Anatomical site-specific response to DNA damage is related to later tumor development in the rat azoxymethane colon carcinogenesis model
Mee Young Hong1,
Robert S. Chapkin1,
Jeffrey S. Morris2,
Naisyin Wang2,
Raymond J. Carroll1,2,
Nancy D. Turner1,
Wen Chi L. Chang3,
Laurie A. Davidson1 and
Joanne R. Lupton1,4
1 Faculty of Nutrition and
2 Department of Statistics, Texas A&M University, College Station, TX 77843-2471, USA
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Abstract
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There is now general agreement that the etiology of proximal and distal colon cancers may differ, thus prompting renewed interest in understanding anatomical site-specific molecular mechanisms of tumor development. Using a 2x2x2 factorial design with male SpragueDawley rats (corn oil, fish oil; pectin, cellulose; plus or minus azoxymethane injection) we found a greater than 2-fold difference (P < 0.001) in tumor incidence proximally versus distally (prox/dist ratio: corn oil, 2.25; fish oil, 2.61). The purpose of the present study was to determine if the higher degree of proximal versus distal tumors in our model system could be accounted for by differences between these two sites in initial DNA damage, response to that damage or an effect of diet at one site but not the other. DNA damage was assessed by quantitative immunohistochemistry of O6-methylguanine adducts; repair by measurement of O6-methylguanine-DNA alkyltransferase and removal was determined by measurement of targeted apoptosis. Although overall initial DNA damage was similar at both sites, in the distal colon there was a greater expression of repair protein (P < 0.001) and a greater degree of targeted apoptosis (P < 0.0001). There was also a reduction in DNA damage in the distal colon of rats consuming fish oil. Together, these results suggest that the lower tumor incidence in the distal colon may be a result of the capacity to deal with initial DNA damage by the distal colon, as compared with the proximal colon. Therefore, the determination of site-specific mechanisms in tumor development is important because distinct strategies may be required to protect against cancer at different sites.
Abbreviations: AOM, azoxymethane; ATase, O6-methylguanine-DNA-alkyltransferase; O6-dmetG, O6-deoxymethylguanine.
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Introduction
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Recent reports in humans suggest that after the age of 70, proximal (right sided) colon tumors predominate over distal (left sided) colon tumors (1). Importantly, people with proximal cancers are likely to have more advanced disease (1). A shift in incidence from distal to proximal colon tumors has also been reported in a large number of studies (14). There is now general agreement that the etiology of proximal and distal colon cancers may differ, thus prompting renewed interest in the understanding of the anatomical site-specific molecular mechanisms of tumor development.
Carcinogen-induced colon cancer in rodent models has been used extensively to delineate molecular mechanisms of colon tumorigenesis. Using this model, investigators have reported a variety of anatomical distributions of tumors (5,6), with location most probably dependent, in part, on diet. For example, in our studies of high-fiber/low-fat diets we often find a greater proportion of tumors in the proximal colon (5), the site of maximum fermentation (7). In contrast, low or no fiber diets combined with high fat (more typical of the US diet) may result in a greater proportion of distal tumors (6), presumably due to both lack of fermentation proximally and a higher concentration of fat in the distal colon contents once water has been extracted (8). In a recent study using a 2x2x2 factorial design (corn oil, fish oil; pectin, cellulose; plus or minus carcinogen), we reported a protective effect of fish oil on colon tumor incidence (9). We now describe the location of these tumors. The purpose of the present study was to determine if the anatomical site-specific distribution of proximal versus distal tumors in our model system could be accounted for by differences between the two sites in initial DNA damage, response to that damage or an effect of diet at one site but not the other.
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Materials and methods
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Animals and study design
There are two parts to this study: (i) the analysis of site-specific distribution of tumors and (ii) a tumor initiation study of methylation-induced DNA damage and response to that damage during the first 12 h post carcinogen injection. Animal use protocols were approved for both studies by the University Laboratory Animal Care Committee of Texas A&M University.
The tumor study has been previously described in detail (9) except for the spatial distribution of the tumors which are reported here. Briefly, 260 male SpragueDawley rats in a 2x2x2 factorial design (corn oil, fish oil; pectin, cellulose; plus or minus carcinogen) were used. Rats received two injections of azoxymethane (AOM) (15 mg/kg body weight) separated by 1 week and were terminated 36 weeks after the second AOM injection. The location of each tumor within the colon was determined and statistically evaluated using Chi Square analysis.
The initiation study used the same sex, strain and age of rats as the tumor study and the rats were provided with diets identical to those in the first study. Thirty male weanling SpragueDawley rats (Harlan SpragueDawley, Houston, TX) were individually housed and maintained in a temperature and humidity-controlled animal facility with a daily 15 h light/9 h dark photoperiod. The rats were acclimatized to the facilities for 1 week prior to receiving the defined diets, and then stratified by body weight so that mean initial body weights did not differ between groups. After acclimatization, rats were provided with defined diets for 2 weeks. Subsequently, AOM was injected (15 mg/kg body weight) and rats were terminated at four time points post carcinogen injection (3, 6, 9 and 12 h). A zero time point was used as a negative control (no injection).
Diets
The two defined diets (Table I
) for the initiation and tumor studies differed only in the type of fat (corn oil or fish oil) (9). The major differences between the fatty acid composition of the two lipid sources have been previously reported (10). Dietary fat was provided at 15 g/100 g diet. The fish oil diet contained 3.5 g corn oil/100 g diet to ensure that essential fatty acid requirements were met. Rats were provided with fresh diet every day and the feeders were removed and washed daily. Animals had free access to food and water at all times. Forty-eight hour food intakes were measured after 1 week of receiving the diets. Body weights were recorded each week.
Tissue collection
Rats were killed by CO2 gas overdose, followed by cervical dislocation. The colon was immediately resected and the first 2 cm of the proximal colon and the last 2 cm of the distal colon were subsequently isolated for immunohistochemistry. Each 2 cm length of colon was divided in half lengthwise; one-half was fixed in 70% ethanol for immunohistochemical analysis of O6-deoxymethylguanine (O6-dmetG) and one-half in a 4% paraformaldehyde solution for apoptosis and DNA repair protein assay as previously described (11).
In vivo measurement of O6-deoxymethylguanine
O6-dmetG DNA adducts were measured immunohistochemically using a mouse monoclonal O6-dmetG antibody (provided by Dr C.Wild, University of Leeds, UK) as previously described (11). Tissue sections were deparaffinized, rehydrated and incubated with 0.3% H2O2 in methanol to block endogenous peroxidase followed by 0.05 N NaOH in 40% ethanol to denature DNA. Sections were incubated with the primary antibody followed by peroxidase-conjugated, rabbit anti-mouse IgG (Jackson, West Grove, PA) as the secondary antibody. The antibodyantigen complex was made visible by diaminobenzidine tetrahydrochloride (DAB; Sigma, St Louis, MO). Specificity of the monoclonal primary antibody has been previously demonstrated (12,13). Liver O6-dmetG DNA adducts in AOM-injected animals were used as a positive control (14). Omission of primary antibody was used as a negative control and 0 h time point animals were used as non-injected controls. At least 20 crypt columns in each proximal and distal colon per animal were randomly chosen for analysis according to previously established criteria (15). The staining intensity was assessed by cell position within the crypt using an Image Analysis System (NIH Image, version 1.61) as previously described (11). Images of colonic crypts were captured from a MICROSTAR IV, Reichert microscope using a Sony DXC-970MD 3CCD camera and a Power Macintosh computer. Optimum offset and gain were determined by pre-analysis of multiple darkly and lightly-stained tissues to maximize the distribution of stain intensity so that small differences in staining were quantifiable. For accurate and consistent results, once established, the settings remained constant for all images. Background staining intensity was determined on 10 randomly obtained images per animal and subtracted from the staining intensity of target cells.
In vivo measurement of repair protein expression
Expression of O6-methylguanine-DNA-alkyltransferase (ATase) (EC 2.1.1.63) was determined by quantitative immunohistochemistry using rabbit anti-rat alkyltransferase as the primary antibody (provided by Dr R.H.Elder, Manchester, UK) as previously described (11). The paraformaldehyde-fixed tissue sections were deparaffinized and rehydrated, before the endogenous peroxidase was quenched by immersing the sections in 3% H2O2 in methanol. Antigen accessibility was enhanced by microwave treatment in a 0.1 M sodium citrate solution (pH 6.0). To block non-specific binding, tissue sections were incubated with avidin, biotin (Avidin/Biotin Blocking kit; Vector, Burlingame, CA) and TNB buffer (0.1 M TrisHCl pH 7.5, 0.15 M NaCl, 0.5% blocking reagent; NEN Life Science Products, Boston, MA). Specificity of the primary antibody has been previously described (16). Biotinylated goat anti-rabbit IgG (Jackson, West Grove, PA) was the secondary antibody. The antigenantibody complex was visualized using the Tyramide Signal Amplification System (NEN Life Science Products). Omission of the primary antibody was used as a negative control. At least 20 crypt columns per animal, per site, were evaluated. The staining intensity was assessed within the crypt using an Image Analysis System (NIH Image, version 1.61) as previously described (11). Epithelial cells in the left crypt column of at least 20 well-oriented crypts per animal were captured and the staining intensity was determined for each cell within the crypt. Background staining intensity was determined on 10 randomly obtained images per animal and subtracted from the staining intensity of target cells.
In vivo apoptosis measurement
This assay is performed using a kit from Oncor (Gaithersburg, MD) which is based on TdT (terminal deoxynucleotidyl transferase)-mediated dUTP-biotin nick end-labeling (17). Tissue sections incubated with DNase I (Ambion, Austin, TX) were used as positive controls and sections without TdT enzyme were used as negative controls. A minimum of 20 well-oriented crypts were randomly chosen for these analyses. Crypt height in number of cells and the number and location of apoptotic cells were recorded. The apoptotic index was determined by dividing the number of apoptotic cells by the total number of cells in the crypt column and multiplying by 100.
Statistical analyses
DNA adducts, apoptosis and DNA repair protein were analyzed using two-way ANOVA to determine the effect of fat, time and fatxtime interaction. When P values for the interaction were <0.05, means of all diet groups were separated using StudentNewmanKeuls (SNK) multiple range tests. When P values were <0.05 for the effects of fat, time or carcinogen but not for the interaction, overall means for fat, time or carcinogen treatment groups were separated by SNK multiple range tests. Comparisons between proximal and distal colon on DNA adducts, apoptosis and DNA repair protein were analyzed using paired t-tests.
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Results
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Difference in tumor incidence between the proximal and distal colon
There was a greater than 2-fold difference in adenocarcinoma incidence proximally versus distally (P = 0.001) (Table II
). Diet did not affect proximal versus distal distribution of the tumors (P = 0.1868) (Table II
).
Site specific DNA damage
Figure 1
is a representative image of colonic crypts assayed for DNA adducts at 0 h (Figure 1; A, proximal; B, distal
) and 12 h (Figure 1; C, proximal; D, distal
) after carcinogen injection. Overall adduct levels were similar in the proximal and distal colon (Figure 1
) and increased with time at both sites (P < 0.001) (Figure 2
). Throughout the entire 12 h of the study, fish oil suppressed DNA adduct levels in the distal colon (P < 0.001) (Figure 3
), but there was no diet effect proximally.

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Fig. 1. Photomicrograph (x40) of colonic crypts stained for DNA adduct levels (O6-deoxymethylguanine). (A) Proximal colon, non-injected control (0 h); (B) distal colon, non-injected control (0 h); (C) proximal colon tissue taken 12 h after carcinogen injection and (D) distal colon tissue taken 12 h after carcinogen injection. DNA adduct levels did not differ between the proximal and distal colon at time 0 h (no carcinogen injection) and 12 h post carcinogen injection.
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Fig. 2. DNA adduct levels (O6-dmetG) in colonocytes over time after azoxymethane injection. DNA adduct levels increased throughout the 12 h time course but did not differ between the proximal and distal colon.
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Fig. 3. DNA adduct levels (O6-deoxymethylguanine) in colonocytes as a function of anatomical site and diet. In the proximal colon there was no effect of diet on DNA adduct levels. In contrast, in the distal colon, fish oil resulted in lower adduct levels than did corn oil (P < 0.001).
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Site-specific DNA repair enzyme expression
DNA repair protein (ATase) expression was greater in the distal colon compared with the proximal colon over time (P < 0.001) (Figure 4
). Fish oil feeding resulted in a greater amount of ATase expression in the proximal colon (P < 0.05) (data not shown), but there was no diet effect distally.

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Fig. 4. DNA repair protein expression (O6-methylguanine alkyltransferase) in colonocytes over time after azoxymethane injection. There was greater expression of DNA repair protein in the distal colon compared with the proximal colon (P < 0.001).
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Site-specific apoptosis
There was a much higher level of apoptosis after carcinogen injection in the distal colon relative to the proximal colon (P < 0.0001) (Figure 5A
). Apoptosis reached a peak at 9 h in the distal colon and then decreased (P < 0.001). In the proximal colon there was an interaction between time and diet (P < 0.05) in which apoptosis peaked by 9 h in corn oil-fed rats and then declined (Figure 5B
). In contrast, fish oil feeding resulted in an apoptotic index in the proximal colon that continued to increase throughout the 12 h time period (Figure 5B
) (P < 0.001). Non-carcinogen-injected animals had fewer than one apoptotic cell in the crypt in both the proximal and distal colon (Figure 6A and B
, respectively). After carcinogen injection, there was a relatively small increase in the number of apoptotic cells in the proximal colon (Figure 6C
), yet a large increase in apoptotic cell number was observed in the distal colon (Figure 6D
).

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Fig. 5. (A) Apoptotic index over time in the proximal and distal colon. Apoptotic index increased until 9 h post azoxymethane injection (P < 0.01) and was higher in the distal colon compared with the proximal colon (P < 0.0001). (B) Apoptotic index over time in the proximal colon as a function of diet. In the proximal colon there was an interaction between time and diet in that the corn oil diet peaked by 9 h and declined, whereas fish oil resulted in an apoptotic profile that continued to increase over time (P < 0.05).
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Fig. 6. Photomicrograph (x40) of apoptotic cells. (A) Proximal colon, non-injected control rat; (B) distal colon, non-injected control rat; (C) proximal colon tissue taken 12 h after carcinogen injection and (D) distal colon tissue taken 12 h after carcinogen injection. Apoptotic index increased after carcinogen injection compared with no carcinogen injection and was much higher in the distal colon compared with the proximal colon.
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Discussion
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Slattery et al. (1) reported that both men and women have more proximal cancers with advancing age, which are associated with more advanced disease. In our rat diet/colon tumorigenesis model system we found more than 2-fold the level of tumors in the proximal versus the distal colon at 36 weeks post carcinogen injection. To determine if these site specific differences in tumor development could be explained by events during the earliest stages of tumor initiation (012 h post carcinogen injection) we measured a specific DNA adduct (O6-dmetG), known to be the most persistent and the most likely to cause a mutation (18); a DNA repair protein specific for this adduct and apoptosis which could result in the removal of DNA damaged cells. The localization of DNA damage, repair and targeted apoptosis within the colonic crypt were detected using computerized quantitative immunohistochemistry with digital image analysis. This powerful technique allowed for the simultaneous analysis of all three parameters in vivo in proximal and distal colons of the same rats.
There were three major findings with respect to differences between the proximal and distal colon, each of which is discussed below.
Initial DNA damage
Initial DNA damage was similar between the proximal and distal colon. There were no overall differences in DNA damage between the proximal and distal colon even though later tumor development at 36 weeks post AOM injection was much greater proximally than distally. This disparity between initial DNA damage and later tumor development is also documented in the early literature on DNA adduct formation as a result of AOM or 1,2-dimethylhydrazine administration. Initial DNA damage was higher in rat liver than in rat colon; however, liver tumors were rare (19). DNA damage persisted in rat colon, but not in rat liver (19). This means that the ability to remove or repair the initial damage is probably more important than the damage itself.
DNA repair and apoptosis
Both DNA repair and apoptosis were greater distally than proximally. O6-Deoxymethylguanine adducts may be removed by the specific repair protein for these adducts (ATase) and by eliminating DNA-damaged cells through targeted apoptosis. The importance of the repair protein (ATase) to AOM-induced colon tumors in rats has been recently demonstrated by Wali et al. (20), who provided rats with a potent inhibitor of ATase and detected a significant increase in tumor incidence (65.9% with inhibitor, versus 30.8% in the group not receiving the inhibitor). In our study, DNA repair protein expression was always greater in the distal colon compared with the proximal colon (P < 0.001) (Figure 4
). This suggests that over time more of the DNA damage would be repaired in the distal colon than in the proximal colon. Figure 2
shows that at 12 h (the last measurement time in our study) the curves for DNA damage crossed, with the distal colon showing fewer adducts than that seen proximally. Whether or not this pattern continues after 12 h remains to be tested in a future study.
Similarly, apoptosis in response to AOM injection was higher in the distal colon than in the proximal colon (P < 0.0001) (Figure 5A
). Again, this suggests that over time there should be a greater removal of cells containing DNA adducts in the distal colon than that seen proximally. Since there was a much heightened response to DNA damage in the distal colon versus the proximal colon with a greater expression of ATase and a higher level of apoptosis this would suggest that there would be a lower rate of tumor formation in the distal colon compared with that seen in the proximal colon. Our finding of less than half the number of tumors in the distal versus the proximal colon is consistent with the greater removal of DNA damage seen distally.
Diet effects on DNA damage
Fish oil resulted in lower levels of DNA damage in the distal colon compared with corn oil. Fish oil supplementation resulted in a greater expression of ATase in the proximal colon than did corn oil (P < 0.05). However, it should be noted that overall, the levels of ATase expression were much lower proximally than distally so the small effect of fish oil on repair enzyme was unlikely to have had a significant biological effect. In contrast, in the distal colon, fish oil resulted in lower levels of DNA adducts (P < 0.001). If diet continued to have an effect during promotion and progression this would predict for fewer tumors in the distal colon of fish oil fed rats compared with those fed corn oil. Indeed, 27.0% of the rats provided corn oil had distal colon tumors compared with 19.7% tumor incidence in fish oil supplemented rats. The finding of an effect of dietary fat on the distal but not proximal colon, is consistent with epidemiological findings (2). For example, colon cancer incidence in Japan has increased, coincident with a progressive Westernization of the diet (21). Importantly, only distal colon cancer has increased, and proximal colon cancer has not changed. From these data Weisburger (2) has concluded that proximal colon cancer may not be directly related to diet, a finding supported by our study.
In summary, the goal of this study was to determine if site-specific tumor development could be predicted by an analysis of initial DNA damage, repair and apoptosis during the first 12 h post administration of the carcinogen azoxymethane. The tumor data showed that there was a lower proportion of rats fed fish oil with tumors than rats fed corn oil (fish oil, 56.1%; corn oil, 70.3%; P < 0.05) (9). There was twice the tumor incidence in the proximal versus the distal colon, independent of the diet (P < 0.001). The important diet effect observed within the first 12 h post AOM injection was a lower level of DNA adducts in the distal colon with fish oil feeding. With respect to tumor location (proximal versus distal) three factors observed during the initiation phase favored lower tumor development in the distal colon: (i) A higher level of DNA repair protein expression in the distal colon; (ii) a higher level of apoptosis in the distal colon and (iii) a positive effect of fish oil on DNA adduct levels in the distal colon. Collectively, these data suggest that the response to DNA damage induced by the methylating agent azoxymethane may predict for future tumor development. The determination of site-specific differences in tumor development is important because right and left sided colon cancer appear to have different etiologies (22,23) and thus different strategies may be required to protect against cancer at different sites.
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Notes
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3 Present address: Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA 
4 To whom correspondence should be addressed Email: jlupton{at}tamu.edu 
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Acknowledgments
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The authors thank Dr C.Wild (University of Leeds, UK) for supplying anti-O6-dmetG; Dr R.H.Elder (Manchester, UK) for supplying anti-O6-methylguanine-DNA-alkyltransferase and Ms S.Taddeo for technical support throughout the studies. This work was supported by grants NIH CA61750, NIH CA59034, NIH CA57030 and NIEHS P30 ES 09106.
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Received March 1, 2001;
revised May 29, 2001;
accepted June 8, 2001.