Primary culture of adult rat hepatocytes has been considered as a ideal model for toxicological studies because cultures hepatocytes maintained many liver-specific functions. The most well characterized one is hormonal response. In this study this system was applied to toxicological study of T-2 toxin which is mycotoxin produced mainly by Fusaria. T-2 toxin caused severe inhibition effect on hormonal induction of α-aminoisobutyric acid (AIB) transport and tyrosine aminotransferase (TAT) activity in culture hepatocytes. This effect was observed at low concentration of toxin (up to 100 ng/ml) and dose-response relationship was shown. Pretreatment also elicit the inhibition but the extent was decreased. Along with these inducible parameters, as noninducible parameter, ouabain uptake which is active membrane transport was also tested. But at 100ng/ml, this was not effected by T-2 toxin even if treatment duration was increased.
To elucidate the mechanism by which T-2 toxin cause damage in inducible functions, cellular macromolecular synthesis was measured. Protein synthesis was decreased to about half at concentration of 25 ng/ml but RNA synthesis was not significantly inhibited. Because hormonal induction of AIB uptake and TAT activity require de novo synthesis of protein, protein synthesis inhibition was proposed as the mechanism of hepatotoxicity.
When the treatment time was elongated, culture hepatocytes overcame protein synthesis inhibition and its rate was accelerated by removing toxin from culture medium. This phenomenon was reappeared by preincubation of T-2 toxin with S-9 fractions. In this repair process, carboxyesterase seemed to function mainly because incubation without NADPH exerted major portion of activity. But the inclusion of conjugation substrates, UDP-glucuronic acid and glutathione did not show any effect. In standpoint of repair, liver seems to be active because liver microsome has 10 times stronger carboxyesterase activity than that of spleen.
Primary hepatocyte culture can be used in toxicological studies using stable parameters mentioned above. But its drug metabolizing capacity decrease sharply during cultivation and this is a main problem in application of cultured hepatocytes. The effects of $\underline{in vivo}$ and $\underline{in vitro}$ inducer treatments on four cytochrome P-450 related monooxygenase activities, 7-ethoxycoumarin O-deethylase, aryl hydrocarbon hydroxylase, biphenyl 4-hydroxylase, aminopyrine N-demethylase, were measured. Phenobarbital and 3-methylcholanthrene show characteristic induction pattern. $\underline{In vivo}$ inducer treatment did not maintained monooxygenase activities but for some time cultured hepatocytes had stronger activities than initial control hepatocytes. In the case of $\underline{in vitro}$ induction, 7-ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities could be increased to higher and similar level of initial but biphenyl 4-hydroxylase and aminopyrine N-demethylase activities did not increased to initial level even if induction effects could be seen.
Several trials were applied to maintain cytochrome P-450 and associated enzyme activities. Methods to decrease lipid peroxides and radicals did not show maintenance effects. The effect of substrates was also examined. Addition of hexobarbital (1 mM) to the culture medium of rat hepatocytes protected against the rapid decline in the level of cytochrome P-450 and the activities of four drug metabolizing enzymes. While the hepatocytes cultured for 72 hr without hexobarbital and only 30% of its original level of cytochrome P-450 the cells maintained under hexobarbital had 75% of the initial level of the hemoprotein. After 72 hr in culture, the activities of aminopyrine N-demethylase and biphenyl 4-hydroxylase were 22-24% of the original rate for the nontreated cells and 73-78% for the hexobarbital-treated cells, respectively. The activities of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase in the cultures of treated cells were even higher than those of the freshly isolated hepatocytes. Additions of other substrates of hepatic mixed function oxidase to the culture medium did not protect from the loss of cytochrome P-450 and enzyme activities.
본 연구에서는 일차 간세포 배양의 독성학적 이용도를 증대시키기 위하여, 잘 알려진 기능을 이용하여 T-2 toxin 의 간독성 유발및 그 회복에 대해 연구하였으며, 배양중 감소하는 약물대사 능력을 개선 시키고자 $\underline{in vivo}$ 또는 $\underline{in vitro}$로 inducer 들을 처리하거나 배지에 항산화제 및 효소 기질을 첨가하여 그것으로 부터 유발되는 효과를 검증하였다.
간독성이 많이 알려지지 않은 T-2 toxin은 일차 배양 간세포 에서 AIB uptake 및 TAT 활성에 대한 호르몬의 작용을 저해하였으며, 호르몬에의해 효과 받지 않은 ouabain uptake 는 저해치 못하였다. 그 주된작용 기전은 단백질및 RNA 합성 실험을 통해 단백질 합성 저해에 의해 기인되는 것으로 생각된다. 간세포에서 유발된 T-2 toxin 의 단백질합성 저해는 시간이 경과함에 따라 극복 되었으며, 이와 같은 독성 감소는 간의 S-9 fraction 과의 반응으로도 얻어질 수 있으므로 회복의 주된 기전 은 약물 대사로 생각된다. T-2 toxin 의 대사는 carboxyesterase 와 cytochrome P-450 에 의해 이루어 진다고 알려져 있는데 이 두효소의 조효소 필요성을 통해 독성 회복에 있어서 그 기여 정도를 검증하였다. 회복의 많은 부분이 carboxyesterase 에 의해 얻어졌으며, 또한 간에서는 spleen 보다 10배 이상의 이 효소가 존재하므로 이것이 간에서의 독성이 잘 알려지지 않은 이유라고 생각된다.
간세포에서의 약물대사 능력은 7-ethoxycoumarin 0-deethylase, aryl hydrocarbon hydroxylase, biphenyl 4-hydroxylase, aminopyrine N-demethylase 의 4 가지로 검증하였는데 배양 중에 모두 현저한 감소를나타내었다. Inducer로 처리된 간세포는 그 사용된 inducer 에 따라 특징적인 증가를 나타내었으나 시간이 경과함에 따라 급격히 심지어는 더욱빠른 속도로 감소하였다. 그러나 얼마간은 처리되지 않은 간세포 보다 높은능력을 가진 상태에서 배양할 수 있었다. 배양액에 직접 첨가된 inducer들도 약물대사 능력을 증가시켰는데 그 양상은 분석된 효소의 종류및 사용한 inducer 에 따라 차이가 있었다.
또한 배양중에 감소하는 약물대사 능력을 초기 수준으로 유지하고자 여러가지 실험을 수행하였는데, 과산화 반응을 감소시키는 방법에서는 효과를 얻지 못하였다. 그러나 배양액에 약물대사 효소의 기질을 첨가시킨 실험에서 우리는 hexobarbital 이 약물대사 능력을 유지한다는 것을 발견하였다. 이 효과는 위에서 언급한 4가지 효소에 모두 나타났으며, cytochrome P-450 의 양에 있어서도 상당한 유지 효과를 나타내었다. 그러나 hexobarbital 이외의 다른 기질은 효과가 없는 것으로 보아 hexobarbital 의 독특한 기능으로 생각된다.
본 연구를 통해 일차 배양 간세포는 간 독성의 model 로 또한 대사활성을 필요로 하는 독성학 연구의 수단으로 그 이용도가 증대 하였다고 생각된다.
