The causative role of oxidative stress in esophageal adenocarcinogenesis has been observed in both human and animal studies.13
In patient samples, it has been reported that glutathione content is progressively decreased in the esophagitis-metaplasia-dysplasia-adenocarcinoma sequence, while myeloperoxidase activity is higher than in controls, plateauing at the stage of BE. Glutathione content is negatively correlated with DNA adducts.23
An oxidative DNA damage marker, 8-hydroxydeoxyguanosine, is significantly increased in the distal esophagus with Barrett's epithelium and high-grade dysplasia, as well as in EAC.24
Expression of manganese superoxide dismutase is significantly reduced in esophageal tissues of BE, low-grade dysplasia, high-grade dysplasia, and EAC when compared with normal esophagus.25
These findings indicate that oxidative stress is an important event in esophageal adenocarcinogenesis.
Antioxidants have been studied as potential cancer chemopreventive agents. One population-based case-control study in Sweden suggest that subjects with a high intake of vitamin C, beta-carotene, and α-tocopherol have 40-50% reduced risk of EAC.26
We chose α-tocopherol and NAC as our EAC inhibitory agents in the present study based on the different antioxidative mechanisms these two presented. α-Tocopherol functions as lipid peroxidation inhibitor in cell membrane by its chain-breaking and free radical scavenger actions. In contrast, NAC is a small water soluble molecule directly providing SH-groups for adduction or oxidation, and is a precursor of glutathione.15
With possible future applications in mind, we chose these agents because of their low toxicity and low cost. α-Tocopherol alone or in combination with NAC showed significant inhibitory effect on EAC in our EGDA model. The highest doses in diet are equivalent to the commonly used supplementation dose of vitamin E and NAC on market (400 IU and 500 mg, respectively). The calculation is based on allometric scaling.27
For example, for a rat that consumes 20 g of diet daily, the diet contains 80 kcal and 20 mg NAC (for a diet containing 1,000 ppm NAC). The calorie-based dosage equals to 20 mg/80 kcal or 0.25 mg/ kcal. For a person with a caloric requirement of 2,000 kcal/day, this is equivalent to 0.25 × 2,000 = 500 mg/day. The daily dose of NAC ranges from 250 mg to 1,500 mg clinically for patients with chronic pulmonary diseases. Due to the extensive first-pass metabolism, oral administration of NAC results in low plasma and tissue levels, but plasma levels are dose dependent.15, 28
Considering the dose used on clinical trial, the doses of NAC used in this study are relatively low.29
α-Tocopherol supplementation significantly increased the serum level of α-tocopherol, but reduced the serum level of γ-tocopherol. This phenomenon is consistent with previous reports.30
The tumor inhibitory effect of α-tocopherol we observed in this study was dose-dependent with significant inhibition (from 84% to 59%) at the highest dose (778 ppm). NAC alone did not significantly reduce tumor incidence. The highest dose of NAC (1,000 ppm) achieved less inhibition than low dose α-tocopherol. In contrast to NAC treatment alone, the combination of 500 ppm NAC and 389 ppm α-tocopherol inhibited tumor incidence from 84% to 55%. It is possible that α-tocopherol played a major role in the combination treatment. As with many nutrients, the inhibitory effect of α-tocopherol shown in this study was not dramatic (less than 50% inhibition). However, the results suggest that the risk of EAC may decrease by increasing dietary α-tocopherol intake.
Omeprazole at 1,400 ppm reduced EAC incidence from 84% to 64% in the current study, which suggested a weak tumor inhibitory effect. We did not observe any unusual weight loss or high death rate in this group of animals. The pH data showed that omeprazole effectively inhibited acid secretion at the given dose. The data we showed here support our hypothesis that omeprazole does not promote EAC and may have weak tumor inhibitory effect. There have been concerns on the side effects of long term PPI treatment related side effect. Hypergastrinemia may induce epithelial proliferation, and bile acids in a neutral refluxate may induce DNA mutations in esophageal epithelium.20, 21
PPI treatment may also induce squamous re-epithelialization which covers more advanced malignancies in Barrett's glands located in submucosa.31, 32
Our data showed that PPI itself was not a strong chemopreventive agent. In order to achieve more pronounced chemopreventive effect, PPI may be applied in combination with other agents. The ongoing ASPECT Trial (Aspirin Esomeprazole Chemoprevention Trial) using PPI in combination with aspirin may answer this question.33
4-HNE is an extensively studied lipid peroxidation product, which is diffusible and can react with DNA bases and proteins.34
It is also an inducer of cyclooxygenase-2 and a mediator of oxidative stress.35, 36
It has been reported that enhanced lipid peroxidation and 4-HNE production may play a role in the recruitment of inflammatory cells.37
4-HNE has also been reported to form DNA adducts at codon 249 of the p53
gene and to inhibit nucleotide excision repair through interaction with cellular repair proteins.38, 39
In the present study, we found strong positive staining in the infiltrating inflammatory cells. We only found background staining in the nearby epithelial cells. This phenomenon may be explained by: 1) lipid peroxidation level is higher in the infiltrating inflammatory cells than the epithelial cells; 2) the method of immunohistochemistry we used is not sensitive enough to detect the 4-HNE adduct in the epithelial cells; 3) high level of 4-HNE in the infiltrating inflammatory cells may act on the nearby epithelial cells by diffusion.
In summary, our results lend support to the oxidative stress hypothesis of esophageal adenocarcinogenesis. α-Tocopherol alone or in combination with NAC significantly reduced tumor yield. The cancer preventive activities of tocopherols and NAC warrant further investigations.