Studies On Domoic Acid Detoxification-related Liver Genes And The Regulation By TBHQ In Red Sea Bream Pagrus Major

Harmful algal blooms (HABs) are a global phenomena and recent evidence indicates that their frequency and intensity are increasing. They are a serious threat to human health, aquaculture, fisheries, and ecosystem health. Amnesic shellfish poisoning (ASP) toxin belongs to a marine neurotoxin which may cause permanent short-term memory loss. The principal toxin responsible for ASP is domoic acid (DA).To investigate the molecular detoxification mechanism of marine algae toxin DA detoxinfication-related genes (AHR, ARNT, CYP1A1, GSTA1, GSTA2, GSTR and HSP70) in marine fish liver, the phaseⅠxenobiotic metabolizing enzymes (XMEs) CYP1A1 and ARNT cDNA sequences were cloned from the liver of red sea bream Pagrus major; the promoter regions of phaseⅠXMEs (CYP1A1) and phaseⅡXMEs (GSTA2 and GSTR) gene of red sea bream were also obtained and characterized; and finally, the putative regulatory elements were predicted. In addition, the comprehensive and comparative analysis of the amino acid sequence similarity, conserved domains, three-dimensional molecular model, gene phylogenetic tree, cis-acting elements, gene and genomics structure of the DA detoxinfication-related liver genes among the economic fishes, model animal (zebrafish) and mammals (human, mouse, and rat), which would provide abundant basic information on the functional research of detoxinfication-related genes.To elucidate the role of the detoxinfication-related liver genes in the DA toxin metabolism of the major cultivated carnivorous fish, red sea bream intracoelomically injected with DA, were investigated. Experimental fish were administered with one injection of DA (2 mg/kg wet weight) or PBS as control. After 24 h, fish were killed and hepatic RNA was isolated. Usingβ-actin as an external control, the relative liver AHR, ARNT, CYP1A1, GSTA1, GSTA2, GSTR and HSP70 mRNA abundance of red sea bream were determined by semi-quantitative RT-PCR within the exponential phase. The result showed the significant induction of hepatic CYP1A1 in response to DA indicates an important role for phase I XMEs in DA metabolism. In contrast, the transcription of three major phaseⅡXMEs GSTs as well as HSP70 was not affected significantly by DA exposure, which suggests that a possible role of CYP1A after DA exposure in the toxin metabolism of marine fish, possibly through the AHR/ARNT signaling pathway.We assume that the high induction of CYP1A1 may decrease the toxicity of DA in fish, whereas GSTs expression were not significant in response to DA, thus DA couldn't be excreted out of fish effectively. If exsit some regulation materials, which could increase the DA excretion by inducing GSTs expression, was deserved further research. The influences of the food antioxidant, tert-butylhydroquinone (tBHQ), on DA metabolism and detoxification-related gene transcription were investigated both in vivo and in vitro. Oral administration of tBHQ (100 mg/kg) resulted in significant decreases of DA accumulations in liver tissues in our experimental fish which were fed with a single dose of 10 mg DA/kg body weight (bwt) 24 h before tBHQ treatment. Real-time RT-PCR further revealed that the mRNA level of GSTA2 and the mRNA levels of AHR, ARNT, CYP1A1, GSTA1 and GSTR were upregulated in the above liver tissues at 24 and 72 h post tBHQ treatment, respectively. In consistence, increased mRNA expressions of GSTA1, GSTA2 and GSTR were also observed in cultured hepatocytes that were exposed to 40 or 60μM tBHQ. Collectively, our findings in this research suggested that the dietary intake of tBHQ accelerated DA metabolism in fish, through mechanisms involving altered transcriptions of detoxification-related liver genes.In conclusion, the study has demonstrated a significant stimulatory effect of DA exposure on the transcription of phase I XMEs CYP1A1 possibly through AHR/ARNT signaling pathway in the liver of red sea bream. The dietary intake of tBHQ can stimulate DA metabolism effectively possibly by increasing the transcription of detoxification-related liver genes in fish. The metabolic adaptation may be based on coordinate regulation of a set of phase I and II XMEs, which would provide abundant evidences to develop new dietary additives to stimulate detoxification-related genes expression levels in aquaculture.