Kitano M, Ichihara T, Matsuda T, Wanibuchi H, Tamano S, Hagiwara A, Imaoka S, Funae Y, Shirai T, Fukushima S
First Department of Pathology, Osaka City University Medical School, Osaka, Japan.
Carcinogenesis. 1998 Aug;19(8):1475-80. doi: 10.1093/carcin/19.8.1475.
The dose dependence of the hepatopromoting effects of phenobarbital (PB) was investigated in a rat liver medium-term bioassay (Ito test) to elucidate a practical threshold level. F344 rats were given a single i.p. injection of diethylnitrosamine (200 mg/kg body wt) and subjected to two-thirds partial hepatectomy at week 3. Commencing 2 weeks from the start, PB at doses of 0, 1, 2, 4, 7.5, 15 or 500 p.p.m. in experiment 1 and 0, 0.01, 0.1 or 0.5 p.p.m. in experiment 2 were fed to the rats for 6 weeks. Experiment 3 was conducted to confirm previous data using the same medium-term bioassay, with PB at doses of 0, 1, 2, 4, 7.5, 15, 30, 60, 125, 250 or 500 p.p.m. fed to the rats. All surviving animals were killed at week 8 in these experiments and their livers were immunohistochemically examined for expression of glutathione S-transferase placental form (GST-P). Quantitative values for GST-P-positive foci in the liver were increased dose dependently in rats given 60-500 p.p.m. PB. However, those for doses in the range 1-7.5 p.p.m. demonstrated a decrease as compared with the control group (0 p.p.m.), with significant differences observed for 1 and 2 p.p.m.. The results for 15-30 and 0.01-0.5 p.p.m. were comparable with the control values. Examination of transforming growth factor-alpha (TGF-alpha)-positive foci also produced similar results to those for GST-P in experiment 1. Immunohistochemical staining of TGF-alpha and GST-P using serial liver sections demonstrated that the TGF-alpha-positive foci comprised a sub-population of the GST-P-positive lesions, being approximately 1/8-1/10th as common in livers of animals treated with PB. TGF-alpha-positive foci were almost always negative on immunostaining for TGF-beta. Western blotting for proteins CYP2B1, 2C6 and 3A2 revealed a good correlation between changes in GST-P-positive foci and CYP3A2 protein expression. The finding of inhibition effects at low doses of PB confirms the presence of a threshold level for promoting effects by PB on liver carcinogenesis in rats.
在大鼠肝脏中期生物测定（伊藤试验）中研究了苯巴比妥（PB）肝促进作用的剂量依赖性，以阐明实际阈值水平。给F344大鼠单次腹腔注射二乙基亚硝胺（200mg/kg体重），并在第3周进行三分之二部分肝切除术。从开始后的第2周起，在实验1中，以0、1、2、4、7.5、15或500ppm的剂量，在实验2中以0、0.01、0.1或0.5ppm的剂量给大鼠喂食PB，持续6周。进行实验3以使用相同的中期生物测定法确认先前的数据，给大鼠喂食0、1、2、4、7.5、15、30、60、125、250或500ppm剂量的PB。在这些实验中，所有存活的动物在第8周处死，对其肝脏进行免疫组织化学检查，以检测谷胱甘肽S-转移酶胎盘型（GST-P）的表达。给予60-500ppm PB的大鼠肝脏中GST-P阳性灶的定量值呈剂量依赖性增加。然而，1-7.5ppm剂量组与对照组（0ppm）相比呈下降趋势，1和2ppm组观察到显著差异。15-30ppm和0.01-0.5ppm组的结果与对照值相当。对转化生长因子-α（TGF-α）阳性灶的检查在实验1中也产生了与GST-P类似的结果。使用连续肝脏切片对TGF-α和GST-P进行免疫组织化学染色表明，TGF-α阳性灶是GST-P阳性病变的一个亚群，在用PB处理的动物肝脏中其出现频率约为GST-P阳性病变的1/8-1/10。TGF-α阳性灶在TGF-β免疫染色中几乎总是呈阴性。对CYP2B1、2C6和3A2蛋白进行蛋白质免疫印迹分析显示，GST-P阳性灶的变化与CYP3A2蛋白表达之间存在良好的相关性。低剂量PB存在抑制作用这一发现证实了PB对大鼠肝癌发生促进作用存在阈值水平。
