Vol. 12, 559 –565, June 2003
Cancer Epidemiology, Biomarkers & Prevention
Vitamin C Intake and Apoptosis in Normal Rectal Epithelium1
Alexandra E. Connelly,2 Jessie Satia-Abouta,
Christopher F. Martin, Temitope O. Keku,
John T. Woosley, P. Kay Lund, and Robert S. Sandler
colorectal cancer risk, vitamin C supplements may be
contraindicated for patients with a history of adenomas.
Departments of Epidemiology [A. E. C., C. F. M., R. S. S.], Nutrition [J. S-A.],
Medicine [C. F. M., T. O. K., R. S. S.], Pathology [J. T. W.], and Molecular
and Cellular Physiology [P. K. L.], University of North Carolina at Chapel
Hill, Chapel Hill, North Carolina 27599-7555
Introduction
Apoptosis, or programmed cell death, is the ability of a cell to
self-destruct by activating an intrinsic cell suicide program.
This mechanism evolved in multicellular organisms as a way to
eliminate genetically damaged cells (1). Various illnesses may
develop because of increased apoptosis, such as neurodegenerative diseases and immunodeficiency disorders (2). Others,
such as cancer and autoimmune disorders, may be a consequence of decreased apoptosis (3– 6).
Apoptosis is accomplished through a multistage chain of
reactions mediated by ROS (7). Apoptotic signals, which arise
in genetically damaged cells, promote the accumulation of p53
protein, which triggers mitochondrial release of ROS3, cytochrome c, and other regulators. These regulators activate a
cascade of caspases (proteolytic enzymes) that digest a number
of critical cell proteins and promote a caspase-activated Dnase,
eventually resulting in apoptotic cell death (8).
Tissue homeostasis is a balance between maturation of new
cells and elimination of older or genetically damaged cells.
Apoptosis plays a key role in this process, routinely responding to
stimuli such as irreparable cell damage. Neoplasia results from a
disruption of this balance and may arise when there is an abnormal
accumulation of altered or genetically damaged cells, either due to
increased proliferation or decreased apoptosis (4, 5). Apoptosis
provides a protective mechanism to remove these genetically damaged cells, decreasing the odds that abnormal cells will proliferate
(5, 6). In fact, evasion of apoptosis has been described as one of the
“six capabilities” of the cancer cell (9). In addition, apoptosis may
increase the body’s ability to rid itself of tumor cells that have
gained access to the circulation (10).
Antioxidant vitamins such as vitamin E, vitamin C, selenium,
and zinc have been suggested as potential anticancer agents because they fight free radicals, which may cause oxidative damage
to DNA, possibly leading to development of cancer. This hypothesis was central to several cancer chemoprevention trials of vitamin C, vitamin E, and ␤-carotene in the late 1980s and early
1990s, including the ␣-Tocopherol, ␤-Carotene Cancer Prevention
Study (11) and the ␤-Carotene and Retinol Efficacy Trial (12).
More relevant to the current study are several trials that tested the
efficacy of antioxidants to prevent adenomatous polyps, such as
the Polyp Prevention Trial (13) and the Australian Polyp Prevention Project (14), and a randomized trial from Toronto (15). Most
of these trials, unexpectedly, did not see a reduction in cancer
incidence or polyp recurrence in the antioxidant-supplemented
group (11–15).
Recent research has suggested a possible explanation for
Abstract
Apoptosis, or programmed cell death, may lower the risk
of neoplasia by removing genetically damaged or mutated
cells. A high rate of apoptosis has been linked to a
reduced risk of colorectal adenomas; therefore, it is
important to understand factors that impact apoptosis.
Antioxidants (e.g., vitamin C) protect cells from harmful
oxidation processes but may interfere with apoptosis by
protecting genetically damaged cells from reactive oxygen
species-dependent cell death. The objective of this study
was to evaluate the association between vitamin C
intake and apoptosis in normal rectal mucosa. Study
participants were part of a large, cross-sectional study,
the Diet and Health Study III. Participants were
recruited from consecutive, consenting patients who
underwent colonoscopy at University of North Carolina
Hospitals between August 1, 1998 and March 4, 2000.
Vitamin C intake, obtained from a food frequency
questionnaire, included both dietary sources and vitamin
supplements. Apoptosis was measured by morphological
evaluation of H&E-stained sections obtained from pinch
biopsy samples of normal rectal mucosa in consenting
participants (n ⴝ 503). The relationship between vitamin
C and apoptosis varied by adenoma status. Among
individuals with adenomas, there was an inverse linear
association between apoptosis and total vitamin C intake.
Similarly, individuals with adenomas in the highest
quintile of total vitamin C intake were substantially less
likely than those in the lowest quintile to have increased
colonic apoptosis (odds ratio, 0.05; 95% confidence
interval, 0.01– 0.46). Vitamin C was not significantly
associated with apoptosis in adenoma-free patients. High
vitamin C intake was associated with reduced colorectal
apoptosis among individuals with adenomas in this study
population. Given that high apoptosis may lower
Received 9/13/02; revised 3/4/03; accepted 3/10/03.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported in part by NIH Grants R01 CA44684, T32 DK 07634, and P30
DK34987.
2
To whom requests for reprints should be addressed, at Department of Epidemiology, CB# 7555, University of North Carolina, Chapel Hill, NC 275997555. Phone: (919) 843-3649; Fax: (919) 966-7592; E-mail: aconnell@
e-mail.unc.edu.
3
The abbreviations used are: ROS, reactive oxygen species; OR, odds ratio; CI,
confidence interval; TUNEL, terminal deoxynucleotidyl transferase-mediated
nick end labeling; DHS III, Diet and Health Study III; UNCH, University of North
Carolina Hospital; FFQ, food frequency questionnaire; BMI, body mass index.
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the null or negative results from antioxidant chemoprevention
trials. Although free radicals (cellular oxidants, a subset of
ROS) are usually described as harmful to cellular systems, they
are also critical mediators of antimicrobial phagocytosis, detoxification reactions, and apoptosis (8). Because apoptosis is a
pathway by which precancerous and other potentially dangerous cells are eliminated, antioxidants could interfere with the
natural elimination of harmful or unhealthy cells by preventing
ROS-dependent apoptosis. Therefore, high antioxidant levels
might actually reduce anticancer apoptotic activity, particularly
in persons with low levels of endogenous ROS (7, 8, 16 –19).
The notion that antioxidants may interfere with the apoptotic
process may explain, at least in part, why recent chemoprevention trials have yielded surprisingly null results.
The purpose of the present analysis was to investigate the
association between vitamin C, a potent antioxidant, and apoptosis. Specifically, we undertook an analysis to examine
whether high intake of vitamin C is associated with reduced
apoptosis in normal colonic mucosa.
Materials and Methods
Study Population. The study population was the DHS III, a
large, cross-sectional study conducted at the UNCH (Chapel
Hill, NC). UNCH is a 700-bed university hospital serving as the
primary health care facility for residents of Orange and adjacent
counties and as a referral center for much of the state. Patients
are diverse with respect to socioeconomic status and race/
ethnicity.
The DHS III Study was designed to investigate dietary and
lifestyle factors associated with colorectal adenomas, generally
believed to be precursors of colorectal adenocarcinomas. Study
participants were recruited from consecutive, consenting patients who underwent colonoscopy between August 1, 1998 and
March 4, 2000. Eligibility requirements included: age ⱖ 30
years; ability to understand and complete an interview in English; and no evidence of familial polyposis, colitis, previous
colonic resection, or previous colon cancer or adenoma. Patients who did not satisfactorily prepare the colon for the
procedure were excluded, as were those with incomplete visualization from the colon to the cecum. Eligible patients were
asked to allow the colonoscopist to obtain rectal biopsy specimens from normal mucosa, in addition to subsequently completing a telephone-administered interview.
Data Collection. During the enrollment period, 2452 outpatient colonoscopies were performed at UNCH; 1526 potential
participants were excluded based on eligibility or an unsatisfactory procedure. Reasons for exclusion were not mutually
exclusive and included previous adenomas (n ⫽ 615), colitis
(n ⫽ 405), previous colon resection (n ⫽ 311), younger than
age 30 years (n ⫽ 267), previous colon cancer (n ⫽ 220),
incomplete examination (n ⫽ 182), inability to give informed
consent (n ⫽ 156), unsatisfactory preparation (n ⫽ 149), polyposis (n ⫽ 6), and other factors (n ⫽ 73). Among the 926
remaining eligible participants, 57 (6.2%) refused, and 66
(7.1%) were not asked because the research assistant was unavailable, leaving 803 (93.4%) who consented to participate in
the study. Of the 803 consenting patients, 503 also agreed to the
rectal pinch biopsies. Therefore, the final sample size for this
analysis was 503.
All biopsies were obtained 8 –10 cm from the anal verge
using standard (8-mm wing) disposable, fenestrated colonoscopy forceps with a central spike (Wilson-Cook, WinstonSalem, NC), yielding a specimen 3– 4 mm in diameter. Biopsies
were taken from normal-appearing mucosa, avoiding raised
lesions or larger blood vessels.
Apoptosis was measured from biopsy specimens in two
ways: (a) strict morphological criteria (examination of H&Estained sections under light microscopy); and (b) TUNEL staining. In the first method, two biopsies were placed in 10%
buffered formalin for routine histology and prepared with H&E
stain. Each prepared slide had five levels of tissue sectioned at
least 50 ␮m apart, from which 8 –12 well-oriented colonic
crypts were scored (per biopsy). Apoptotic cells were identified
by chromatin condensation at the nuclear boundary, membrane
blebbing, and cytoplasmic shrinkage. An experienced technician, blinded to adenoma status, scored all sections. The scoring
technique had a reproducibility of 99%. Apoptosis was scored
as the number of apoptotic cells/colonic crypt. For each patient,
the mean number of apoptotic cells was derived by taking the
average number of apoptotic cells from the two biopsy specimens (i.e., from a total of 16 –24 well-oriented crypts). A
second method, TUNEL staining, was also used; however,
recent studies have cited problems with this assay (20, 21).
Prolonged tissue fixation, tissue handling, increased nonspecific staining, need to optimize (to avoid over- and understaining), and specificity are some of the problems that may interfere
with the TUNEL assay and lead to inaccurate determination of
apoptosis. For these reasons, we feel that morphological identification of apoptosis is a better reflection of apoptosis than
TUNEL staining, and therefore we used the morphological
measure of apoptosis in the present analysis. We also note that
the relationship between vitamin C and apoptosis was essentially the same when apoptosis was measured with TUNEL as
it was when measured using histological measures.
Dietary information (i.e., dietary vitamin C and total energy intakes) was obtained using a modified version of the
Block-NCI FFQ (22). The version of the Block quantitative
FFQ used in this study was specially modified for use in North
Carolina to include regional foods (23). The FFQ was administered by telephone within 12 weeks of colonoscopy, and the
reference period for dietary intake was in the past year, to
account for seasonal variations in diet. We elected to examine
vitamin C rather than other antioxidants (e.g., vitamin E or
selenium) because intake of vitamin E from foods is relatively
small, and selenium consumption may not be well captured by
FFQs (24).
Supplement use was assessed with closed-ended questions
about the use of multiple vitamins (e.g., Centrum and other
antioxidant combinations) as well as single vitamin C supplements. We inquired, specifically, about duration (in years),
frequency (days/week), and usual dose (e.g., 100, 250, 500,
750, or 1000 mg) of vitamin C supplements over the previous
year. Dietary vitamin C intake, calculated by the BLOCK
analysis program, was combined with supplemental intakes to
obtain total vitamin C intake.
Statistical Analysis. Univariate analysis was used to examine
the distributions, delete outliers, and identify implausible values of apoptosis and vitamin C intake. The linear relationship
between vitamin C and apoptosis was investigated using multiple linear regression analysis. Candidate confounders for potential inclusion in models were identified by comparison of
means of vitamin C intake and apoptosis between levels of each
potential confounder (age, sex, race, nonsteroidal anti-inflammatory drug use, smoking, total energy, and BMI) in bivariate
analyses. If a statistically significant difference in mean vitamin
C intake and apoptosis across levels of the confounder was
observed, that variable was further assessed, using a change in
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Cancer Epidemiology, Biomarkers & Prevention
estimate approach, in subsequent modeling. We also evaluated
effect measure modification by presence or absence of adenoma. Multiple linear regression modeling was conducted
using a backward elimination strategy. Apoptosis and total
vitamin C intake were log-transformed to fulfill the normality
assumption of the linear regression model.
Stratified and multiple logistic regression analyses were
conducted to investigate the relationship between vitamin C
intake and apoptosis. Logistic regression modeling was done by
backwards elimination. Potential confounders identified from
bivariate and stratified analysis were assessed as confounders of
the multivariate model using a change in main effect estimate
of 20%. The log likelihood test was used to identify effect
modifiers. Logistic regression was used to model the odds of
high apoptosis (higher than the study population median value
for apoptosis) in comparison with low apoptosis. Exposure
effects were separately assessed for dietary and for total vitamin
C intake in quintiles derived from the distribution of all participants in our study sample. The standard multivariate technique was used to adjust for total energy intake (25).
All analyses were conducted in SAS, version 8.1 (SAS
Institute, Cary, NC).
Results
The mean age of participants was 56 years (SD ⫽ 11 years),
36% were 60 years or older, 40% were male, almost threequarters were white, and 31% were obese (BMI ⱖ 30 kg/m2).
One hundred and seventy participants (35%) had at least one
colorectal adenoma. The mean total vitamin C intake was 313
mg/day (range, 20 –1589 mg/day), and the mean dietary vitamin C intake was 191 mg/day (range, 19 – 656 mg/day). The
median score for apoptotic cells/crypt was 2.74 (range, 1.3–
5.3). There is no standard for high or low apoptosis in human
tissue; therefore, high or low apoptosis was defined as above or
below this study sample median.
Table 1 gives mean total and dietary vitamin C intake and
mean number of apoptotic cells/crypt, stratified by demographic and health-related characteristics. There was a statistically significant difference in mean apoptosis between categories of total energy intake by adenoma status. Specifically,
respondents who consumed fewer than 1500 kcal/day (excluding alcohol) had lower apoptosis than those consuming more
than 1500 kcal/day. Participants with one or more adenomas
had lower rates of apoptosis than those without adenomas. In
addition, obese, overweight, and normal weight participants
had substantially lower rates of apoptosis than underweight
participants, all P ⱕ 0.05.
Mean total vitamin C intake also differed by certain participant characteristics. African Americans had statistically significantly lower mean daily vitamin C intakes than whites; men
consumed less total vitamin C than women; and current smokers reported lower intakes than former or never smokers. The
most striking difference was for BMI: participants with normal
BMI had significantly higher mean total vitamin C intakes (368
mg) in comparison with underweight or obese participants (194
and 251 mg, respectively). Respondents who consumed fewer
than 1000 kcal/day (excluding alcohol) also had lower intakes
of total vitamin C (208 mg) than those consuming more than
1000 kcal/day (338 mg).
Neither total nor dietary vitamin C intake was correlated
with apoptosis in the crude (unadjusted) analysis (Pearson’s r ⫽
0.03 and r ⫽ – 0.01, respectively). Bivariate analyses revealed
a U-shaped relation between quintiles of total vitamin C and
apoptotic activity; however, this association was not statistically significant (P ⫽ 0.55).
Total energy intake was the only confounder in both the
multiple linear and logistic regression models. After adjustment
for total energy, there was a decrease in apoptosis with increasing total vitamin C intake, but only among participants with
adenomas (␤ ⫽ ⫺0.0443; P ⫽ 0.06; Fig. 1). Among individuals
without adenomas, there was no significant linear association
between total vitamin C intake and apoptosis (␤ ⫽ 0.0059; P ⫽
0.70). Similar results were observed for dietary vitamin C
(adenoma, ␤ ⫽ ⫺0.0570 and P ⫽ 0.08; adenoma free, ␤ ⫽
⫺0.0023 and P ⫽ 0.92).
Table 2 shows the results from multivariate logistic regression analysis, adjusted for total energy intake. As in the
multiple linear regression analysis, the association between
vitamin C and apoptosis was confounded by energy intake and
modified by adenoma status. (Unadjusted results were in the
same direction but of lesser magnitude than energy-adjusted
results.) Among participants with adenomas, odds of high apoptosis decreased with increasing quintiles of total vitamin C
intake. The odds of high apoptosis were significantly lower in
the highest versus the lowest quintile of total vitamin C intake
(OR for the highest quintile, 0.05; 95% CI, 0.01– 0.46). Although most ORs were not statistically significant, higher total
vitamin C intake was associated with lower odds of high
apoptosis in both adenoma and adenoma-free participants. Patterns in dietary vitamin C intake were less clear.
Discussion
In this study of 503 men and women in North Carolina, we
found that total vitamin C intake was inversely associated with
apoptosis among participants with adenomas. Previous research
has suggested an association between inhibition of apoptosis
and colorectal cancer development (26, 27). Additionally, in a
previous analysis of this study population, low apoptosis was
associated with a 7-fold increased odds for the presence of
colorectal adenomas (28). This result implies that apoptosis
may be a very strong predictor of adenoma development, a
precursor lesion of colorectal cancer. Given this finding, it is
important to identify modifiable factors that influence human
apoptotic activity to develop risk reduction strategies for colon
cancer and possibly other cancers. Furthermore, many chemotherapy treatments work through apoptotic pathways, thus our
results could also be relevant to cancer treatment.
In light of our findings, the null results observed in three
published antioxidant polyp prevention trials (13–15) are not
surprising. Specifically, our results suggest that persons who
tend to develop adenomas may be more susceptible to a reduction in apoptosis following high antioxidant consumption. One
explanation for this effect could be lower levels of endogenous
ROS in persons prone to developing adenomas (8). If this is the
case, high antioxidant levels might actually reduce anticancer
apoptotic activity (i.e., ROS-dependent apoptosis) because of a
high ratio of antioxidants to oxidants in these individuals.
Because we had no measurements of endogenous ROS
levels, we could not investigate low ROS status as a possible
explanation for the stronger inverse association between vitamin C and apoptosis in those with adenomas. It is possible that
those at high risk of eliminating too many free radicals (because
of lower endogenous ROS levels) are at increased risk for
cancer at high levels of antioxidant consumption. This pathway
warrants future investigation.
We measured apoptosis in normal rectal mucosa, rather
than in the adenomatous tissue. The primary reason for this was
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Table 1
Mean apoptosis and total vitamin C intake by demographic and health-related characteristics of the DHS III population (n ⫽ 503)
Characteristic
N (%)
Mean no. of
apoptotic cells/crypt
Mean total
vitamin C intake
(mg/day)a
Mean dietary
vitamin C
intake (mg/day)
Age (yrs)
⬍45
45–59
ⱖ60
Pb
74 (14.9)
242 (48.8)
180 (36.3)
2.86
2.75
2.72
0.10
269
334
306
0.21
199
192
179
0.18
Ethnicity
African American
White
Other
Pb
119 (23.8)
364 (72.7)
18 (3.6)
2.79
2.75
2.78
0.84
210
346
332
⬍0.01
173
197
186
0.09
Gender
Male
Female
Pb
202 (40.2)
301 (59.8)
2.70
2.80
0.28
281c
335
0.04
201
184
0.08
Smoking status
Never smoker
Former smoker
Current smoker
Pb
245 (48.8)
167 (33.3)
90 (17.9)
2.76
2.70
2.88
0.12
330
341
215c
⬍0.01
193
208
143c
⬍0.0001
BMI (kg/m2)
⬍18.5 (underweight)
18.5–24.9 (normal)
25.0–29.9 (overweight)
ⱖ30.0 (obese/morbidly obese)
Pb
13 (2.6)
165 (33.3)
160 (32.3)
158 (31.9)
3.19c
2.80
2.76
2.70
0.08
194c
368c
330
251
⬍0.01
185
199
194
178
0.33
Total energy intake (kcal/day)
⬍1000
1000–1500
1501–2000
⬎2000
Pb
107 (21.3)
168 (33.4)
131 (26.0)
97 (19.3)
2.69
2.67
2.85
2.84
0.05
208c
331
361
332
⬍0.01
114c
179c
224
252
⬍0.0001
NSAID use (during past 5 yrs)d
Occasional/never
Regular user
Pb
224 (45.2)
272 (54.8)
2.81
2.72
0.17
314
313
0.96
191
190
0.91
Adenoma status
At least one adenoma
No adenomas
Pb
173 (35.0)
321 (65.0)
2.46c
2.93
⬍0.01
290
329
0.15
193
185
0.41
Colonoscopy preparation
Golytely
Phosphosoda
Pb
158 (31.6)
342 (68.4)
2.83
2.73
0.14
314
313
0.95
194
189
0.63
a
Vitamin C from dietary plus supplemental sources.
P for the overall F-statistic.
Statistically significant difference in means at the P ⬍ 0.05 level between this category and all other categories for this characteristic with Bonferroni correction for
multiple comparisons.
d
NSAID (nonsteroidal anti-inflammatory): regular use ⫽ 3 or more times/week, occasional use ⫽ less than 3 times/week.
Notes: numbers do not sum to 503 for all characteristics due to missing data; data in this table are unadjusted for other covariates.
b
c
to address one of the objectives of the DHS III Study: to
compare apoptosis in those with and without adenomas. Apoptosis was measured in tissue obtained from the same rectal
location in all participants, irrespective of whether or not they
had an adenoma. Previously, we found that low apoptosis was
strongly related to the presence of adenoma(s) in the colon (28).
This suggests a “field effect” in the colon because low apoptosis was associated with the presence of adenomas located
anywhere in the colon. Thus, a low rate of apoptosis may
indicate a higher risk for developing adenomas.
Findings from our previous work suggest that low apoptosis in normal rectal mucosa is a physiologically inappropri-
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Cancer Epidemiology, Biomarkers & Prevention
Table 2 Odds of high apoptosis (above the median) by quintile of total
vitamin C and of dietary vitamin C consumption
Total vitamin Cb
Q1 (20–119 mg/day)c
Q2 (⬎119–188 mg/day)
Q3 (⬎189–257 mg/day)
Q4 (⬎257–415 mg/day)
Q5 (⬎415–1589 mg/day)
Dietary vitamin Cd
Q1 (19–98 mg/day)c
Q2 (⬎98–143 mg/day)
Q3 (⬎144–201 mg/day)
Q4 (⬎201–273 mg/day)
Q5 (⬎273–656 mg/day)
Adenomas
No adenomas
Adjusted ORsa
(95% CI)
Adjusted ORsa
(95% CI)
1.00 (ref)
0.57 (0.20–1.62)
0.45 (0.16–1.34)
0.45 (0.14–1.43)
0.05 (0.01–0.46)
1.00 (ref)
0.61 (0.28–1.34)
0.64 (0.30–1.41)
0.50 (0.22–1.11)
0.89 (0.40–2.00)
1.00 (ref)
1.85 (0.59–5.78)
1.17 (0.37–3.65)
0.31 (0.07–1.30)
0.28 (0.07–1.21)
1.00 (ref)
0.77 (0.36–1.64)
0.97 (0.43–2.16)
0.79 (0.36–1.76)
0.68 (0.30–1.54)
a
Adjusted for total energy consumption using the standard multivariate approach.
Vitamin C from dietary plus supplemental sources.
c
Cutpoints derived from distribution of vitamin C intake among all participants.
Note: RDA vitamin C for men, 90 mg/day; for women, 75 mg/day. Consumption
above Q1 is above the RDA.
d
Vitamin C exclusively from dietary sources.
b
Fig. 1. Predicted linear regression line for total vitamin C and apoptosis presented by adenomas status.
ate state and disadvantageous with regard to adenoma risk (28).
On the other hand, it is possible that in some individuals,
reduced apoptosis in normal rectal tissue is physiologically
appropriate (i.e., an indication of a low stress environment
receiving low rates of DNA hits). We have no means of identifying a low stress environment in the current study; however,
this hypothesis could be evaluated in future studies.
It is also conceivable that antioxidants repair damage
caused by ROS, thereby reducing the number of cells that need
to be eliminated by the apoptotic mechanism. Low apoptosis
would not be considered an undesirable condition, in this scenario, because it results from a reduced need for cell elimination. However, in terms of adenoma risk, low apoptosis in
normal rectal mucosa appears to be disadvantageous (28).
Bivariate analyses (Table 1) show an unexpected relationship between apoptosis and energy consumption. Some studies
have suggested that low energy intake is associated with a
reduced risk of cancer (29 –31). In our study, participants with
lower total energy intake, in which one would expect a reduced
risk for adenoma, also had lower apoptosis levels. On the other
hand, as one would expect, those with very low BMI had high
rates of apoptosis, whereas those with high BMI had lower rates
of apoptosis. Because overweight individuals tend to underreport energy intake (32), it is conceivable that there was
misclassification of energy intake among those with low rates
of apoptosis (i.e., high-BMI individuals).
Results of studies examining associations of antioxidants
with apoptosis are mixed. Some experimental studies have
found that antioxidants reduce apoptosis (19, 33–36), whereas
others have not (37, 38). Very few published studies have
specifically examined the association between vitamin C intake
and apoptosis in humans; however, in a study of patients with
congestive heart failure, Rossig et al. (39) found that the administration of vitamin C significantly reduced apoptosis.
It has been proposed that the cancer-protective effect of
vegetable and fruit consumption is partly due to antioxidant
properties that allow scavenging of free radicals that, in turn,
prevent DNA damage and subsequent mutation (40). Several
randomized clinical trials of ␤-carotene and other antioxidant
supplements have attempted to identify the specific components of fruits and vegetables that might cause a reduction in
cancer risk. Five well-known trials showed that these vitamin
supplements did not reduce the incidence of adenomas and
other cancers (11–15); in fact, in the ␤-Carotene and Retinol
Efficacy Trial, ␤-carotene supplements increased lung cancer
risk in smokers (12). It is important to note that dietary vitamin
C may have a different effect than supplemental vitamin C
because of other components in the fruits and vegetables containing the vitamin (41). In our analyses, results for total and
dietary vitamin C were generally similar; however, total and
dietary vitamin C were only modestly correlated in our population (r ⫽ 0.45; P ⬍ 0.01).
Our findings have potential implications for cancer patients because many radiation and chemotherapeutic agents
work by inducing apoptosis (5, 42, 43). If vitamin C protects
cancer cells from ROS-dependent apoptosis, high vitamin C
intake during treatment could potentially reduce the efficacy of
chemotherapy treatments. There is evidence, however, that not
all apoptosis involves ROS and not all chemotherapy agents
enhance apoptosis by ROS-dependent mechanisms (44). Also,
vitamin C may provide health benefits by mechanisms distinct
from its antioxidant properties. Vitamin C may, for example,
modify the metabolism of polycyclic hydrocarbons, leading to
reduced mutagenic activity (45). Therefore, even if vitamin C
reduces therapy-induced apoptosis of tumor cells through prevention of oxidation, the efficacy of all chemotherapies might
not be influenced by high vitamin C intake. Our findings
suggest that interactions between vitamin and C and therapeutic
agents are worthy of further investigation.
There are several features that make our study notable.
Most importantly, this is one of very few studies that have
examined apoptosis in human volunteers, and we were able to
evaluate the distribution of apoptosis across various demographic and behavioral characteristics. Most studies of apoptosis have used animal or cell models. We also had a large sample
size for this type of biological data, in conjunction with carefully collected diet and lifestyle information.
Certain limitations must be acknowledged. First, estimates
of vitamin C from FFQs are not as precise as would be ideal;
biological measures may be more desirable. However, validation studies have reported modest correlations between vitamin
C intakes derived from FFQs and plasma or leukocyte vitamin
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Vitamin C and Apoptosis in Normal Rectal Epithelium
C [r ⫽ 0.43 and r ⫽ 0.31, respectively (46)], and several other
studies have used vitamin C estimates from FFQs to study diet
and cancer/adenoma associations (47–50). In addition, a recent
study found that plasma ascorbic acid provided a similar ranking of individual intake compared with FFQ-derived vitamin C
intake (51).
Second, the relationship of vitamin C with apoptosis may
be modified by other antioxidants such as vitamin E and selenium status, but a larger study population or better assessment
of these nutrients is needed to adequately explore these pathways. Third, we have no knowledge of daily, weekly, or
monthly variation of apoptotic rates in humans, nor do we know
how sensitive apoptosis is to changes in diet or other healthrelated factors. Also, recent changes in vitamin C consumption,
which could have a direct effect on apoptosis, could not be
evaluated. In future studies, it would be useful to obtain repeated measures of both vitamin C and apoptosis to assess
longitudinal patterns of apoptosis in response to environmental
changes.
In conclusion, vitamin C intake was inversely associated
with apoptosis among participants with adenomas. Because
high apoptosis has been shown to be strongly associated with a
reduced presence of adenomas, vitamin C supplementation may
negatively impact the pathway of colorectal neoplasia, particularly among persons with adenomas. It also appears that there
may be a different mechanism of antioxidant action for certain
subgroups of the population. Because of conflicting results
regarding the benefits of antioxidants on chronic disease initiation or progression, persons at higher risk of adenomas should
be encouraged to maintain a healthy diet, including regular
consumption of fruits and vegetables (52). Our results suggest,
however, that in terms of colorectal cancer risk, consumption of
excessive vitamin C supplements may be contraindicated
among individuals with a history of adenomas.
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565
Vitamin C Intake and Apoptosis in Normal Rectal Epithelium
Alexandra E. Connelly, Jessie Satia-Abouta, Christopher F. Martin, et al.
Cancer Epidemiol Biomarkers Prev 2003;12:559-565.
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