Transcript variations, phylogenetic tree and chromosomal localization of porcine aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) genes.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating xenobiotic-induced toxicity. AhR requires aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription complex and promote the activation of genes which have dioxin responsive element in their regulatory regions. The present study was performed to determine the complete cDNA sequences of porcine AhR and ARNT genes and their chromosomal localization. Total RNA from porcine livers were used to obtain the sequence of the entire porcine transcriptome by next-generation sequencing (NGS; lllumina HiSeq2500). In addition, both, in silico analysis and fluorescence in situ hybridization (FISH) were used to determine chromosomal localization of porcine AhR and ARNT genes. In silico analysis of nucleotide sequences showed that there were two transcript variants of AhR and ARNT genes in the pig. In addition, computer analysis revealed that AhR gene in the pig is located on chromosome 9 and ARNT on chromosome 4. The results of FISH experiment confirmed the localization of porcine AhR and ARNT genes. In the present study, for the first time, the full cDNAs of AhR and ARNT were demonstrated in the pig. In future, it would be interesting to determine the tissue distribution of AhR and ARNT transcript variants in the pig and to test whether these variants are associated with different biological functions and/or different activation pathways.

Mammalian Per-Arnt-Sim proteins in environmental adaptation.

The Per-Arnt-Sim (PAS) domain is conserved across the kingdoms of life and found in an ever-growing list of proteins. This domain can bind to and sense endogenous or xenobiotic small molecules such as molecular oxygen, cellular metabolites, or polyaromatic hydrocarbons. Members of this family are often found in pathways that regulate responses to environmental change; in mammals these include the hypoxia, circadian, and dioxin response pathways. These pathways function in development and throughout life to regulate cellular, organ, and whole-organism adaptive responses. Remarkably, in the case of the clock, this adaptation includes anticipation of environmental change. In this review, we summarize the roles of PAS domain-containing proteins in mammals. We provide structural evidence that functionally classifies both known and unknown biological roles. Finally, we discuss the role of PAS proteins in anticipation of and adaptation to environmental change.

Amnesic shellfish poisoning toxin stimulates the transcription of CYP1A possibly through AHR and ARNT in the liver of red sea bream Pagrus major.

To investigate the role of detoxification-related liver genes in amnesic shellfish poisoning toxin metabolism, red sea bream Pagrus major were exposed to domoic acid (DA, 2mugg(-1) wet weight) for 24h. Hepatic mRNA expression levels of AHR, ARNT, CYP1 and GSTs were determined by semi-quantitative RT-PCR. The cytosolic factors aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mRNA levels of DA exposure group were substantially enhanced by 113.3% and 90.9%, respectively. Consistent with this result, the phase I xenobiotic metabolizing enzyme (XME) cytochrome P-450 1A (CYP1A) was significantly induced. In contrast, the transcriptions of three major phase II XME glutathione S-transferases as well as heat shock protein 70 were not significantly affected by DA exposure. These results suggest a possible role of CYP1A after DA exposure in the toxin metabolism of marine fish, possibly through the AHR/ARNT signaling pathway.

Identification of zebrafish ARNT1 homologs: 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish requires ARNT1.

To use the zebrafish (Danio rerio) as a model to study 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) developmental toxicity, it is essential to know which proteins are involved in mediating toxicity. Previous work has identified zfAHR2 as the receptor that binds TCDD mediating downstream responses. Although zfARNT2b can form a functional heterodimer with zfAHR2 in vitro, zfarnt2 null mutants show no protection against endpoints of TCDD developmental toxicity, demonstrating that zfARNT2b cannot be the physiological dimerization partner for zfAHR2 mediating responses to TCDD in zebrafish embryos. The purpose of the current study was to identify an alternate dimerization partner(s) for zfAHR2 that may function to mediate TCDD developmental toxicity. By searching zebrafish genomic sequence and using the polymerase chain reaction-based rapid amplification of cDNA ends technique, three forms of cDNA that seem to be alternate mRNA splice variants of a zebrafish homolog of ARNT1 were detected. Analysis of the zfARNT1 proteins in vitro demonstrates that the two longest forms of zfARNT1, zfARNT1b and zfARNT1c, can form functional heterodimers with zfAHR2. However, the shortest form, zfARNT1a, seems to be nonfunctional with zfAHR2 in vitro. To determine whether a zfARNT1 protein functions with zfAHR2 in vivo, a morpholino targeted against the 5' end of zfARNT1 (zfarnt1-MO) was used. Injection of the zfarnt1-MO before TCDD treatment significantly decreases the induction of zfCYP1A mRNA and protein. In addition, zfarnt1 morphants show complete protection against TCDD-induced pericardial edema and show partial protection against reduced blood flow and craniofacial malformations caused by TCDD, demonstrating the role of zfARNT1 proteins in mediating these responses.