Interactions Of Pig(sus Domesticus)organic Anion Transporting Polypeptide 1a2 With Three Groups Of Antibiotics

Organic anion transporting polypeptides?human:OATPs,animal:Oatps?are important cell membrane transporters.They are widely distributed in different tissues and organs such as the liver,kidney,small intestine,testis and blood-brain barrier,affecting the absorption and distribution of a broad spectrum of drugs and are considered as important determinants for drug bioavailability.Substrates of OATPs include endogenous compounds such as steroids,bile salts,thyroid hormones as well as many toxins and drugs.Antibiotics such as fluoroquinolones and?-lactam agents have been found to be transported by OATP family members.Tetracyclines,macrolides and?-lactam antibiotics are broad-spectrum antibiotics that are widely utilized to treat animal diseases caused by bacterial infections.However,large quantities of these drugs are often applied due to their low bioavailability,which in turn may lead to drug resistance and metabolic residues.With food safety issues becoming more and more important,drug residues problems result from over-use of veterinary drugs in food animals need to be taken into consideration.The improvement of drug efficacy and hence reducing the use of veterinary drugs may be an important way to solve such a problem.OATP1A2 is the first human OATP family member that was cloned and has been found to be expressed in various tissues such as the brain,small intestine,liver,and kidney.OATP1A2 is involved in the transport of various structurally diverse drugs and is essential for cellular accumulation of these drugs.Pig?Sus domesticus?Oatp1a2?sOatp1a2?shares high amino acid sequence homology with OATP1A2 and is considered to be a functional homolog of OATP1A2.Previous studies in our laboratory found that sOatp1a2 is abundantly expressed in the pig liver.Since many drug-metabolizing enzymes exist in the liver,the organ is considered as a major site for drug metabolism.The abundant expression of sOatp1a2 in pig liver suggests that the transporter may play an important role in drug absorption by the liver.In this study,we investigated the interaction between sOatp1a2 and tetracyclines,macrolides,and?-lactams using the HEK293?human embryonic kidney 293?cell line stably expressing sOatp1a2.The results obtained are as follows:?1?Immunostaining was used to analyze protein level and expression sites of Slco1a2 in the stable clones previously selected in our laboratory.It was found that sOatp1a2 was correctly targeted to the cell surface,and exerted uptake function for OATP family prototypic substrates taurocholate and estrone-3-sulfate?ES?.The Km value of the taurocholate and ES is 29.2±4.5?M and 17.3±3.2?M,respectively.?2?Inhibition experiments demonstrated that tetracyclines such as tetracycline,chlortetracycline,oxytetracycline and doxycycline significantly inhibited taurocholate and estrone-3-sulfate uptake by sOatp1a2.Concentration-dependent analysis revealed that these tetracyclines inhibited taurocholate transport in a concentration-dependent manner.The IC₅₀value of tetracycline,chlortetracycline,oxytetracycline,and doxycycline for taurocholate transport is 50.7±22.9?M,20.4±11.9?M,1.40±0.67?M,and 18.7±8.5?M,respectively.LC/MS analysis revealed that tetracycline and chlortetracycline may be directly transported by sOatp1a2,exhibiting a 1.4-fold and 1.5-fold increase of uptake,respectively,compared to cells transfected with empty vector.On the other hand,no significant change was observed for oxytetracycline and doxycycline uptake by cells expressing sOatp1a2compared to empty vector,suggesting that these drugs may merely serve as inhibitors but not substrates of sOatp1a2.?3?Studies of tilmicosin,tylosin,erythromycin,and clarithromycin in the macrolide family found that these drugs also significantly inhibit sOatp1a2 transport of taurocholate and estrone-3-sulfate.Further studies of tilmicosin and tylosin showed that they inhibited taurocholate uptake at a concentration-dependent manner,and their IC₅₀0 for taurocholate transport is 10.5±4.8?M and 4.30±1.62?M,respectively.LC/MS analysis showed that no significant change was observed for the absorption of tilmicosin and tylosin by sOatp1a2,suggesting that tilmicosin and tylosin may only be inhibitors of sOatp1a2.?4?Inhibition experiments showed that the?-lactam drugs penicillin,cefquinome sulfate,and amoxicillin exhibited significant inhibitory effects on sOatp1a2 transport of taurocholate and estrone-3-sulfate.Further studies of penicillin showed that the antibiotic inhibited taurocholate uptake at a concentration-dependent manner,its IC₅₀0 for taurocholate transport is 33.3±13.4?M.?5?Soybean meal,which is an important vegetable protein source in swine feed,contains large amount of isoflavones.Many kinds of isoflavones have been shown to affect transporter functions.Here we also found that genistein and daidzein,two major types of isoflavones in soybean meal,inhibited transport function of taurocholate and ES.Interestingly,preliminary study with a construct containing 5'-UTR region of sOatp1a2coding gene Slco1a2 revealed that long-term treatment of genistein and daidzein may increase expression of Slco1a2.