance our understanding of both the adaptive molecular mechanisms that marine consumers use to cope with environmental chemical pressures and the evolutionary history of allelochemical-metabolizing enzymes in the CYP superfamily.