Arizona Cancer Center, University of Arizona, USA.
Gut. 2010 Dec;59(12):1606-16. doi: 10.1136/gut.2010.213686. Epub 2010 Sep 28.
Barrett's oesophagus is a premalignant disease associated with oesophageal adenocarcinoma. The major goal of this study was to determine the mechanism responsible for bile acid-induced alteration in intracellular pH (pH(i)) and its effect on DNA damage in cells derived from normal oesophagus (HET1A) or Barrett's oesophagus (CP-A).
Cells were exposed to bile acid cocktail (BA) and/or acid in the presence/absence of inhibitors of nitric oxide synthase (NOS) or sodium-hydrogen exchanger (NHE). Nitric oxide (NO), pH(i) and DNA damage were measured by fluorescent imaging and comet assay. Expression of NHE1 and NOS in cultured cells and biopsies from Barrett's oesophagus or normal squamous epithelium was determined by RT-PCR, immunoblotting or immunohistochemistry.
A dose dependent decrease in pH(i) was observed in CP-A cells exposed to BA. This effect of BA is the consequence of NOS activation and increased NO production, which leads to NHE inhibition. Exposure of oesophageal cells to acid in combination with BA synergistically decreased pH(i). The decrease was more pronounced in CP-A cells and resulted in >2-fold increase in DNA damage compared to acid treatment alone. Examination of biopsies and cell lines revealed robust expression of NHE1 in Barrett's oesophagus but an absence of NHE1 in normal epithelium.
The results of this study identify a new mechanism of bile acid-induced DNA damage. We found that bile acids present in the refluxate activate immediately all three isoforms of NOS, which leads to an increased NO production and NHE inhibition. This consequently results in increased intracellular acidification and DNA damage, which may lead to mutations and cancer progression. Therefore, we propose that in addition to gastric reflux, bile reflux should be controlled in patients with Barrett's oesophagus.
巴雷特食管是一种与食管腺癌相关的癌前疾病。本研究的主要目的是确定胆汁酸诱导的细胞内 pH 值（pH(i)）变化的机制及其对来源于正常食管（HET1A）或巴雷特食管（CP-A）的细胞中 DNA 损伤的影响。
细胞暴露于胆汁酸鸡尾酒（BA）和/或酸中，同时存在/不存在一氧化氮合酶（NOS）或钠-氢交换器（NHE）抑制剂。通过荧光成像和彗星试验测量一氧化氮（NO）、pH(i)和 DNA 损伤。通过 RT-PCR、免疫印迹或免疫组织化学测定培养细胞和巴雷特食管或正常鳞状上皮活检中的 NHE1 和 NOS 的表达。
暴露于 BA 的 CP-A 细胞观察到 pH(i)呈剂量依赖性下降。BA 的这种作用是 NOS 激活和 NO 产生增加的结果，导致 NHE 抑制。食管细胞暴露于酸与 BA 的联合作用下协同降低 pH(i)。这种降低在 CP-A 细胞中更为明显，并导致 DNA 损伤增加超过 2 倍，与单独酸处理相比。对活检和细胞系的检查显示巴雷特食管中 NHE1 的表达强烈，但正常上皮中不存在 NHE1。
本研究的结果确定了胆汁酸诱导的 DNA 损伤的新机制。我们发现反流物中的胆汁酸立即激活 NOS 的所有三种同工酶，导致 NO 产生增加和 NHE 抑制。这继而导致细胞内酸化和 DNA 损伤增加，这可能导致突变和癌症进展。因此，我们提出，除了胃反流外，还应控制巴雷特食管患者的胆汁反流。
