Siomek Agnieszka, Brzoska Kamil, Sochanowicz Barbara, Gackowski Daniel, Rozalski Rafal, Foksinski Marek, Zarakowska Ewelina, Szpila Anna, Guz Jolanta, Bartlomiejczyk Teresa, Kalinowski Bartlomiej, Kruszewski Marcin, Olinski Ryszard
Nicolaus Copernicus University, Department of Clinical Biochemistry, Bydgoszcz, Poland.
Acta Biochim Pol. 2010;57(4):577-83. Epub 2010 Nov 9.
Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.
i）铜锌超氧化物歧化酶缺乏会导致氧化应激和致癌作用；ii）核因子-κB（NF-κB）信号通路失调可介导包括癌症在内的多种疾病。因此，我们首次决定检测超氧化物歧化酶1（SOD1）基因敲除小鼠、杂合子小鼠和野生型小鼠的氧化性DNA损伤水平以及NF-κB蛋白的DNA结合活性。使用高效液相色谱-气相色谱-质谱联用仪（HPLC-GC-MS）和高效液相色谱-电化学检测器（HPLC-EC）分析了两种氧化性损伤DNA的生物标志物：8-氧代脱氧鸟苷（8-oxodG）和8-氧代鸟嘌呤（8-oxoGua）的尿排泄量以及氧化性损伤DNA的水平。使用NF-κB p50/p65 EZ-TFA转录因子检测法评估核提取物中p50和p65蛋白的DNA结合活性。在SOD1基因敲除小鼠、杂合子小鼠和野生型小鼠的脑、肝、肾及尿液中测定了这些参数。基因敲除小鼠肝脏和肾脏中DNA的8-oxodG水平高于野生型小鼠。野生型小鼠和SOD1缺陷型动物之间8-oxoGua和8-oxodG的尿排泄量未发现差异。SOD1缺陷型小鼠肾脏中p50蛋白的活性较高，但令人惊讶的是肝脏中没有，而p65的活性没有任何变化。我们的结果表明，在铜锌超氧化物歧化酶缺乏的动物中，氧化性DNA损伤水平和NF-κB1活性仅在某些器官中升高，这可能为器官特异性活性氧诱导的致癌作用提供一些解释。
