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The effect of BHT and its metabolite on the uptake of taurocholate into primary culture of rat hepatocytes

The effect of BHT and its metabolite on the uptake of taurocholate into primary culture of rat hepatocytes
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대학원 약학과
BHTmetaboliteuptaketaurocholateprimary culturerat hepatocytes
이화여자대학교 대학원
The effect of butylated hydroxytoluene (BHT) and its major metabolite, 3,5-ditert-4-hydroxybenzoic acid (BHT-acid) on the uptake of taurocholate into hepatocytes was studied using the primary culture of rat hepatocytes. Hepatocytes were isolated by an in situ collagenase perfusion technique and maintained as a monolayer in serum free media for 24 hours before use. The uptake of taurocholate waa saturable with an apparent Km of 12.8±2.2 UM and maximal velocity V of 0.18±0.01 nmol/mg protein.min. Both BHT and BHT-acid inhibited that hepatocellular uptake of taurocholate when they were added to the culture. BHT-acid caused more inhibition in taurocholate uptake than BHT itself, and the inhibition was competitive with a Ki of 376.7±23.1 UM. The results of current study suggest that the decreased bile acid excretion reported previously in BHT treated rats is due to an inhibition of taurocholate uptake by BHT and/or its metabolite, and the increase of bile flow poduced by BHT is due to the osmotic choleresis releated to the secretion of BHT and/or its metabolites into the bile.;식품의 산화방지제로 사용되고 있는 butylated hydroxytoluene(BHT)와 BHT의 주 대사산물인 3,5-ditert-4-hydroxy benzoie acid(BHT-acid)가 담즙산인 taurocholate가 간세포내로 유입되는 과정에 끼치는 영향을 일차 간세포 배양을 이용하여 연구하였다. 간세포는 collagenase perfusion기술에 의하여 분리하였으며, 24시간동안 serum free WO/BA-M_(2) 배지에서 monolayer로 유지시켰다. Taurocholate가 간세포내로 유입되는 ㎞과 최대속력 V는 각각 12.5±2.2㎛과 0.18±0.01 nmol/㎎ protein·min이었다. BHT와 BHT-acid를 배양세포에 첨가하였을때 taurocholate가 간세포내로 유입되는 것이 저해되었다. 또한 BHT 그 자체보다도 BHT의 주 대사산물인 BHT-acid가 taurocholate가 간세포내로 유입되는 과정을 좀 더 저해하였다. 이때 BHT-acid는 taurocholate의 유입을 경쟁적으로 저해하였으며, Ki값은 376.7 ±23.1㎛이었다. 본 실험의 결과는 BHT를 처리한 쥐의 bile flow의 증가는 BHT와 BHT 대사산물이 담즙산으로 배설됨으로써 osmotic choleresis에 의한 것으로 나타났고, 감소된 담즙산염의 량은 담즙산의 enterohepatic circulatin과정중의 하나인 담즙산염이 간세포로 유입되는 과장이 BHT나 BHT-대사산물에 의하여 저해되기 때문이라는 것으로 나타났다.
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