Epithelial apoptosis in the gastro-intestinal tract is normally restricted to superficial cells but in pathological states of inflammation or infection, apoptotic cell death can be far more expansive. Under these conditions apoptosis may result from the production of cytokines 1, cell activation 234, infective agents 5 and adverse responses to pharmaceuticals 67. Depending on the agonist or eliciting milieu, apoptotic cell death is accompanied by the activation of various cell death pathways including caspases, ceramide, altered gene expression, mitochondrial dysfunction and consumption of ATP e.g., with DNA repair, that result in histone-associated DNA fragmentation 89. Many of these cell death pathways are also associated with the secondary production of oxidants 10 which appears to contribute to the magnitude of cell death. For example, over-expression of antioxidants like intracellular SOD reduces cell death 11, or alternatively, depletion of antioxidant reserves exacerbates cell death to a variety of stimuli 12.

Despite the knowledge that apoptosis is increased in gut inflammation, either from idiopathic causes like inflammatory bowel disease 89 or associated with infections like H. pylori13, little has been done to examine the effects of antioxidants on epithelial cell death. We have previously demonstrated an excessive degree of apoptosis in patients with gastritis induced by H. pylori, and that the degree of apoptosis could be reduced by dietary supplementation with vitamin C, irrespective of whether the infection was cleared or not 13. This observation was suggestive that dietary antioxidants could limit the gut pathology by limiting cell death mechanisms. We have also demonstrated that mesalamine (5-aminosalicylic acid), used as a frontline treatment for inflammatory bowel disease, could prevent apoptotic epithelial cell death in response to peroxynitrite 14. Peroxynitrite is a potent oxidant; formed from two free radicals – nitric oxide and superoxide – that catalyzes the nitration of tyrosine residues. The resultant nitrotyrosine is used as a marker of peroxynitrite or at least nitration reactions in gastritis 13, inflammatory bowel disease 1516 as well as general inflammation 17. While we have demonstrated that peroxynitrite-induced apoptosis of macrophages and colon epithelial cells is reduced by ascorbic acid 18, it is not known if other dietary antioxidants exert similar benefits or if they are present in epithelial cells from the upper gastrointestinal tract. However, flavonoids and related phenolic compounds have been described to be more effective than vitamin C in limiting DNA damage induced by nitrating/nitrosating species in cell free systems 19.

Altered intracellular redox balance with depletion of antioxidant reserves is associated with the activation of transcription factors like NF-κB, and genes (TNFα, IL-1β) associated with apoptosis, proliferation and inflammation 3489. Indeed, dietary antioxidants like resveratrol 20, green tea 21, cucumin 22 and cat's claw 2324 have been shown to inhibit gene expression associated with inflammation and states of immune activation. From this one could postulate that the benefits of diets rich in antioxidants could be in part due to a reduction in signals leading to apoptosis and cell activation. This possibility was addressed in this study by examining the ability of three antioxidants, vitamin C and two decoctions commonly used in Asia and South America (green tea and cat's claw) to limit epithelial cell apoptosis in response to a variety of oxidative stresses. The latter two forms of antioxidants were chosen because the antioxidant and anti-inflammatory phytochemical constituents (polyphenols, flavanols) are distinct from ascorbate but yet there is a growing appreciation for their role in managing disorders associated with oxidative stress.

The South American ethnomedicine, Cat's claw (Vinicol™), Uncaria guianensis, was obtained as a freeze dried concentrate from Rainforest Phytoceuticals, LLC, Delmar, NY. This concentrate is prepared in accord with ethnomedical traditions, where a decoction is made from the bark, and the solutes concentrated by freeze drying the decoction. The subspecies Uncaria guianensis was used over Uncaria tomentosa, as it is a more potent anti-inflammatory agent and has negligible amounts of alkaloids. A green tea extract was prepared from commercial sources by a decoction method. Briefly, dry leaves (2.5 g) were added to 100 ml of boiling water and steeped for 15 min. The infusion was cooled down to room temperature, decanted and total solids separated by filtration with a Whatman N° 4 filter paper. The filtrate was then freeze-dried using a Freezemobile 6 concentrator (Virtis, Gardiner, NY). The light brown solid extract was then used for all subsequent studies. For spectrophotometric analysis and cell culture studies the extract was filtered at 20 μm and 0.2 μm, respectively. Vitamin C (ascorbic acid) was purchased from Sigma Chemical Company, St. Louis, Missouri, USA.

From this study it is clear that concentrations of oxidants that elicit minimal to minor degrees of epithelial cell necrosis can promote significant apoptotic cell death. This result is not specific to a particular oxidant, as a comparable response was observed with the three distinct oxidants – peroxynitrite, hydrogen peroxide, and the free radical DPPH. In each case, these oxidants caused significant apoptosis and cell death which was dramatically reduced by the addition of dietary antioxidants. With the exception of vitamin C, the potency of these antioxidants as cytoprotective (anti-apoptotic) agents, appears to exceed their inherent ability to quench the specific oxidant stimulus. In other words, at concentrations of green tea or cat's claw that had minimal quenching effects on the oxidant signal itself, a significant benefit to cellular systems was seen. This may reflect the combined contributions of intracellular antioxidants with the exogenous antioxidant. However, if that were the sole explanation, the rank order of potency would not change when switching from cell free to cellular systems. Ascorbate would remain more effective, rather than being the weakest of the antioxidants studied to limit apoptosis (in AGS cells). It appears that other factors influence the anti-apoptotic activity of antioxidants.

We chose to evaluate cat's claw and green tea for several reasons. Beyond being extracts and hence they are made up of a collection of phytochemicals, in contrast to vitamin C, these "teas" are used for health maintenance in various cultures. Recently, both green tea and cat's claw have been reported to reduce the expression of inflammatory genes; responses that are linked to apoptosis and proliferation 2324252627. Indeed, cat's claw has been described as a potent inhibitor of the transcription factor NF-κB activation, preventing the expression of TNFα and inducible nitric oxide synthase 2324. These immune/inflammatory products are associated with gastritis and epithelial cell death 56713 and hence cat's claw may have utility in limiting gut inflammation 23. Indeed, this matches its ethnomedical use in South America where it is used to treat gastritis and other forms of chronic inflammation like arthritis 28. Green tea, and its constituent polyphenols, has been associated with reduced expression of inflammatory genes and signal transduction processes leading to cell death 21252627.

We focused on epithelial cells of the upper gastrointestinal tract because the sites of highest epithelial exposure to these dietary antioxidants would be the stomach and small intestine following ingestion. While it is likely that these antioxidants are absorbed and exert actions from the vascular space systemically (hence applications in arthritis), knowledge of their pharmacokinetics is limited. Given that gastritis, whether it is induced by infection with H. pylori or toxicity responses to NSAIDs, is associated with NF-κB activation, TNFα expression and apoptosis in epithelial cells 567, strategies to limit these responses may have significant therapeutic potential. This is particularly true if the therapeutic modalities are available to a wide section of the population for little expense. However, the doses of antioxidants used in this study are above that noted with diet or simple tea consumption and are more reflective of therapeutic intervention based on dietary supplementation.

Gastritis associated with persistent H. pylori infection is associated with an increased incidence of gastric cancer. Whether dietary antioxidant supplementation retards this expression has been an appealing hypothesis. Several lines of evidence support this contention. Correa et al 29 have clearly shown in a large trial conducted over six years that dietary antioxidant supplementation alone (vitamin C or beta-carotene), without antimicrobial therapy, can limit the progress of gastric cancer and actually may promote regression of precancerous states. Additionally, it appears that the population sub-groups at greatest risk for gastric cancer have a limited ability to secrete ascorbic acid from the plasma through the mucosal and into the gastric lumen 30. In a related study, supplementation with vitamin C reduces the degree of apoptosis and nitrotyrosine immunohistochemistry 13, a marker of peroxynitrite formation 15. NF-κB is activated in gastric epithelia with H. pylori infection, and clearance of this infection reduced NF-κB levels as well as expression of inducible nitric oxide 513, thereby reducing the mucosal burden of nitric oxide and possibly peroxynitrite. While clearance of the infection is curative, much of the world's population cannot afford the therapy, and further clearance of the infection with antimicrobial therapy is not guaranteed. Thus, treating the host response to the infection with common, readily available antioxidants may afford some degree of protection. In contrast, a recent study in Japan indicated that the consumption of green tea was not associated with either a reduction or an increase in gastric cancer rates 31. This raises concern as to the ability of polyphenols derived from green tea to afford protection from mutagenic substances or limit the progression of established cancer. However, the discrepancy is more likely a reflection that higher doses of polyphenols are required to achieve benefit than that which is achieved with simple ingestion of tea. This is evident when one evaluates the concentrations required to achieve anticancer, cytoprotective actions in vitro 1921252627. More recently in the Apc^min mouse model of familial adenomatous polyposis, a condition with a massive induction of polyps in the gastrointestinal tract, green tea extracts reduced polyp load and potentiated the effects of sulindac, a cyclo-oxygenase inhibitor used to treat gastrointestinal cancer 32. This further affirms the potential of this strategy in managing disorders characterized by epithelial cell death and transformation.

The gastric mucosal responses to non-steroidal anti-inflammatory drugs also involve the activation of NF-κB, generation of cytotoxic cytokines like TNFα and epithelial apoptosis 67. Administration of low concentrations of nitric oxide by an ester linkage reduces this effect, suggesting that nitric oxide was offering benefit by quenching other radicals involved in cell activation. Under these circumstances, could co-administration of dietary antioxidants also limit the toxicity of the pharmaceutical? If so, this approach could offer significant cost savings over the use of newer cyclo-oxygenase 2 (COX2) inhibitors. In a recent paper by Laine 33, it was calculated that although COX2 inhibitors reduced the incidence of gastrointestinal damage when compared to non-selective COX inhibitors, these benefits were achieved at a significant increase in health care costs (approximately $560 per patient per year). In contrast, we have reported that cat's claw prevents the significant enteropathy associated with sustained use of indomethacin, a non-selective COX inhibitor 23, and this combination could alleviate the gastrointestinal damage without the high costs of COX2 inhibitors.

In the present study, oxidant stress was induced by signals that are largely hydrophilic. In other words apoptosis was not provoked with lipid peroxides. As the chosen antioxidants were hydrophilic, a significant interaction was anticipated. However, these antioxidants may be less effective against a lipid peroxide generating system, and under those conditions an antioxidant like vitamin E may be more effective. However, lipid hydroperoxide-induced apoptosis is accompanied by an alteration in the intracellular redox balance leading to a depletion of intracellular thiols and subsequent activation of caspases and PARP cleavage 33, responses that would be attenuated by hydrophilic antioxidants.

Peroxynitrite is a potent oxidant that can induce lipid peroxidation and as such may provide important insight. However, endogenous production of peroxynitrite remains controversial. Nitration of tyrosine residues is used as an index of peroxynitrite formation but it is not specific 13. However, the purpose of using peroxynitrite in this study was to address the effects of a potent oxidant and not to evaluate the role of peroxynitrite in pathological states. Indeed some oxidants may be environmental and not produced endogenously but yet antioxidant supplementation may provide therapeutic utility.

In the present study, AGS cells (human gastric epithelial cells) were used. AGS are a transformed cell line, consequently, one could argue that dietary antioxidants could prevent the elimination of these transformed cells by the mucosal immune response. Indeed that is a concern that has been raised following the failure of prospective antioxidant trials in smokers to limit the incidence/progression of lung cancer 35 and the recent epidemiological analysis of gastric cancer incidence and green tea consumption 32. However, other evidence has indicated that the antioxidants, vitamin E or N-acetylcysteine, enhanced the ability of the chemotherapeutic agent 5-fluorouracil to induce apoptosis in colon cancer cells 36. These in vitro observations are supported by the Apc^min animal model 32 and sustained clinical trial in patients with H. pylori gastritis 30 suggesting that dietary antioxidants do not exacerbate precancerous states but may indeed promote regression. In addition, cells of a lymphoid origin where NF-κB is constitutively activated, inhibition of NF-κB confers complete protection to hydrogen peroxide and pervanadate, but rendered HeLa cells more susceptible to apoptosis induced by TNFα 37.

Dietary antioxidants confer significant protection to gut epithelial cells from pro-apoptotic oxidant stress. The phytochemical mixtures found in the teas, cat's claw and green tea, appear to be more effective than vitamin C in some cell lines and at concentrations that suggest that they may be acting at levels distinct from the mere scavenging of the oxidant signal. Diet supplementation with these or related antioxidants may prove valuable in limiting the pathophysiology of numerous disorders associated with gut inflammation.

There are no competing interests for BK Reuter or F. Angeles. Both MJS Miller and M Sandoval have financial interests in Rainforest Phytoceuticals, LLC who supplied the cat's claw used in this study.