Inhibition of chronic ulcerative colitis-associated colorectal adenocarcinoma development in a murine model by N-acetylcysteine
Darren N. Seril,
Jie Liao,
Kwok-Lam K. Ho,
Chung S. Yang,1 and
Guang-Yu Yang,1
Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA
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Abstract
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Long-term ulcerative colitis (UC) patients are at increased risk for developing colorectal cancer. In order to develop strategies for preventing UC-associated carcinogenesis, we studied the effect of the antioxidant N-acetylcysteine (NAC) on UC-associated cancer development in a mouse model. Female C57BL/6J mice were subjected to long-term administration of dextran sulfate sodium (DSS) in the drinking fluid and 2-fold iron-enriched AIN76A diet, with or without NAC. In the DSS-plus-2-fold iron positive control group, gross tumor incidence was 88.5% (23/26 mice) after 12 DSS cycles (1 DSS cycle = 7 day DSS treatment period followed by 10 day recovery period). The tumor multiplicity was 2.1 ± 0.2 tumors/tumor-bearing mouse, and the tumor volume was 0.054 ± 0.019 cm3. With 0.2% NAC administration, tumor incidence was significantly reduced (68%, 17/25 mice; P < 0.05), as was the tumor multiplicity (1.5 ± 0.1 tumors/tumor-bearing mouse; P < 0.05). The tumor volume was lower (0.014 ± 0.004 cm3), but not significantly decreased. The proliferation index was significantly decreased in non-cancerous epithelia (48.5 ± 6.0% vs 32.0 ± 3.7%; P < 0.05), but not in tumor cells. NAC significantly induced apoptosis in both non-cancerous epithelia and colorectal adenocarcinoma. The number of cells immunostained-positive for nitrotyrosine was markedly decreased in the non-cancerous mucosa of NAC-treated mice (102.4 ±16.6 positive cells/mm2 mucosa vs 53.6 ± 14.9 cells/mm2; P < 0.05). In addition, the number of inducible nitric oxide synthase (iNOS)-positive inflammatory cells in the non-cancerous mucosa of the distal colon was markedly decreased by NAC. This study indicates that the antioxidant NAC has the potential to serve as a preventive agent for UC-associated colorectal cancer, possibly via inhibition of cellular proliferation and nitrosative stress-caused cellular damage.
Abbreviations: AC, adenocarcinoma; DSS, dextran sulfate sodium; iNOS, inducible nitric oxide synthase; NAC, N-acetylcysteine; NO, nitric oxide; PCNA, proliferation cell nuclear antigen; RNS, reactive nitrogen species; ROS, reactive oxygen species; SCC, squamous cell carcinoma; UC, ulcerative colitis.
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Introduction
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Ulcerative colitis (UC) is an idiopathic inflammatory disease of the large intestine that is quite common in the United States and other Western countries (1). Long-term UC patients are at greater risk for developing colorectal adenocarcinoma (AC) than the general population (24). The factors contributing to UC-associated carcinogenesis are not firmly established, although it is generally assumed that chronic inflammation is the primary driving force (5,6). It is essential to gain a clearer understanding of the mechanism of inflammation-driven colorectal carcinogenesis in order to design effective strategies for blocking the progression from chronic UC to colorectal dysplasia and carcinoma.
Reactive oxygen and nitrogen species (ROS and RNS) are overproduced in the colon of UC patients (714). Oxidative and nitrosative stress may contribute to the increased colorectal cancer risk in these individuals (15). Molecules such as the hydroxyl radical and nitric oxide (NO) can react with most cellular constituents, and have been shown to be mutagenic in in vitro assays. Oxidative and nitrosative damage may take part in the initiation stage of cancer development, possibly by altering oncogenes and tumor suppressor genes (1618). For example, the p53 tumor suppressor gene is frequently mutated in the non-cancerous colonic mucosa of UC patients (19), and is putatively susceptible to oxidative and nitrosative DNA damage (20). Oxidative and nitrosative stress may also serve a tumor-promoting role during UC-associated cancer development by stimulating cellular proliferation (18). The inhibition of colonic oxidative and nitrosative stress may therefore be an effective approach to preventing colorectal AC development in association with chronic UC.
N-acetylcysteine (NAC) is a thiol-containing species that has been used clinically as a mucolytic and as an antidote for acetaminophen toxicity (21). NAC is regarded as a promising cancer chemopreventive agent based on its anti-mutagenic and anti-carcinogenic activities (22). Several studies in animal models have demonstrated that NAC can inhibit carcinogen-induced tumorigenesis at a number of different organ sites (2226). NAC can directly scavenge ROS and RNS, and also serve as a precursor to reduced glutathione, the primary cellular antioxidant (21). These antioxidant activities may contribute to the therapeutic and chemopreventative effects of NAC. While it has been shown to attenuate acute colitis induced in rats (27), the use of NAC in the chemoprevention of inflammation-caused malignancies, including UC-associated colorectal cancer, has not been studied.
Various genetic and chemical-induced models of colitis have been employed recently to study the process of colitis-associated carcinogenesis (2830). High molecular weight dextran sulfate sodium (DSS) administration through the diet or drinking fluid induces colonic inflammation in rodents with clinical and histopathological similarity to human UC (31). DSS-induced chronic UC is also similar to the human disease in that it is complicated by the development of colorectal AC arising via the dysplasia-carcinoma sequence (29,32,33). Iron-deficiency anemia is a frequent complication of UC due to chronic colorectal bleeding. These patients are managed clinically by iron supplementation (34). We have found that dietary iron supplementation enhances the development of colorectal dysplasia and carcinoma in the DSS model in mice. The histopathogenesis of colorectal tumor development in this model is similar to that seen in humans (35). In the present study, we used the DSS-induced and iron-enhanced model of UC-associated carcinogenesis to examine the efficacy of the antioxidant NAC in the prevention of UC-associated cancer in mice. We further studied the effect of NAC on inflammation, cell proliferation, apoptosis, and nitrosative cellular damage using histopathological and immunohistochemical approaches.
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Materials and methods
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Animals and chemicals
Female C57BL/6J mice, 6 weeks of age, were obtained from The Jackson Laboratory (Bar Harbor, ME), and were used for the induction of chronic UC. The mice were housed five per cage, and maintained in air-conditioned quarters with a room temperature of 20 ± 2°C, relative humidity of 50 ± 10%, and an alternating 12 h light/dark cycle. The animals were acclimated to the animal room conditions at Laboratory Animal Services on the Rutgers University campus for 2 weeks, and administered tap water ad libitum until the start of the experiment. Body weights and food consumption were measured approximately every other week for the duration of the experiment.
DSS (MW: ~40 000) was obtained from ICN Pharmaceuticals (Costa Mesa, CA). NAC, hematoxylin, eosin, and 3,3'-diaminobenzidine were purchased from Sigma Chemical Co. (St Louis, MO). Antigen Unmasking Solution, and avidin/biotinylated horseradish peroxidase macromolecular complex kits were from Vector Laboratories (Burlingame, CA). Mouse monoclonal anti-proliferation cell nuclear antigen (PCNA) antibody was from Oncogene (Cambridge, MA), monoclonal rat anti-leukocyte common antigen (CD45) antibody was from BD Pharmingen (San Diego, CA), polyclonal rabbit anti-nitrotyrosine antibody was from Upstate Biotechnology (Lake Placid, NY), and polyclonal rabbit anti-inducible nitric oxide synthase (iNOS) antibody was from Santa Cruz (Santa Cruz, CA). AIN76A-based diet containing twice the normal amount of iron (2x Fe diet; AIN76A diet supplemented with 45 mg iron per kg diet) and 2x Fe diet containing 0.2% NAC (wt/wt) (2x Fe-NAC) were purchased from Research Diets, Inc. (New Brunswick, NJ).
Animal experiment
A total of 80 mice fed the 2x Fe or 2x Fe-NAC diets were distributed into four groups. Group 1 (n = 10 mice) was administered water as a negative control. Group 2 (n = 10 mice) was given 2x Fe-NAC diet (0.2% NAC, an approximate daily intake of 200 mg NAC/kg body weight). Group 3 (n = 30 mice) was given 0.7% DSS (wt/vol) as the sole source of drinking fluid and 2x Fe diet. Group 4 (n = 30 mice) was given 0.7% DSS solution and 2x Fe-NAC diet. Based on our previous studies, 0.7% high molecular weight DSS induces mild UC in C57BL/6J mice, characterized by mild to moderately severe colonic inflammation with small, scattered regions of ulceration. DSS was administered in 17-day `DSS cycles' that consisted of 7 days of DSS administration, followed by 10 days of ordinary tap water administration (31). This schedule was used to simulate the cycle of acute flare-ups alternating with periods of disease inactivity observed in human UC patients. Control animals were administered distilled water for 7 days, followed by tap water for 10 days. All groups were subjected to 12 consecutive DSS cycles, which in our experience is the number of cycles yielding a gross tumor incidence of 70 to 90% in control animals given low-dose DSS and 2x Fe diet. The animals in Group 2 and Group 4 were administered 2x Fe-NAC diet during the 10-day recovery periods.
Tissue preparation and histopathological evaluation
Following the 12th cycle of DSS administration, the mice were killed by CO2 asphyxiation. The colon was inflated by in situ intraluminal perfusion with 10% neutral buffered formalin, removed, opened longitudinally, and examined for gross abnormalities. Gross tumors were counted, and the diameter of the tumors was measured in three perpendicular directions using calipers. The gross tumor volume was calculated using the equation V = 4/3
r3, where r was the average tumor radius. The colon was then attached to a plastic sheet and fixed in 10% neutral buffered formalin for 24 h before being transferred to 80% ethanol. The tissues were prepared for routine processing and embedding as a `Swiss roll'. Serial tissue sections (5 µm) were made and mounted on glass slides. Serial tissue slides were routinely stained with hematoxylin and eosin for histopathological analysis, or used for immunohistochemical analyses.
UC-associated tumors in the colon were analyzed microscopically and diagnosed as colorectal AC or anal squamous cell carcinoma (SCC) based on previously described criteria (35). AC was further categorized as tubular or mucinous well differentiated based on the glandular architecture and mucin secretion. Tumors diagnosed as anal SCC were further classified as well- or poorly-differentiated based on the presence or absence of keratinized cells, respectively.
Histopathological grading of chronic UC
Hematoxylin and eosin-stained serial tissue sections were used for histological grading of inflammation in the proximal, middle, and distal regions of the colon. The grading for UC was based on inflammation severity, ulceration, hyperplasia, and area of inflammatory involvement, as described previously (36). The score for each criterion was the mean of the scores of 89 mice from each treatment group. A total score, or UC index, was generated for each sample by adding the individual criterion scores for that sample. The mean UC index was generated from the mean of the UC indices from 89 mice per group. The criterion and UC index scores for the entire colon were obtained by averaging the scores for the three colon regions.
Immunohistochemistry
Immunohistochemical analyses using antibodies against PCNA, CD45, nitrotyrosine, and iNOS were performed using the avidinbiotinperoxidase complex method as described previously (37). Paraffin-embedded tissue sections were routinely dewaxed and rehydrated before endogenous peroxidase activity was quenched with 1% H2O2. When necessary, antigen was retrieved by microwave pretreatment in citrate buffer (Antigen Unmasking Solution, Vector). After blocking non-specific proteinprotein interactions with diluted normal serum, the slides were incubated with primary antibody (2.5 mg/ml for 2 h at room temperature for anti-PCNA; 5 mg/ml overnight at 4°C for anti-CD45; 1 mg/ml for 2 h at room temperature for anti-nitrotyrosine; 1 mg/ml for 2 h at room temperature for anti-iNOS), followed by the appropriate secondary antibody for 45 min at room temperature, and ABC reagent for 45 min at room temperature. Diaminobenzidine was used as the chromagen. Slides were thoroughly washed with PBS between incubations, and counterstained with Mayer's hematoxylin. Negative control slides were established by replacing the primary antibody with PBS and normal serum. Staining was classified as positive or negative based on the presence or absence of brown color precipitate.
Immunostained-positive cell number and mucosal area were quantified in the proximal, middle, and distal regions of the colon using a Nikon DX research microscope and Nikon DM1200 digital camera in conjunction with an Image-Pro Plus computerized image analysis system (Media Cybernetics, Silver Spring, MD). The PCNA proliferation index was determined in non-cancerous epithelia, or in colorectal AC, in serial tissue slides from four to five randomly selected mice per group. Greater than 1000 cells were counted per colon region or tumor. The PCNA labeling index was calculated as the ratio of the number of PCNA-immunostained-positive cells and the total number of cells multiplied by 100. For nitrotyrosine, iNOS and CD45 staining, the number of immunostained-positive cells in non-cancerous mucosa or colorectal AC was counted in four to five microscope fields (20x objective) per colon region (total area analyzed:
0.5 mm2 per colon region). The slides analyzed were from four to five randomly selected mice from each group. The data were expressed as the number of immunostained-positive cells per area of mucosa or tumor. All parameters were quantified without knowledge of the animal treatment.
Apoptosis
Hematoxylin and eosin-stained slides were analyzed for apoptosis in non-cancerous epithelia and in colorectal AC based on morphological criteria. Apoptotic cells were defined as those exhibiting cell shrinkage, small, hyperchromatic nuclei, nuclear debris, and eosinophilic cytoplasm. To assess apoptosis in non-cancerous epithelia, >5000 epithelial cells were counted per colon region per slide. Four to five mice were randomly selected from each group for the analysis. Apoptosis in colorectal AC was assessed in 10 tumors from 8 to 9 mice per group. Three thousand to 4000 cells were counted per tumor. The apoptosis index was expressed as the ratio of the apoptotic cell number to the total number of cells counted multiplied by 100.
Statistical analysis
Data were expressed as the mean ± SE, and were analyzed using SigmaStat Version 1.01 statistical software (Jandel Scientific, San Rafael, CA). The effect of NAC treatment on UC-associated colorectal tumor incidence was analyzed by McNemar's test. Analysis of normally distributed data was performed using Student's t-test. Non-parametric data were analyzed using MannWhitney Rank Sum test. Body weights were compared among treatment groups by one-way ANOVA with post-hoc analysis using Bonferroni/Dunn tests. Values were taken to be statistically significant at P < 0.05.
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Results
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General observations
As we observed previously, the mice in the DSS-treated groups exhibited bloody, mucoid diarrhea and reductions in body weight during the DSS treatment periods as compared with non-treated controls. However, body weights returned to non-DSS treated control levels within 7 to 10 days of the cessation of DSS treatment (data not shown). As shown in Table I
, the mean body weights did not differ between treatment groups at the end of the experiment. NAC diet did not affect food consumption after 12 DSS cycles with or without DSS treatment. Similarly, DSS treatment had no effect on food consumption at the end of the experiment as compared with the non-treated controls (data not shown).
Four out of 30 (13%) mice in Group 3 and 5/30 (17%) mice in Group 4 died prior to the completion of 12 DSS cycles, exhibiting bloody diarrhea and excessive weight loss. Upon necropsy, all of the deaths were determined to be due to severe colitis. One of the Group 3 mice harbored a colorectal tumor. However, all of the animals that died prior to the completion of 12 DSS cycles were excluded from the results of the carcinogenesis experiment.
Effect of NAC on chronic UC-associated colorectal tumor incidence, multiplicity, and volume
We have observed the development of colorectal AC arising via the dysplasia-carcinoma morphological sequence in our previous studies using the DSS-induced model of UC in mice, and that dietary iron supplementation significantly enhances the carcinogenesis process (35). Nineteen percent (19%) of the animals treated long-term with DSS without iron supplementation developed colorectal tumors, whereas 88% of the mice administered DSS plus 2x iron diet manifested colorectal tumors. In the present study, colorectal tumors were not observed in the non-DSS treated, negative control animals that were administered either 2x Fe (Group 1) or 2x Fe-NAC diet (Group 2). Macroscopic tumors were observed in the colons of 23 out of 26 (89%) mice in Group 3 subjected to 12 DSS cycles (Table I
). These mice frequently harbored multiple tumors, with a mean multiplicity of 2.1 ± 0.2 (mean ± SE) tumors per tumor-bearing animal. The mean tumor volume was 0.054 ± 0.019 cm3, with a range of 0.0005 cm3 to 0.763 cm3. Group 4 mice were subjected to 12 DSS cycles and administered 0.2% NAC in the diet (an approximate daily intake of 200 mg NAC per kg body weight) during the recovery period. Group 4 mice exhibited a significantly lower tumor incidence (17/25, 68%; P < 0.05) and tumor multiplicity (1.5 ± 0.1 tumors per tumor bearing mouse; P < 0.05). The mean tumor volume was lower in Group 4 (0.014 ± 0.004 cm3; range: 0.0005 cm3 to 0.080 cm3) compared with Group 3, but the difference did not reach statistical significance (P = 0.107, MannWhitney Rank Sum test) (Table I
). The tumors were further categorized as small (<0.01 cm3), medium (0.010.05 cm3), and large (>0.05 cm3). Group 4 exhibited fewer medium- and large-sized colorectal tumors (Figure 1A
). The average volume for medium- and large-sized tumors combined was significantly lower in the Group 4 mice as compared to Group 3 (Figure 1B
).

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Fig. 1. The effect of NAC on UC-associated colorectal tumor volume. (A) Distribution of small- (<0.01 cm3), medium- (0.010.05 cm3) and large-sized (>0.05 cm3) tumors in mice administered DSS and control diet (AIN76A + 2x Fe: Group 3) or NAC diet (Group 4) for 12 cycles. (B) Average tumor volume for small-sized tumors (<0.01 cm3) and medium- and large-sized tumors combined (>0.01 cm3) and the affect of NAC diet, in Group 3 and Group 4 mice. Data are expressed as the mean tumor volume in cm3. Error bars represent SE. *Significant difference from Group 3 (P < 0.05, MannWhitney Rank Sum test).
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The tumors observed macroscopically were confirmed to be AC by histopathology, and were categorized as either mucinous or tubular well-differentiated AC (Figure 2
). Most of the tumors observed in this model were well-differentiated, mucinous AC, exhibiting invasive cancer nests with mucin lakes (Figure 2A and 2B
). A few of the tumors were categorized as tubular well-differentiated AC, which showed a polypoid growth pattern (Figure 2C and 2D
). Mucinous AC accounted for 45/49 (91.8%) and 24/25 (96.0%) AC in Group 3 and Group 4, respectively. Four out of 49 (8.2%) and 1/25 (4.0%) AC were classified as tubular-differentiated in Group 3 and Group 4, respectively. There was no difference in the distributions of AC types between Groups 3 and 4. Analrectal well-differentiated SCC was also observed at a high rate in both treatment groups. The SCC incidence was 13/26 (50%) in Group 3, and 13/25 (52%) in the NAC group. NAC treatment had no effect on the incidence of analrectal SCC.

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Fig. 2. Types of colorectal AC observed in the DSS-induced and iron-enhanced UC model. (A) Colonic mucinous well-differentiated AC invading into and beyond the muscle layer (original magnification: 25x). (B) Higher magnification of a cancer cell nest with a mucin lake in the tumor shown in (A) (250x). (C) Colonic tubular well-differentiated AC exhibiting a polypoid growth pattern (25x). (D) Higher magnification of tubular AC shown in (C) (250x).
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Effect of NAC on DSS-induced chronic UC
The colons from Group 1 and Group 2 negative control mice were morphologically normal, showing no inflammation or ulceration. The colons from all of the DSS-treated mice exhibited mild to moderately severe inflammation upon histopathological examination, characterized by mucosal erosion, ulceration, and epithelial hyperplasia. The effect of NAC treatment on chronic UC at the end of the experiment was assessed histopathologically, and the results for lesion severity, ulceration, hyperplasia, area of inflammatory involvement, and total histological score (UC index) are shown in Table II
. In Group 3, the mean histological scores for UC were highest in the distal region of the colon, and increased from the proximal colon to the distal colon. The lesion severity tended to be mild in the proximal colon, with small regions of inflammation and epithelial hyperplasia. Ulceration was infrequently observed in this region, and hyperplasia was usually classified as mild (thickening of the colonic mucosa with morphologically normal epithelial cells). In the middle and distal colon, UC was moderately severe, exhibiting more extensive areas of inflammation and epithelial cell injury. Ulcers were more frequently observed, and moderate to severe hyperplasia (characterized by marked mucosal thickening, nuclear atypia and crypt branching) was common. Similar to Group 3, the highest UC index scores occurred in the distal colon of Group 4 mice, but most of the histological scores were lower than those of Group 3 (Table II
). Statistically significant reductions were observed in the UC index of the proximal region of the colon and in the average area of inflammatory involvement for the entire colon in the NAC-treated group (Table II
).
Effect of NAC on cell proliferation and apoptosis
The colonic PCNA labeling index was determined immunohistochemically in areas of non-cancerous epithelia to assess the effect of NAC on epithelial cell proliferation. In Group 1 and Group 2, PCNA-positive cells were mostly confined to the proliferation compartment in the lower half of the colonic glands. Fewer proliferating cells were observed in the upper half of the glands (containing the differentiation compartment). There was no difference in the colonic epithelial proliferation index between Group 1 and Group 2 (Figure 3A
). The colonic epithelia of Group 3 mice showed elongated glands, with increased numbers of PCNA-positive cells in both the upper half and lower half of the colonic glands (Figure 4A and 4B
). As shown in Figure 3A
, the epithelial proliferation rates were significantly decreased in the colons of Group 4 versus Group 3 mice. All regions of the colon exhibited reduced proliferation rates with NAC treatment, with statistically significant reductions in the proximal and distal thirds of the colon. Group 4 mice exhibited significantly fewer proliferating cells in the glandular proliferation compartment, as well as fewer PCNA-positive cells in the upper half of the glands in the proximal and distal colon (Figure 4C and 4D
). Proliferation was reduced, but not significantly so, in these epithelial compartments in the middle region of the colon (Figure 3A
). In colorectal AC, the tumor cell proliferation rate was lower in the NAC-treated mice (Group 4 vs Group 3: 51.9 ± 3.3% vs 56.50 ± 1.4%), but the difference was not significant (Figure 3B
).


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Fig. 3. The effect of NAC on cell proliferation and apoptosis in non-cancerous epithelia and colorectal AC of control and DSS-treated mice. (A) The percentage of PCNA-immunostained positive cells was determined in non-cancerous epithelia of the proximal, middle and distal regions of the colon and in the colon overall, from all groups. The figures show the mean PCNA labeling indices for the upper 50% of colonic glands (upper half), the lower 50% of colonic glands (lower half) and the entire gland. Error bars represent SE (n = 45 mice). *Significant difference from respective Group 3 value (P < 0.05, Student's t-test). (B) PCNA labeling index in colorectal AC from Group 3 and Group 4 mice. Error bars represent SE (n0= 5 mice). (C) The percentage of apoptotic cells was determined in non-cancerous colorectal epithelia in the proximal, middle and distal parts of the colon and in the entire colon from all groups. Apoptosis was determined in colorectal AC (Groups 3 and 4) as well. Data are expressed as the mean apoptosis index. Error bars represent SE (non-cancerous colorectal epithelia: n = 45 mice; colorectal AC, n = 10 tumors from 89 mice). Significant difference from respective region of: *Group 1; **Group 3 (P < 0.05, Student's t-test); ***Group 3 (P < 0.05, MannWhitney Rank Sum test).
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Fig. 4. Representative micrographs of cell proliferation in non-cancerous epithelia, as detected by PCNA immunohistochemistry. (A) In an area of regenerative and mildly hyperplastic non-cancerous epithelia from a Group 3 mouse, a large number of PCNA-positive cells were observed in the glandular crypts (proliferation compartment) and throughout the glands (original magnification: 250x). (B) Higher magnification of the area shown in (A): numerous proliferating cells were located in the upper half and on the surface of the hyperplastic epithelia (500x). (C) PCNA-positive proliferative cells were mainly located in the glandular crypts in the colon of a Group 4 mouse (250x). (D) Higher magnification of the area shown in (C): few proliferative cells were observed in the upper regions of the glandular epithelia in a Group 4 NAC-treated mouse (500x).
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Apoptosis was assessed morphologically in normal appearing epithelia in order to avoid misidentification of apoptotic cells due to the presence of epithelial injury and necrosis. Apoptotic cells, exhibiting condensed nuclei and eosinophilic cytoplasm, were mainly observed in the upper half of the glands, frequently at the surface of the glands and at gland junctions. Apoptotic cells occasionally appeared in the lower half of the glands, in the proliferation compartment (crypt region). Figure 3C
shows the apoptosis index results for the non-cancerous epithelia in control- and DSS-treated mice with and without NAC administration. The apoptosis index was significantly induced in the normal mucosa of the proximal colon, and the entire colon (mean ± SE, 0.177 ± 0.011% vs 0.126 ± 0.012%; P < 0.05), in Group 2 mice compared with Group 1 mice. The apoptosis index was also significantly induced in the non-cancerous epithelia of Group 4 mice as compared with Group 3 (0.194 ± 0.015% vs 0.110 ± 0.006%; P < 0.05). NAC treated mice more frequently exhibited apoptotic cells at the gland surface, as well as in the proliferation compartment. Figure 3C
also shows the results for apoptosis in colorectal AC and the effect of NAC administration. AC from Group 4 mice exhibited a significantly greater apoptosis index as compared to those from Group 3 (0.475 ± 0.057% vs 0.261 ± 0.063%; P < 0.05).
Effect of NAC on nitrotyrosine-, iNOS-, and CD45-positive cell number
Immunohistochemical analysis of nitrotyrosine accumulation was performed to determine the effect of NAC treatment on inflammation-caused nitrosative damage. The colorectal mucosa of Group 1 mice contained scattered nitrotyrosine-positive inflammatory cells in the lamina propria. As shown in Figure 6A
, Group 2 mice fed NAC diet exhibited significantly fewer nitrotyrosine-positive cells in the colon. The non-cancerous mucosa of Group 3 mice contained a large number of intensely stained, nitrotyrosine-positive inflammatory cells in the lamina propria, adjacent to the colonic epithelia (Figure 6A
). Epithelial cells with nitrotyrosine staining were also frequently observed (Figure 6B
). The number of nitrotyrosine-positive cells was decreased in the colonic mucosa of Group 4 NAC-treated mice (Figure 6C and 6D
). Quantitative analysis confirmed that the number of nitro-tyrosine-positive cells per area of mucosa was significantly reduced in the proximal, middle, and distal regions of the colon in DSS + NAC-treated mice, as well as in the colon overall, as compared to Group 3 (Figure 5A
). The number of nitrotyrosine-positive cells was not significantly decreased in AC from Group 4 mice compared with Group 3 (Figure 5A
).

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Fig. 6. Representative micrographs of nitrotyrosine- and iNOS-immunostaining in non-cancerous colonic mucosa. (A) In a mouse treated with DSS and 2x Fe diet (Group 3), numerous nitrotyrosine-positive cells were observed in the non-cancerous mucosa (original magnification: 125x). (B) Higher magnification of the region shown in (A): numerous nitrotyrosine-positive inflammatory cells, as well as a positively stained epithelial cell (arrow), are visible (250x). (C) Fewer nitrotyrosine-positive cells were observed in the non-cancerous mucosa of a mouse from Group 4 (125x). (D) Higher magnification of region shown in (C), with fewer nitrotyrosine-positive inflammatory cells adjacent to the epithelia in an NAC-treated, Group 4 mouse. (E) Numerous infiltrating iNOS-positive cells were observed in the non-cancerous mucosa of a Group 3 mouse (125x). (F) Higher magnification shows iNOS-expressing cells in a region of epithelial regeneration (250x). (G) With NAC treatment, the iNOS-positive cell number was reduced in the non-cancerous mucosa of the distal colon (125x). (H) Higher magnification of the area shown in (G): a few iNOS-positive cells were seen adjacent to the colonic glands in a Group 4 NAC-treated mouse (250x).
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Fig. 5. Effects of NAC on nitrosative damage and infiltration of iNOS- and CD45-positive inflammatory cells in control and DSS-treated mice. (A) The number of nitrotyrosine immunostained-positive cells per area of colorectal mucosa was determined in the three colon regions, in the colon overall and in colorectal AC. Data are expressed as the mean number of nitrotyrosine positive cells per mm2 mucosa or tumor. Error bars represent SE (for colorectal mucosa, n = 45 mice; for colorectal AC, n = 5 tumors from five mice). Significant difference from respective region of: *Group 1; **Group 3 (P < 0.05, Student's t-test). (B) The number of iNOS-positive cells per area of mucosa in the proximal, middle and distal colon, in the colon overall and in colorectal AC in mice fed control or 0.2% NAC diet. Data are expressed as the mean iNOS-immunostained positive cell number per mm2 mucosa or tumor. Error bars represent SE (n = 45 mice). *Significant difference from distal colon of Group 3 (P < 0.05, Student's t-test). (C) CD45-positive inflammatory cell number per area of colorectal mucosa or colorectal AC, determined in control and DSS-treated mice fed AIN76A/2x Fe or 0.2% NAC diet. Data are expressed as the mean number of CD45 immunostained-positive cells per mm2 mucosa or tumor. Error bars represent SE (n = 45 mice).
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Immunohistochemical staining for iNOS was performed in order to determine if the reduction in nitrotyrosine-positive cells by NAC was due to effects on iNOS expression. Similar to the staining pattern for nitrotyrosine, positivity for iNOS was found in lamina propria inflammatory cells. However, iNOS staining was not observed in colonic epithelial cells. There was no difference in the number of mucosal iNOS-positive cells between Groups 1 and 2 (Figure 5B
). Group 3 showed an intense mucosal infiltrate of iNOS-positive inflammatory cells, particularly in the distal colon and rectum (Figure 6E and 6F
). As shown in Figure 6G and 6H
, and in Figure 5B
, the number of iNOS-positive cells in the non-cancerous mucosa was significantly reduced in the distal colon of NAC-treated animals as compared with the same colon region in Group 3 (mean ± SE, 302.5 ± 24.2 cells/mm2 mucosa vs 455.4 ± 30.6 cells/mm2; P < 0.05). NAC treatment had no affect on the number of iNOS-positive cells in the other regions of the colon, and no discernible difference in the staining intensity of iNOS was noted between groups. Similarly, NAC consumption did not have an affect on the infiltration of iNOS positive cells into colorectal AC (Figure 5B
). Analysis of CD45-positive cell infiltration showed that the number of mucosal CD45-positive inflammatory cells was slightly decreased in Group 2 versus Group 1 (Figure 5C
). However, there was no difference in the number of mucosal CD45-positive inflammatory cells between Group 4 and Group 3. The number of CD45-positive cells within colorectal AC was also unaffected by NAC treatment (Figure 5C
).
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Discussion
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The pathological processes underlying UC-associated colorectal cancer development are still relatively unclear, perhaps because of the lack of suitable model systems. Recently, several of the genetic and chemical-induced models of colitis in rodents have been utilized to study inflammation-driven colorectal carcinogenesis (2830), which should facilitate mechanism studies and the search for effective prevention modalities. We have been studying a model of UC-associated carcinogenesis in which mice are subjected to long-term, cyclic administration of DSS together with iron supplementation through the diet. The clinical and histopathological features of this model are similar and relevant to those of chronic UC and colorectal carcinoma development observed in human UC patients (35). Chronic UC is commonly complicated by iron-deficiency anemia, which is remedied by iron supplementation. It is suspected that inflammation-caused oxidative/nitrosative cellular damage contributes to the development of UC-associated colorectal neoplasms. Iron supplementation and other sources of relatively high amounts of dietary iron, such as the Western diet, may represent risk factors for UC-associated cancer in humans by augmenting oxidative and nitrosative stress (15,35). The DSS-induced and iron-enhanced UC carcinogenesis model in mice was used in the present study to assess the potential use of the antioxidant NAC in the prevention of UC-associated cancer. It was shown that NAC treatment does inhibit colorectal cancer development in this mouse model, an effect that was associated with decreases in inflammation-caused epithelial cell proliferation and nitrosative damage, and an induction of apoptosis.
The decrease in inflammation-caused epithelial cell proliferation observed in this study may be an important mechanism by which NAC inhibited UC-associated colorectal AC development. It is thought that intermittent colonic epithelial damage and restitution caused by chronic inflammation contribute to the increased cancer risk in long-term UC patients. The elevated rate of cell turnover associated with the epithelial damage-restitution cycle may increase the occurrence of mitotic aberrations and other genetic and epigenetic changes, as well as take part in the promotion stage of cancer development (6). The effect of NAC on cell proliferation observed in the present study is consistent with other findings, which have shown that NAC decreases the proliferation of cancer cell lines, as well as colonic epithelial cells in human tissue samples from individuals at high risk for colorectal cancer (3841). The observations that NAC can block cell cycle progression by inhibiting topoisomerase II
activity, possibly via glutathione, and by inducing the expression of cyclin-dependent kinase inhibitors, may provide mechanistic bases for the antiproliferative effect (39,40). In addition to directly or indirectly inhibiting the machinery of cell cycle progression, NAC could also decrease the proliferating cell population by inducing programmed cell death or differentiation. Indeed, deregulation of the colonic crypt maturation program is thought to be an important characteristic of colorectal cancer development (42,43). Depending on the study, NAC is known to both inhibit and induce apoptosis, and has been shown to induce the expression of genes that take part in the differentiation process, including p16INK4a and p21Waf1 (22). In this study, NAC treatment was associated with an induction of programmed cell death in the inflammation-caused hyperproliferative colorectal epithelia, which may have contributed to the decrease in proliferation and possibly tumor development. Interestingly, NAC had differing effects on non-cancerous epithelia and colorectal tumor cells. The NAC treated mice exhibited smaller tumors, but the rate of tumor cell proliferation was unaffected by NAC treatment. However, NAC consumption increased the rate of apoptotic tumor cell death, which may have functioned to reduce tumor size independent of cellular proliferation. The reason for the disparity between the effects of NAC on non-cancerous epithelia and colorectal AC is unknown, but it may be due to differences in the proliferation and apoptosis control mechanisms present in non-cancerous and cancerous cells. Another interesting observation in our study was the induction of cell apoptosis in the colons of normal control mice by NAC. This phenomenon has also been observed in rat and human vascular smooth muscle cells (44), which are involved in the pathogenesis of atherosclerosis, a chronic inflammation-associated process. Although the mechanism by which NAC induces apoptosis is unknown, regulation of the cellular redox system, including glutathione levels, and modulation of the cell maturation process may be involved. The apoptosis-inducing effects of NAC require further study.
Some effects of NAC described here and elsewhere may be due to antioxidant activities. Oxidants have been shown to regulate cell growth, differentiation, and inflammation via redox-sensitive transcription factors, the most notable of these being NF-
B (4548). ROS and RNS can also induce the release of inflammatory mediators and cytokines (49), which have the potential to promote the proliferation of epithelial cells and induce resistance to apoptosis (50,51). NAC may counteract the mutagenic effects of ROS and RNS (21). Colonic oxidative stress and nitrosative stress are characteristic of chronic UC, which has led to the notion that oxidative/nitrosative-caused cellular and genetic damage contribute to the initiation of UC-associated neoplasms. In this scenario, ROS and RNS elaborated by infiltrating inflammatory cells drive carcinogenesis by mediating oncogene and tumor suppressor gene alterations, as well as an overall reduction in genomic stability, in adjacent epithelial cells (1618). In the present study, immunostaining for nitrotyrosine, a marker of NO-mediated protein damage, was decreased in the non-cancerous mucosa of the colon in NAC-treated mice. This inhibition of inflammation-caused nitrosative damage may be due to direct or indirect radical scavenging effects. The iNOS-expressing cells in this model are predominately macrophages, as indicated by immunostaining for Mac-3 (a macrophage-specific antigen) in our previous study (35). NAC consumption did not alter the infiltration of CD45-positive inflammatory cells, most of which are lymphocytes. However, a reduction in the involvement of iNOS-expressing cells was observed in the distal colon (where inflammation was more severe) and may have contributed to a reduction in nitrotyrosine-positive cells and the macrophages-associated inflammatory response. Dietary iron supplementation in the DSS model is associated with an enhanced mucosal infiltration of iNOS-positive inflammatory cells, as well as increased nitrotyrosine staining (35). It is not known if NAC has direct effects on the infiltration or activity of macrophages. The effects of NAC on macrophage activity and iNOS, and the contribution of macrophages to inflammation-associated carcinogenesis, require closer examination.
NAC has many favorable properties of an effective chemopreventive agent, including ease of administration, low cost, and relatively low toxicity (52,53). NAC is currently the subject of clinical trials, including for the prevention of colon polyps (54,55). These studies utilize oral doses of 600 to 800 mg NAC per day, a daily intake of roughly 10 mg per kg body weight for a 75 kg individual (41,54,55). It has been shown that long-term NAC consumption is well-tolerated at a daily dose of 20 mg/kg body weight, and minimal adverse effects are evident at 40 mg/kg body weight (52). The appropriateness of the NAC doses used in animal studies for human use is an important concern. In prevention studies using rats, weight loss and reduced food intake have been observed with long-term consumption of diets containing from ~2000 to 13 000 p.p.m. NAC (26,56). However, a study in mice showed that the prolonged administration of 5000 p.p.m. NAC diet had no effect on weight gain or food consumption (57), similar to the present findings using 2000 p.p.m. NAC diet. Thus, the tolerable dose of NAC in rodents is most likely species- and strain-dependent. Due to large differences in metabolic rate, it is convenient to use the concept of nutrient density when comparing the dietary requirements of different species, such as rodents and humans (58). The NAC doses used in the clinical studies mentioned above represent nutrient densities of 0.3 to 1.5 mg NAC per kcal for a 75 kg person consuming 2000 kcal per day. In this study, the NAC dose was 200 mg/kg body weight/day. For a 20 g mouse consuming 2.0 g AIN76A diet (3.6 kcal/g) per day, this is an NAC nutrient density of 0.6 mg/kcal. Thus, the dose of NAC used in the present study is comparable with the non-toxic doses used in humans.
In conclusion, this study showed that NAC significantly inhibited the development of UC-associated colorectal tumors in mice, in association with suppression of nitrosative cellular damage and inflammation-caused hyperproliferation. The present findings suggest that NAC consumption may benefit chronic UC patients by decreasing the occurrence of colorectal cancer and the requirement for prophylactic surgery, especially in those at particular risk for developing malignancies (i.e. individuals with UC for >10 years and pancolitis).
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Notes
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1 To whom correspondence should be addressed Email: gyyang{at}rci.rutgers.edu or csyang{at}rci.rutgers.edu 
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Acknowledgments
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The authors would like to thank Mr Asim Warsi for assisting in the maintenance of the animals in this study. This work was supported by a research grant from the American Cancer Society to G.-Y.Y. (# RPG-00-034-01-CNE). D.N.S. was supported by a predoctoral fellowship award from the New Jersey Commission on Cancer Research. C.S.Y. is a member of the Environmental Health Center of New Jersey and the Cancer Institute of New Jersey, which made shared facilities available for this study (Center Grant ES0052).
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Received September 4, 2001;
revised February 6, 2002;
accepted March 1, 2002.