Slaus K, Coughtrie M W, Sharp S, Vanhaecke T, Vercruysse A, Rogiers V
Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
Biochem Pharmacol. 2001 May 1;61(9):1107-17. doi: 10.1016/s0006-2952(01)00598-6.
The expression of sulfotransferase and steroid sulfatase was studied in rat liver using the most promising culture models of hepatocytes, including monolayer culture with a pyruvate (30 mM) enriched medium, co-culture with rat epithelial cells from primitive biliary origin and collagengel sandwich culture. In the latter, addition of dexamethasone (1 microM) to the medium was examined. Phenol sulfotransferase enzymes (SULT1) were studied by measuring activities towards 4-methylphenol and estradiol, hydroxysteroid sulfotransferase (SULT2A) activity was determined towards dehydroepiandrosterone (DHEA). Microsomal steroid sulfatase activity was measured towards estrone sulfate. Western blot analysis was carried out using polyclonal antibodies raised against rat phenol sulfotransferase SULT1A1 (ASTIV), estrogen sulfotransferase SULT1E1 (EST) and hydroxysteroid sulfotransferase (HST). SULT2A activity towards DHEA was maintained at a high level during the whole culture time. In the co-culture it even reached the level of freshly isolated cells. Addition of pyruvate had no positive effect on the activity measured in monolayer cultures. High SULT1A1 activity towards 4-methylphenol was found in the co-culture system. In the monolayer culture, the activity initially decreased with 35% but was then kept at a constant level, while in the sandwich culture low activities were measured. For dexamethasone, an inducing effect on the various SULT activities could not be detected. Independently of the culture model used, the SULT1E1 activity towards estradiol decreased to 20% and 5% of the initial activity after four and seven days of culture, respectively. Microsomal steroid sulfatase activity was best maintained in collagengel sandwich cultures. During the first four days in culture it retained 73% of the initial activity, afterwards it decreased to 40% of the activity found in freshly isolated hepatocytes, irrespective of the culture conditions. High expectations exist for collagengel sandwich cultures, however, in our study the results were rather disappointing. Monolayer is a suitable culture model for short-term purposes. For long-term in vitro biotransformation studies, co-culture is preferred but is rather complex.
利用最有前景的肝细胞培养模型，包括用富含丙酮酸（30 mM）的培养基进行单层培养、与源自原始胆管的大鼠上皮细胞共培养以及胶原凝胶夹心培养，研究了大鼠肝脏中磺基转移酶和类固醇硫酸酯酶的表达。在后者中，研究了向培养基中添加地塞米松（1 μM）的情况。通过测量对4-甲基苯酚和雌二醇的活性来研究酚磺基转移酶（SULT1），通过测定对脱氢表雄酮（DHEA）的活性来确定羟类固醇磺基转移酶（SULT2A）的活性。针对硫酸雌酮测定微粒体类固醇硫酸酯酶的活性。使用针对大鼠酚磺基转移酶SULT1A1（ASTIV）、雌激素磺基转移酶SULT1E1（EST）和羟类固醇磺基转移酶（HST）产生的多克隆抗体进行蛋白质免疫印迹分析。在整个培养期间，SULT2A对DHEA的活性维持在较高水平。在共培养中，其活性甚至达到了新鲜分离细胞的水平。添加丙酮酸对单层培养中测得的活性没有积极影响。在共培养系统中发现SULT1A1对4-甲基苯酚具有高活性。在单层培养中，该活性最初下降了35%，但随后保持在恒定水平，而在夹心培养中测得的活性较低。对于地塞米松，未检测到对各种SULT活性的诱导作用。无论使用何种培养模型，SULT1E1对雌二醇的活性在培养四天和七天后分别降至初始活性的20%和5%。微粒体类固醇硫酸酯酶的活性在胶原凝胶夹心培养中保持得最好。在培养的前四天，它保留了初始活性的73%，之后降至新鲜分离肝细胞中发现的活性的40%，与培养条件无关。人们对胶原凝胶夹心培养寄予厚望，然而，在我们的研究中结果相当令人失望。单层培养是用于短期目的的合适培养模型。对于长期体外生物转化研究，共培养更受青睐，但相当复杂。
