Effect Of Arecoline On The Expression Of Rat Hepatic CYP2E1 And Hepatic And Renal Transporters In Vivo

Objective: Betel quid is regarded as the fourth widely used food additives ranking only second to Tobacco,Alcohol and Caffeine and it is chewed by approximately 600 million people all over the world.Arecoline is reported to be the most abundant alkaloid in the areca nut and it has been demonstrated various pharmacological activities,such as tapeworm expelling and snail eradication activity,anti-inflammatory and antioxidant activity,anti-atherosclerosis activity and hypoglycemic activity,otherwise,arecoline can also lead to oral submucous fibrosis(OSF),hepatic and renal toxicity,reproductive toxicity,and etc.The hepatic CYP2E1 activity aberrantly increased or overexpression of hepatic CYP2E1 is regarded to be an important factor on the proliferation of Reactive Oxygen Species(ROS),and the oxidative stress caused by ROS is the common physiopathologic mechanism in liver diseases.In addition,the alteration in transporters expression and/or activity is not only related to the occurrence and development of some diseases,also response for the pharmacokinetics,pharmacodynamics and toxicity of clinical drugs,which resulting in drug-drug interactions.For this resaerch,I aim to study the effect of arecoline on the expression of rat hepatic CYP2E1 and hepatic and renal drug transpoters in vivo and offering experimental guidance for clinical safe medicine usage for betel nut aficionados.Methods: Total 24 male wistar rats were randomly averaged divided into 4 groups and numberd consecutively to receive various intragastric administrations.In Group 1-3,rats were orally administered with 4,20 and 100 mg/kg/d arecoline hydrobromide,respectively.In Group 4(vehicle group),rats were intragastric administered with isopyknic physiological saline.Rats were intragastric administrated by arecoline hydrobromide for consecutive 7 days,then rats were sacrifced by cervical dislocation,the hepatic CYP2E1 m RNA expression,CYP2E1 protein expression and CYP2E1 activity were determined by RT-PCR,western blotting and LC-MS/MS method,respectively.Another 24 male wistar rats were randomly averaged divided into 4 groups and numbered consecutively to receive various intragastric administrations,In Group 1-3,rats were orally administered with 0.8,4 and 20 mg/kg/d arecoline hydrobromide,respectively.In Group 4(vehicle group),rats were orally administered with isopyknic physiological saline.After intragastric administration once a day for consecutive 21 days,rats were sacrifced by cervical dislocation,and the relative m RNA expression of 13 vital hepatic and renal transporters(Oct1,Oct2,Oat2,Octn1,Octn2,Oatp1a1,Oatp1a4,Oatp2b1,Mrp2,Mrp4,Mrp5,Bcrp and Mdr1a)were detected by RT-PCR.Results:(1)After oral administration of arecoline hydrobromide to rats for consecutive 7 days,the m RNA expression of hepatic CYP2E1 remained unchanged while the protein content of hepatic CYP2E1 was dose-dependently increased,as for the hepatic CYP2E1 activity,it was induced while the induction was attenuated with dose increasing.(2)After intragastric administration of arecoline hydrobromide to rats for consecutive 21 days,the results from real-time PCR indicated that the m RNA levels of hepatic Octn2,Mrp2 and Mdr1 a were significantly decreased while renal Mrp5 m RNA level was significantly increased at low dose of AH.Moreover,AH treatment at high dose significantly inhibited the m RNA expression of hepatic Oct2,Oat2,Octn2,Oatp1a1,Oatp1a4,Oatp2b1,Mrp2 and Mdr1 a as well as renal Mrp2,Bcrp and Mdr1 a.However,the m RNA expression of renal Octn2,Oatp1a1,Oatp1a4 and Mrp5 were significantly up-regulated following the treatment of high dose of AH,and the AH-induced effect on the above transporters was dose dependent to some extent.Conclusion:(1)our study results indicated that the regulatory mechanism of arecoline on rat hepatic CYP2 El mainly by increasing the stabilization of CYP2E1 protein against degradation or improving translation efficiency of CYP2E1 m RNA rather than transcriptional activation of the gene,additionally,it may involve the post-translational modification of the CYP2E1 protein.Take it by and large,CYP2E1 responsing to xenobiotics is equal among rodent and human,the induction of rat hepatic CYP2E1 by arecoline hydrobromide suggests that there exists a high risk of metabolic interaction among the substrate drugs of CYP2E1 in betel-quid aficionados,and more attention should be paid to.(2)the alteration in expression and function of hepatic and renal transporters are of vital importance with regards to the pharmacokinetics and pharmacodynamic effects of their substrate drugs.Long-term and low-dose uptake of arecoline hydrobromide is closer to the daily intake of betel nut aficionados.In our study,we found that hepatic m RNA expression of Octn2,Mrp2 and Mdr1 a was inhibited and renal m RNA expression of Mrp5 was increased at a low dosage of arecoline hydrobromide,suggestting that more attention should be paid to the clinical treatment of betel nut aficionados to avoid fatal effects caused by adverse drug interactions.