Study On The Roles Of CYP1A1,CYP3A4 In DSP Toxin Metabolism In Perna Viridis

Okadaic acid(OA)is the main component of diarrheic shellfish poisoning toxins(DSP toxins).After feeding on DSP toxins-producing dinoflagellate,bivalve can accumulate the toxin in the body and spread it through the food chain,thus causing human diarrhea poisoning.However,Bivalves have some tolerance to the toxin,and the mechanism is not clear.Cytochrome P450(Cytochrome P450,CYP450)plays an important role in the biotransformation and metabolism of exogenous compounds.To learn the role of cytochrome P450 in DSP toxins metabolic detoxification in bivalves,in this paper,four genes,CYP3A4,CYP1A1,PXR,and AHR were obtained from digestive gland of the mussels Perna viridis by bioinformatics method at first.On this basis,real-time fluorescence quantitative PCR was used to analyze the mRNA expression changes in the four genes after exposure to DSP toxins-producing Prorocentrum lima,and activities of CYP3A4 and CYP1A1,and content of OA were determined in the present of some inhibitors or activators of CYP3A4 and CYP1A1,such as ketoconazole,clotrimazole,rifampicin and ?-naphthoflavone.Finally,HPLC/MS-MS was used to detect the production of OA metabolites in the presence of ketoconazole and rifampicin in vitro.We found that the CYP3A4 was up-regulated,and CYP1A1 and AHR mRNA were downregultaed in the digestive glands of P.viridis after exposoure to P.lima.In the presence of ketoconazole,the CYP3A4 activity and OA content in digestive glands of mussels significantly decreased.After tamalapine was added,the activity of CYP3A4 was not enhanced,but significantly decreased,and the content of OA remained changed,suggesting that species differences of rifampin on the activity of CYP3A4.The activity of CYP1A1 was induced and the OA content significantly increased after the addition of ?-naphthoflavone.However,there was no significant change in CYP1A1 enzyme activity and OA concentration in the presence of rifampicin.These results indicated that CYP3A4 and CYP1A1 were all involved in the metabolic detoxification of DSP toxins in mussels.A metabolite of OA with mother ion for m/z 819,fragment ions for m/z 563 and 255 was detect from the co-culture of digestive gland tissue homogenate of P.viridis and OA by HPLC-MS/MS,suggesting that oxygenation or hydroxylation might be an important form of DSP txoins metabolism in mussel.No detection of the metabolite under the pressure of ketoconazole and rifampicin,when CYP3A4 was inhibited,further indicated that CYP3A4 might play an important role in OA metabolism.In conclusion,our results show that DSP toxin in deed experience some biotransformation in the digestive gland of mussel,and CYP3A4 might play an important role in the metabolism of DSP toxin.