The results from this pilot, randomized, controlled clinical trial suggest that supplementation with calcium or vitamin D3
, but not with both agents combined, may decrease oxidative DNA damage, as indicated by decreased 8-OH-dG immunohistochemical labeling, in the normal-appearing colorectal epithelium of sporadic adenoma patients. These findings are consistent with the hypothesis that high intakes of calcium or vitamin D3
may decrease oxidative stress and oxidative DNA damage in the colon, and, thus, reduce risk for colorectal neoplasms. Our findings also suggest that vitamin D3
combined with calcium may have either a lesser or no treatment effect on 8-OH-dG labeling than does either calcium or vitamin D alone. Consistent with existing animal data (15
), we found evidence that baseline VDR (vitamin D receptor) expression levels may modify treatment effects of calcium and vitamin D3
, such that those with higher colorectal crypt VDR expression may be more strongly responsive to treatment. Finally, the treatment effect of calcium and vitamin D3
tended to be stronger in men and those with higher baseline anti-oxidant relative to pro-oxidant exposures.
Oxidative stress, a condition characterized by an imbalance of pro-oxidants to antioxidants which results in macromolecular damage and disruption of redox signaling and control (28
), may play a role in colon carcinogenesis, inducing protein and DNA damage and lipid peroxidation, and impairing intracellular signaling. Under normal conditions, reactive oxygen species (ROS) have an important role as intracellular signaling molecules that regulate many genes (29
). However, under inflammatory conditions, increased generation of ROS products leads to cell molecule damage such as oxidation of DNA (29
). The most abundant product of oxidative DNA modifications by ROS is 8-hydroxy-2′-deoxyguanosine (8-OH-dG) (30
). This oxidized base is a useful biomarker of oxidative stress that can be measured in urine, blood, and tissues (19
). Several studies demonstrated increased levels of oxidatively modified DNA in colorectal adenocarcinomas when compared to adenomas and adjacent normal epithelium (32
). This suggests that inhibition of oxidative stress in the normal colorectal epithelium may slow down or prevent carcinogenesis, and prompts the development of chemopreventive agents, such as calcium and vitamin D, that target oxidative stress in the colon.
There are several lines of evidence to support our hypotheses that calcium and vitamin D may act as antioxidants and DNA damage reducing agents in the colon. Bile acids damage cell membranes, at least in part through an oxidative mechanism (34
), provoking an inflammatory response and causing DNA damage (36
), and both calcium and vitamin D can reduce the free bile acid load in the colon lumen. Calcium directly binds bile acids, rendering them inert (37
). Vitamin D activation of the ubiquitous vitamin D receptor (VDR) in the colon up-regulates CYP3A4, which in turn catabolizes the secondary bile acid, lithocholic acid (38
). Furthermore, high blood 25-(OH)-vitamin D levels provide a pool of vitamin D that is available for various tissues, such as the colorectal epithelium. In colonocytes, vitamin D increases expression of enzymes involved in antioxidant response, inhibits iron-dependent lipid peroxidation in liposomes, lowers glutathione reductase levels, induces glutathione peroxidase and manganese dependent superoxide dismutase activity, and elevates glutathione levels ((16
), also reviewed in (14
)), thereby decreasing oxidative stress in the colorectal epithelium. The results of this study, combined with the biological evidence, support calcium and vitamin D3
as oxidative DNA damage reducing agents.
Contrary to our original hypothesis and to what has been described in some epidemiologic and clinical studies (41
), we did not observe a treatment effect in the calcium plus vitamin D group. We also previously reported that vitamin D combined with calcium may have lesser treatment effects on colorectal epithelial apoptosis and differentiation than does calcium or vitamin D separately (17
). There are several possible explanations for this finding. Considering the study’s small sample size, the lack of treatment effect in the calcium and vitamin D group may have been due to chance. It is also possible that the two agents may have attenuated the effects of one another. 1,25-(OH)2
regulates calcium homeostasis (47
). As calcium concentration decreases, the production of 1,25-(OH)2
increases, which in turn increases intestinal calcium absorption (47
). Elevated calcium in the diet may suppress 1,25-(OH)2
synthesis at the cellular level, which in turn may also attenuate activation of vitamin D-responsive detoxifying enzymes. One animal study (48
) found that calcium and vitamin D were more potent inhibitors of colon tumorigenesis when given separately, but several other animal studies reported synergistic effects with calcium and vitamin D combined (49
). A large adenoma recurrence trial also supported an enhanced chemopreventive effect of vitamin D with calcium (42
). Taken altogether, the combined effect of calcium and vitamin D on oxidative DNA damage in colorectal epithelium is unclear and will require clarification via larger studies.
In contrast to as in men, there was no evidence for a treatment effect of vitamin D alone and in combination with calcium on colorectal crypt 8-OH-dG labeling levels in women. There are several possible explanations for this finding, including a very low statistical power to detect treatment effects due to the small sample size. Another possible explanation may be that women in our study may have had decreased estrogen levels as the majority of them were postmenopausal and not taking estrogens. The Women’s Health Initiative Hormone Replacement Therapy Trial (51
) found that endogenous estrogen plus progestin therapy, but not estrogen alone therapy, reduced risk for colorectal cancer (52
). However, one human study found that an estrogen intervention activated the VDR pathway, and downregulated inflammatory and immune signaling pathways in the rectal mucosa of postmenopausal women (53
). So, the findings of our study are consistent with the hypothesis that low estrogen levels may interfere with VDR signaling in the colorectal mucosa, resulting in no changes in 8-OH-dG levels after supplementation with vitamin D; however, further studies are needed to clarify these issues.
Epidemiologic studies have not consistently found associations of colorectal neoplasms with individual pro- and anti-oxidant factors despite the strong biological rationale and basic science evidence. Several analyses (25
) suggested that oxidative stress, inflammation, diet, and other risk factors may synergistically or individually affect risk of colorectal neoplasia through multiple pathways. Therefore, we used the OBS to categorize patients into different “oxidative stress” profiles based on multiple determinants of oxidative stress. We hypothesized that patients with different “oxidative stress” profiles may respond differently to calcium and/or vitamin D. Those with a high baseline OBS (higher balance of anti- to pro-oxidant exposures) had greater estimated calcium and calcium plus vitamin D treatment effects on 8-OH-dG labeling than those with a low OBS. A low OBS reflects low total intakes of antioxidants such as vitamin C and carotene, combined with high pro-oxidant exposures such as high fat or iron intakes. In the colon lumen, free calcium directly binds bile acids (37
), thereby reducing pro-carcinogenic effects of bile acids on the colorectal epithelium. Persons with high fat intake have higher colonic lumen levels of deoxycholic and lithocholic bile acids (36
), and may require more calcium to neutralize the DNA damaging bile acids than do persons on a low-fat diet. Antioxidant enzymes in humans function in combination with low weight antioxidant compounds such as vitamin C, α-tocopherol, and β-carotene (54
). In the colorectal epithelium, vitamin D activates the expression of antioxidant enzymes (14
), which may not function properly in the antioxidant-depleted environment. Therefore, it is possible that calcium and vitamin D effects on the oxidative DNA damage marker, 8-OH-dG, are modified by the presence or absence of various pro- or antioxidant exposures. On the other hand, our findings may have been due to chance and further investigations are required to confirm them.
Since complete loss of the VDR significantly increased 8-OH-dG labeling in the mouse colon (15
), we hypothesized that different VDR expression levels in the normal-appearing colorectal mucosa modify vitamin D treatment effects. Consistent with this hypothesis, we observed substantial decreases in 8-OH-dG labeling in study participants with high, but not low, baseline VDR expression.
This study has several limitations. First, treatment effects of vitamin D and calcium on the oxidative DNA damage marker 8-OH-dG in parts of the colon other than the rectum are unclear, as we did not collect tissue biopsies from different parts of the colon and there are no published studies of 8-OH-dG labeling throughout the colon. Another potential limitation of this study is that it is not known whether oxidative stress markers are associated with risk for colon cancer in humans. However, substantial published literature supports the plausibility of an important role for increased oxidative DNA damage in colon carcinogenesis, especially for the transition from colorectal adenoma to carcinoma (32
). Persistent oxidative stress leads to protein and DNA damage and lipid peroxidation which can cause genetic and epigenetic alterations, and may facilitate the development of neoplasia from the normal colorectal mucosa (29
). Therefore, 8-OH-dG in the normal colorectal mucosa may serve as a biomarker of risk for colorectal neoplasms. Finally, the most obvious limitation of the study is the small sample size, which may have increased the probability of chance findings in detecting or not detecting a treatment effect.
The strengths of this study include the randomized, double-blind, placebo-controlled trial design; high protocol adherence by study participants; examination of both the independent and combined effects of calcium and vitamin D3
on an oxidative stress marker; automated standardized biopsy handling and immunostaining procedures; and the use of cutting edge technologies to conduct the quantitative image analyses. Another strength of this study is that we used immunohistochemical detection of 8-OH-dG in the colorectal epithelium as it was important to detect 8-OH-dG in colonocytes, but not in infiltrating lymphocytes or other intermingled cells. Such detection was made possible by the development of a specific monoclonal antibody against 8-OH-dG (19
), and our novel image analysis methods. HPLC (high-performance liquid chromatography), an alternative method of measuring 8-OH-dG in colon tissue, may overestimate oxidative DNA damage in the colonocytes, especially in the presence of inflammation. Finally, this study is the first human study to test the effect of calcium and/or vitamin D3
on an oxidative DNA damage marker in the normal-appearing colorectal mucosa.
Overall, these preliminary results from this pilot clinical trial suggest that calcium and vitamin D, given separately, may decrease oxidative DNA damage in the normal-appearing colorectal epithelium; the treatment effects of calcium and vitamin D on oxidative DNA damage marker 8-OH-dG may be strongest in those with higher vitamin D receptor expression in the colon; 8-OH-dG may be a modifiable biomarker of oxidative stress that can be used in colon cancer-related chemoprevention trials to assess treatment efficacy; and support further investigations of calcium and vitamin D as chemopreventive agents against colorectal neoplasms.