Hodges N J, Orton T C, Strain A J, Chipman J K
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, Safety of Medicines Department, AstraZeneca, Alderley Park, Macclesfield SK10 4TG, UK.
Carcinogenesis. 2000 Nov;21(11):2041-7. doi: 10.1093/carcin/21.11.2041.
A transient induction of S phase DNA synthesis is a common feature of non-genotoxic rodent hepatocarcinogens when administered in vivo. In the present study the ability of phenobarbitone (PB) to induce S phase DNA synthesis in primary cultures of rat hepatocytes was investigated. In the absence of serum or growth factors PB was not a mitogen per se. However, stimulation of S phase DNA synthesis by epidermal growth factor (EGF) was enhanced by co-culture with PB. This effect was both time and concentration dependent. The lowest concentration of PB that significantly enhanced the effect of EGF was 10 microM and the effect was maximal at 1.0 mM. At a concentration of 2.0 mM PB no longer enhanced EGF-induced S phase DNA synthesis. Hepatocyte cultures pretreated with PB (0.1 mM) for 2 days were more responsive to the induction of S phase DNA synthesis by EGF for the subsequent 2 days. Despite the inhibition of PB enhancement of S phase DNA synthesis by the antioxidant dimethylthiourea, reduced glutathione was not depleted by PB treatment nor were oxidized glutathione or lipid peroxides elevated. Western blotting analysis showed that PB had no effect on epidermal growth factor receptor (EGFR) autophosphorylation per se after 1 and 48 h culture, enhanced sensitization of EGFR therefore does not appear to contribute to the enhancement of S phase DNA synthesis by PB. In contrast, treatment of hepatocytes with PB for 12 h resulted in a small but statistically significant activation of p42/44 MAP kinase activity and activation of protein kinase C, as measured by redistribution of enzyme activity from a soluble to a particulate compartment of hepatocytes. Therefore, PB-mediated changes in protein kinase activity may contribute to the potentiation this compound affords.
非遗传毒性啮齿动物肝癌致癌物在体内给药时，S期DNA合成的短暂诱导是其常见特征。在本研究中，研究了苯巴比妥（PB）在大鼠原代肝细胞培养物中诱导S期DNA合成的能力。在无血清或生长因子的情况下，PB本身不是有丝分裂原。然而，与PB共培养可增强表皮生长因子（EGF）对S期DNA合成的刺激作用。这种效应具有时间和浓度依赖性。能显著增强EGF作用的PB最低浓度为10微摩尔，在1.0毫摩尔时效应最大。在2.0毫摩尔浓度下，PB不再增强EGF诱导的S期DNA合成。用PB（0.1毫摩尔）预处理2天的肝细胞培养物在随后2天对EGF诱导的S期DNA合成更敏感。尽管抗氧化剂二甲基硫脲抑制了PB对S期DNA合成的增强作用，但PB处理并未耗尽还原型谷胱甘肽，氧化型谷胱甘肽或脂质过氧化物也未升高。蛋白质印迹分析表明，培养1小时和48小时后，PB本身对表皮生长因子受体（EGFR）自身磷酸化无影响，因此EGFR敏感性增强似乎与PB增强S期DNA合成无关。相反，用PB处理肝细胞12小时导致p42/44丝裂原活化蛋白激酶活性有小幅但具有统计学意义的激活，以及蛋白激酶C的激活，这可通过酶活性从肝细胞的可溶性部分重新分布到颗粒部分来衡量。因此，PB介导的蛋白激酶活性变化可能有助于该化合物发挥的增强作用。
