James Graham Brown Cancer Center, University of Louisville, KY 40202, USA.
Mutat Res. 2012 Mar 18;743(1-2):59-66. doi: 10.1016/j.mrgentox.2011.12.022. Epub 2012 Jan 5.
We investigated the effect of punicalagin (PC) on benzo[a]pyrene (BP)-induced DNA adducts in vitro and in vivo. Incubation of BP (1 μM) with rat liver microsomes, appropriate co-factors and DNA in the presence of vehicle or punicalagin (1-40 μM) showed dose-dependent inhibition of the resultant DNA adducts, with essentially complete (97%) inhibition at 40 μM. However, PC failed to inhibit anti-BPDE-induced DNA adducts when tested in an in vitro non-microsomal system, suggesting that the inhibition of the microsomal BP-DNA adducts occurred due to inhibition of P450 1A1 by PC. To determine its efficacy in vivo, female S/D rats were administered punicalagin via the diet (1500 ppm; approximately 19 mg/day/animal) or subcutaneous polymeric implants (two 2-cm, 200mg with 20% drug load; 40 mg PC/implant) and then treated with continuous low-dose of BP by a subcutaneous polymeric implant (2 cm, 200mg with 10% load; 20mg BP/implant) and euthanized after 10 days. Analysis of the lung DNA by (32)P-postlabeling showed significant (60%; p=0.029) inhibition of DNA adducts by PC administered via the implants; the dietary route showed modest (34%) but statistically insignificant inhibition. Furthermore, total PC administered by implants was approximately 38-fold lower compared with the dietary route. Analysis of the lung microsomes showed significant inhibition of cytochrome P450 1A1 activity and induction of glutathione. Release of PC from the implants was found to be biphasic starting with a burst release, followed by a gradual decline. Ultra performance liquid chromatography analysis showed no detectable PC in the plasma but its hydrolyzed product, ellagic acid was readily detected. The plasma concentration of ellagic acid was over two orders of magnitude higher (589 ± 78 ng/mL) in the implant group compared with diet (4.36 ± 0.83 ng/mL). Together, our data show that delivery of PC by implants can reduce its effective dose substantially, and that the inhibition of DNA adducts in vivo occurred presumably due to the conversion of PC to ellagic acid.
我们研究了鞣花酸（PC）对苯并[a]芘（BP）诱导的体外和体内 DNA 加合物的影响。在存在载体或鞣花酸（1-40 μM）的情况下，用大鼠肝微粒体、适当的辅因子和 DNA 孵育 BP（1 μM）显示出所得 DNA 加合物的剂量依赖性抑制，在 40 μM 时基本完全（97%）抑制。然而，当在非微粒体体系中进行测试时，PC 未能抑制抗-BPDE 诱导的 DNA 加合物，这表明微粒体 BP-DNA 加合物的抑制是由于 PC 抑制 P450 1A1 所致。为了确定其在体内的功效，雌性 S/D 大鼠通过饮食（1500 ppm；约 19 mg/天/动物）或皮下聚合物植入物（两个 2 厘米长，200mg 载药量为 20%；40 mg PC/植入物）给予鞣花酸，并通过皮下聚合物植入物（2 厘米长，200mg 载药量为 10%；20mg BP/植入物）持续给予低剂量 BP 并在 10 天后处死。通过（32）P-后标记法对肺 DNA 进行分析表明，通过植入物给予的 PC 显著（60%；p=0.029）抑制 DNA 加合物；饮食途径显示适度（34%）但无统计学意义的抑制。此外，通过植入物给予的总 PC 大约是饮食途径的 38 倍低。对肺微粒体的分析表明，细胞色素 P450 1A1 活性显著抑制，谷胱甘肽诱导。发现 PC 从植入物中的释放呈两相释放，最初是爆发释放，随后逐渐下降。超高效液相色谱分析显示在血浆中未检测到 PC，但可检测到其水解产物鞣花酸。与饮食组（4.36 ± 0.83ng/mL）相比，植入组血浆中鞣花酸的浓度高出两个数量级以上（589 ± 78ng/mL）。综上所述，我们的数据表明，通过植入物给予 PC 可以大大降低其有效剂量，并且体内 DNA 加合物的抑制可能是由于 PC 转化为鞣花酸所致。
