The estimated yearly number of deaths world-wide from esophageal cancer is 300 034 for men and 142 228 for women[6
], making it the sixth leading cause of cancer deaths among men and women combined. There are two principal histologic types of esophageal cancer, adenocarcinoma and squamous cell carcinoma. In the United States, the incidence of adenocarcinoma has increased four-fold between 1973 and 2002, whereas squamous cell carcinoma has declined 30% over the same period, making adenocarcinoma the predominant form of esophageal cancer[8
]. Barrett’s metaplasia of the esophagus is an important predisposing condition for the development of esophageal adenocarcinoma[9
]. Barrett’s esophagus (BE) is a metaplastic lesion of the distal esophagus, characterized by the replacement of the normal squamous epithelium by columnar intestinal epithelium containing goblet cells. BE is associated with increased duodeno-gastro-esophageal reflux[10,11
], which causes increased exposure of the esophagus to bile acids from the duodenum and acidity (gastric acidity) from the stomach. Individuals with esophageal adenocarcinoma experience even greater exposure to bile than persons with uncomplicated BE[12
]. Expression of bile acid transporter proteins is increased in BE tissues, suggesting that the development of BE metaplasia may be an adaptation to protect cells from bile acids[13
]. Thus progression to BE and to adenocarcinoma may be strongly influenced by bile acid exposure. As discussed next, evidence indicates that short-term exposure of esophageal cells to bile acids induces oxidative stress, DNA damage, mutation and apoptosis; and among surviving cells selects over the long-run for resistance to apoptosis and ultimately cancer.
Five studies have shown that bile acids cause increased production of ROS in esophageal cells, including those from BE metaplasia. A cocktail of five bile acids designed to mimic the bile acids present in gastroesophageal reflux was used to test whether reflux induces ROS[14
]. The five bile acids were glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA) and deoxycholic acid (DCA). This cocktail induced ROS in biopsies from human BE metaplastic tissue. The bile acid cocktail also induced ROS in cultured SV40-transformed squamous esophageal epithelial cells (HET1-A). DCA induced ROS in cultured human esophageal adenocarcinoma cells (OE33) and squamous cell carcinoma cells (KYSE-30)[15
]. GCDCA in acidic media induced ROS in cultured esophageal squamous cell lines derived from patients with gastroesophageal reflux disease (GERD) with BE, or without BE[16
]. When mice were fed a zinc deficient diet containing a DCA supplement, ROS production was increased and BE-like lesions developed[17
Six studies showed that bile acids induce DNA damage in esophageal cells (Table ), and five of these reported evidence for oxidative DNA damage.
Bile acids induce DNA damage in cells of the esophagus
The findings that bile acids induce DNA damage suggest that bile acids may also increase the frequency of mutation, since replication of a damaged DNA template strand often results in a replication error and thus a mutation.
Esophagoduodenostomies were performed on Big Blue F1 lac
I transgenic rats to surgically increase duodeno-gastro-esophageal reflux[21
]. The frequency of lac
I mutant cells proved to be significantly higher in the esophageal mucosa of the surgically altered rats than in the unaltered control rats, indicating that components of refluxate, such as bile acids, increase mutation. Forty-six percent of the mutant cells were altered at CpG dinucleotide sites, and the majority of these mutations (61%) were C to T or G to A transitions. This pattern of mutation is similar to that in human esophageal adenocarcinoma, suggesting that reflux is not only mutagenic, but also carcinogenic. Consistent with these findings, it was found that DCA treatment of cultured esophageal cells cause an increase in the frequency of GC to AT mutations in the p53
]. In addition, increased duodeno-gastro-esophageal reflux was observed to increase mutagenesis using a surgical model in Big Blue mice (rather than rats)[22
Bile acids induce apoptosis in esophageal cells, perhaps through the mediation of damaging ROS. DCA induced apoptosis in esophageal biopsies from normal human squamous epithelium[23
]. Also, five different bile acids [GCDCA, GDCA, TCA, taurochenodeoxycholic acid (TCDCA) and taurodeoxycholic acid (TDCA)] individually, and also in a mixture, induced apoptosis of cultured human normal esophageal mucosal epithelial cells[24
Although a short-term effect of high bile acid exposure is induction of apoptosis, a longer-term effect of repeated high exposure to apoptosis-inducing agents, such as bile acids, appears to be selection for apoptosis resistant cells. When tissue samples from patients with normal esophagus, esophagitis, BE lesions and adenocarcinomas were studied for apoptosis capability, it was found that apoptosis is inhibited early in the dysplasia-carcinoma sequence of BE by over-expression of the anti-apoptotic protein, Bcl-2[25
], presumably as a result of chronic gastroesophageal reflux containing bile acids. BE cells have high levels of the anti-apoptotic proteins IL-6, Bcl-xL and Mcl-1[26
]. Studies of tissues obtained from patient biopsies, indicated that BE cells are resistant to apoptosis induction by DCA compared to esophageal squamous epithelium and normal colon epithelium[23
]. Reduced apoptosis competence may arise by mutation in genes encoding proteins necessary for apoptosis. Since cells resistant to apoptosis have a growth advantage in the presence of agents that ordinarily induce apoptosis, such as bile acids, these cells will tend to proliferate to form a field of apoptosis resistant cells[27
]. Within such a defective field, repeated encounters with bile acids in reflux would cause further DNA damage. Such DNA damage, leading to further mutation, may give rise to malignancy.
Considerable evidence indicates an association of bile acid exposure with esophageal cancer. In rats, reflux of duodenal or gastro-duodenal contents, that include bile acids, induced esophageal carcinoma in the absence of exogenous carcinogen[28
]. Rat surgical models with increased duodenal reflux into the esophagus, but without added carcinogen, caused esophagitis, BE-like lesions and adenocarcinomas[29–32
]. Persons with BE were found to have increased duodenoesophageal reflux and increased exposure to bile acids in their refluxate, suggesting that the BE premalignant lesion is linked to bile acid exposure[10,11
]. In a rat duodenal-contents reflux model, a high animal-fat intake changed the bile acid composition of bile juice and increased the development of BE and esophageal adenocarcinoma[33
In summary, evidence indicates that, in esophageal cells and tissues, bile acids have the short-term effect of inducing oxidative stress, oxidative DNA damage, mutation and apoptosis. Over a longer period, bile acids are implicated in the development of apoptosis resistance and eventually the development of adenocarcinoma.