The colonic response to genotoxic carcinogens in the rat: regulation by dietary fibre
Y. Hu,
J. Martin,
R. Le Leu and
G.P. Young,1
Flinders Centre for Digestive Health and Flinders Medical Research Institute, Flinders University of South Australia, Adelaide 5042, Australia
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Abstract
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The apoptotic response to DNA damage appears to be an innate biological mechanism for protection against tumourigenesis. It is possible that agents that protect against colorectal cancer act by enhancing the apoptotic deletion of cells suffering DNA damage, with consequent removal of those with tumourigenic mutations. We examined the acute apoptotic response to genotoxic carcinogens (`AARGC') in colonic epithelium and the possibility that dietary fibres of different fermentability might regulate AARGC. To fully define the time-course and nature of AARGC in response to the carcinogen azoxymethane (AOM), a single injection of AOM (10 mg/kg) was given to rats and apoptosis monitored in the colon by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling staining over a 72 h period. Having defined the site and time of maximum response, two groups of eight rats were fed diets containing 10% wheat bran fibre (WB; fermentable) or 10% methylcellulose (MC; poorly fermentable) for 4 weeks. Colonic AARGC was compared by light microscopy; lumenal short chain fatty acids (SCFAs) and pH were measured as indicators of the fermentative environment. AOM-induced AARGC was maximal at 8 h and greater in distal compared with proximal colon. Apoptotic cells were situated predominantly in the lower half of the crypt, with the median at position 9 indicating involvement of daughter as well as stem cells. There was no `second wave' of apoptosis within 72 h as follows irradiation in small intestine. Distal colonic AARGC in rats fed WB was twice that in rats fed MC (P < 0.01). Compared with MC, WB significantly lowered lumenal pH and increased all SCFAs including butyrate, while proliferation did not differ between the fibres. Certainly, dietary fibres can regulate AARGC and further studies are warranted to determine if this biological effect is the way in which dietary factors regulate tumourigenesis. Lumenal generation of butyrate may enhance AARGC as butyrate is proapoptotic in vitro.
Abbreviations: AARGC, acute apoptotic response to genotoxic carcinogens; AOM, azoxymethane; CRC, colorectal carcinoma; DMH, dimethylhydrazine; H&E, haematoxylin and eosin; SCFA, short chain fatty acid
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Introduction
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Apoptosis provides an innate cellular defence against tumourigenesis in at least two ways: by removal of cells with genomic instability that has developed during tumourigenesis (1) and by deletion of cells suffering DNA insult due to genotoxic carcinogens (2).
Evidence indicates that the regulatory pathways of apoptosis are frequently disabled in colorectal carcinoma (CRC), with an increased threshold for its activation (1) and a progressive disorder of apoptotic homeostasis during carcinogenesis as genomic instability progressively increases (3,4). As a consequence, genetically defective cells escape apoptotic deletion with the possible survival of clones possessing biologically significant mutations.
Genotoxic carcinogenssuch as azoxymethane (AOM)alkylate DNA, forming DNA adducts and initiating tumourigenesis (5). Some damaged cells are repaired but a few are not (6). This failure to delete such mutated cells may give rise to an abnormal clone with the potential to progress to cancer. It has been proposed that this reactive apoptotic response to DNA damage is the biological mechanism for protection against tumourigenesis (2,7).
Little is known about the potential for exogenous regulation of repair and/or of apoptotic deletion of cells with DNA damage. Dietary and other exogenous factors are known to be capable of regulating colorectal tumourigenesis. Some, such as the short chain fatty acid (SCFA) butyrate produced during fermentation of carbohydrate in the colon (8,9), activate apoptosis of CRC cells in vitro (10,11). This raises the possibility that agents that are protective against CRC, act via enhancement of apoptotic deletion of cells with acute DNA damage with consequent removal of those with potentially tumourigenic mutations.
Certain genotoxic carcinogens such as AOM and 1,2-dimethylhydrazine (DMH) induce the dysplasia-carcinoma sequence in rodents and have been used extensively to evaluate the protective effects of dietary and other protective factors. These carcinogens cause DNA adducts, resulting in initiating mutations and subsequent development of tumours (12). DNA damage due to carcinogens causes `nuclear anomalies' in the proliferative compartment of the crypt (13). Subsequent studies have confirmed an acute apoptotic response to genotoxic carcinogens (`AARGC') in the colon (12,14) and to radiation in the small and large intestine (2). However, the characteristics of AARGC in the colon have not been completely defined and the potential for regulation by protective agents has received little attention. To test the possibility that exogenous factors influence tumourigenesis by regulating apoptosis, reproducible models of apoptosis occurring in response to genotoxic damage are needed in the colon.
In the present study, we have further explored the nature of AARGC in colonic epithelium of rats in terms of its distribution along the colon and the crypt, and the possibility of multiple waves of apoptosis (15). We also compared terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) and morphological haematoxylin and eosin (H&E) staining as methods for detection of apoptosis. Finally, we examined the possibility that dietary fibres of different fermentability might regulate AARGC and how such regulation related to changes in the colonic lumenal environment including butyrate levels.
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Materials and methods
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Animals, diets and tissue sampling
Male SpragueDawley rats (150 g) were housed in cages (3/cage) and maintained in a temperature and humidity-controlled animal facility with a 12 h lightdark cycle. Groups, each of eight rats, were fed ad libitum on either rat chow (Ridley AgriProducts, Murray Bridge, South Australia) or a modified AIN diet (9), depending on the experiment (see below). The AIN diets had a protein:carbohydrate:fat balance of 20:55:20 by weight so as to `humanize' the fat contribution to energy intake to 35% and contained 10% dietary fibre by weight, provided either as wheat bran (WB; Purina Natural Bran, Uncle Toby's, Sydney Australia) or methylcellulose (MC; Dow, Midland, MI). WB is readily fermented while MC is not (8). Components in each of the diets were adjusted as described previously (9) so that the protein:carbohydrate:fat ratio was maintained. Diets were prepared in bulk prior to each experiment and stored at 20°C. Body weights of all rats were recorded weekly.
After 4 weeks of stabilization on the allocated diet, rats received a single subcutaneous injection of AOM (Sigma Chemical, St Louis, MO) (10 mg/kg body wt) at 09:00 h. This dose, when repeated over a few weeks, is sufficient to cause colorectal tumourigenesis (9). Rats in each group were killed by CO2-induced narcosis, at selected intervals from 4 through to 72 h. Immediately after death, the entire colon was rapidly removed and divided into two approximately equal proximal and distal portions; the limit of the proximal portion was defined by the `herring-bone' pattern. These were flushed clean with ice-cold saline. Segments of 2 cm were taken from the proximal end of the proximal portion and the rectal end of the distal portion. These segments were immediately fixed in 10% paraformaldehyde overnight at room temperature and embedded in paraffin. The maximum time elapsed between death and fixation was 23 min. Trans-axial sections of 4 mm thickness were taken for histological examination.
Experimental strategy
Two separate sets of experiments were conducted. In the initial set, rats were fed rat chow and the goals were to examine the time-course of, distribution of, and methodologies for detecting, apoptosis induced by the carcinogen. The second set compared the effects of the two fibres on apoptosis over a more limited time-course; it simultaneously examined changes in lumenal environment in the colon. Thus, in this set it was necessary to make up the diets with defined fibre compositions so as to control all variables.
Detection and measurement of apoptosis
The frequency and distribution of epithelial cells undergoing apoptosis were determined by using paraffin-embedded serial sections stained with H&E or TUNEL methods. The TUNEL method identifies apoptotic cells at the single cell level by reacting to the 3'-OH ends of DNA fragments in apoptotic cells. In brief, paraffin sections were dewaxed, rehydrated through descending concentrations of A-grade alcohol and washed with distilled water. Subsequently, the sections were digested with 20 mg/ml proteinase K (Sigma Chemical) for 20 min, washed three times in distilled water and then treated with 0.3% solution of hydrogen peroxide (H2O2) for 20 min to inhibit endogenous peroxidase activity. The sections were then soaked in Tris buffer (30 mM Trizma base pH 7.2, 140 mM sodium cacodylate, 1 mM sodium chloride) for 10 min and then incubated at 37°C for 60 min in a moist chamber with 50 ml of the Tris buffer containing 8.3 U TDT (Boehringer Mannheim, Mannheim, Germany) and 0.83 nmol biotinylated 16-dUTP (Boehringer Mannheim). The reaction was stopped by soaking sections twice in SSC (30 mM sodium chloride, 30 mM sodium citrate), followed by washing in PBS at room temperature for 15 min. The biotinylated dUTP molecules incorporated into nuclear DNA were visualized by incubation with horseradish peroxidase-conjugated streptavidin (Dako, Glostrup, Denmark) diluted 1:100 at room temperature for 30 min. After further washing in PBS for 15 min, the peroxidase colouring reaction was performed by immersing sections for 10 min in 3',3'-diaminobenzidineH2O2 solution. Serial sections were stained with H&E and analysed under the light microscope.
With H&E, apoptotic cells were identified by characteristic morphology, i.e. by cell shrinkage, nuclear condensation and blebbing, and formation of apoptotic bodies (16). With TUNEL, apoptotic cells showed brown nuclear staining.
Twenty separate crypts from each of the proximal and distal segments were chosen and counted by two observers blinded to the experimental conditions. Crypts were considered suitable for counting when sectioned axially from surface to crypt base, with a distinct crypt lumen and a single column of epithelial nuclei on either side. Each side (`crypt column') was counted separately with the total crypt cell count being defined as the number of nuclei in a column from base to surface. The apoptotic index was the number of apoptotic cells divided by the total cells in a crypt column. The position of apoptotic cells was defined by nuclear position counted up from the base of the crypt.
Detection and measurement of proliferation
The proliferative activity in colon epithelial cells was measured using immmunohistochemical staining with anti-PCNA monoclonal antibody (Santa Cruz Laboratories, Santa Cruz, CA). The expression of PCNA was identified by cell nuclei that stained brown to PCNA. In brief, the sections were first incubated in 0.3% H2O2 for 20 min to inhibit the endogenous peroxidase activity, then incubated in 10% normal horse serum for 30 min to block non-specific staining. PCNA antibody was then applied at 1:200 dilution in 10% normal horse serum for 3 h or overnight at room temperature. The slides were then treated sequentially by: three 5 min washes with PBS; incubation with the biotinylated secondary rabbit anti-mouse IgG (Pharmingen, San Diego, CA) for 30 min at room temperature; washing in PBS; incubation with the tertiary avidinbiotin complex reagent (Vector Laboratories, Burlingame, CA) for 30 min at room temperature; rinsing with PBS; incubation with the chromogen 3,3'-diaminobenzidine (Sigma Chemical) at 1 mg/ml with 0.003% hydrogen peroxide for 310 min; rinsed with distilled water; counterstained with H&E; dehydrated and cover-slipped.
Twenty crypts were selected and scored as for apoptosis.
Faecal pH
Freshly passed faeces were collected from each rat 1 week before the rats were killed, and the pH were measured by pH meter as described in ref. 8. Caecal contents and pieces of faeces within the lumen of the proximal and distal colon were obtained from each rat after death and the pH measured in the same way.
SCFA analysis
Immediately after measuring pH, all the samples were weighed, an aliquot mixed in 2 ml of ice-cold saline and stored frozen at 20°C for SCFA analysis. SCFA concentrations were measured using gasliquid chromatography as described previously (17).
Statistical analysis
Statistical analyses were performed using SPSS for Windows, version 10.0 (SPSS, Chicago, IL). Data are expressed as means with standard errors of mean (SEM). One-way ANOVA was used to examine differences between proximal and distal colon, between different time-points, TUNEL and H&E methods, and WB and MC. A probability value of P < 0.05 was used as the critical level of significance. Regression analysis of TUNEL and H&E methods was performed with SPSS.
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Results
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Time-course of apoptosis
In response to AOM, a significant increase in apoptosis was seen over time (Figure 1
), with very few apoptotic cells seen in normal epithelium not exposed to AOM (i.e. time zero). The highest rate of apoptosis occurred at 8 h in the proximal and distal colon. After the 8 h peak, there was a decline in apoptotic rate out to 72 h with no `second wave' of apoptosis. AOM treatment led to a greater rate of apoptosis in distal compared with proximal colon (Figure 1
). The number of induced apoptotic cells per crypt column at the 8 h time point in the distal colon was 4.2 ± 0.4 (SEM) by H&E, compared with 1.6 ± 0.3 (SEM) in the proximal colon (P < 0.01).

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Fig. 1. Time-course of AOM-induced apoptosis in proximal and distal colon (by H&E). Vertical bars represent SEM.
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Site of apoptotic response to carcinogens
Apoptosis was typically observed in epithelial cells predominantly in the lower half of the crypt, by either method (Figures 2 and 3
). The median (peak) position was nine nuclei from the base (interquartile range 514) for both methods. Nuclear staining by TUNEL corresponded closely to morphological changes shown by H&E although not all cells stained by TUNEL were positive by H&E (Figure 2
). Examination of surface cells adjacent to crypt mouths revealed a very low apoptotic score by either method following administration of AOM; <1/100 cells for H&E and for TUNEL.

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Fig. 2. Photomicrographs of AOM-induced apoptosis at 8 h in the distal colonic epithelium, as seen by H&E (A) and TUNEL (B) in semi-adjacent sections. The arrows point to apoptotic cells, that the case of the left side of the left crypt would have been counted by either method. The arrow for the right crypt points to an apoptotic cell seen by both methods.
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Fig. 3. Crypt distribution of AOM-induced apoptosis at 8 h in distal colon, as seen by H&E and TUNEL. Cell position 1 corresponds to the crypt base. The number of cells represents the cumulative count from 20 crypt columns.
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Comparison of H&E and TUNEL
More cells were apoptotic as shown by TUNEL than by H&E in response to AOM (Figure 4
). Comparable rates at 8 h in distal colon were TUNEL 5.1 ± 2.3 (SEM) versus H&E, 4.2 ± 0.4 (P < 0.05). However, the distribution of apoptotic cells in the crypt was similar for both methods (Figure 3
). There was a positive correlation between TUNEL and H&E: TUNEL = H&Ex1.29; r = 0.866, P < 0.0001 (Figure 5
).

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Fig. 4. Apoptotic index in distal colon at 8 h after administration of AOM as measured by H&E and TUNEL. Vertical bars show SEM. *Significant difference between H&E and TUNEL (P < 0.05).
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Effect of AOM treatment on crypt cellularity
Figure 6A and B
shows effect of AOM on crypt column height. In the distal colon, crypt height decreased by an average of about three cells in the 16 h following injection, after which recovery began. By ANOVA, the effect of time was significant (P < 0.001): comparing 0 with 16 h, P = 0.024. In the proximal colon, crypt height decreased by an average of about two cells and was most evident at 24 h after injection, before recovery occurred. By ANOVA, the effect of time was significant (P < 0.0005): comparing 0 with 24 h, P = 0.027.
Effect of fibre type on histology
There were no significant differences in food intake or weight gain for rats given WB compared with MC (data not shown). Administration of WB or MC did not affect the average number of cells per crypt in distal colon as shown in Figure 7
. Crypt column height was 36.7 ± 1.8 for WB in distal colon, compared with 34.8 ± 2.1 for MC (P = NS). In the proximal colon, the number of cells per crypt were 26.8 ± 0.9 for WB and 25.8 ± 0.6 for MC (P = NS).

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Fig. 7. Effect of dietary fibre type on crypt cellularity (A) and proliferation (B), in distal colon prior to administration of carcinogen. No difference is significant. MC, methylcellulose; WB, wheat bran. Vertical bars represent SEM.
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Effect of fibre type on apoptosis
There was a significant difference between the effects of the fibres on AARGC. At 8 h after AOM (the maximal time-point), AARGC in rats fed WB was almost double that seen in rats fed MC, as shown in Figure 8
. The difference was significant in the distal (P < 0.01) but not the proximal colon (P > 0.05, data not shown). As there was a theoretical possibility that MC might delay the peak for some reason, we included a measurement of AARGC at 12 h. As can be seen from Figure 8
, AARGC did not rise above the 8 h value in the MC fed rats and remained significantly lower than in the WB fed rats (P < 0.05). Baseline apoptosis in the absence of AOM was not affected by the dietary fibre type, with crypt column counts being the same as for rat chow shown in Figure 1
(<0.5 apoptotic cells/crypt column).

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Fig. 8. Effects of fermentable fibre WB and non-fermentable fibre MC on genotoxin-induced apoptosis in response to AOM, in proximal and distal colon, at 8 and 12 h after adminstration of AOM. The difference in effects of fibre on distal colon was significant (P < 0.01). Vertical bars represent SEM.
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Effect of fibre type on proliferation
There was no significant difference between the effects of the fibres on proliferation in distal colon when expressed as the percentage of positively staining cells per crypt column (Figure 7
). Likewise, in the proximal colon there was no difference between the two groups: corresponding labelling indexes for WB and MC were 5.6 and 6.6%, respectively (P = NS).
Effect of fibre type on lumenal events
WB caused a significant reduction in lumenal pH at all points of fecal sampling (Table I
) when compared with MC. The effects of fibre types on faecal SCFA concentration are shown in Figure 9
. Concentrations of the three main SCFAs, acetate, propionate and butyrate, were significantly higher in faeces of rats consuming WB compared with rats consuming MC (P = 0.028, P = 0.00001 and P = 0.0005, respectively). We also measured lumenal parameters of fermentation in rats fed chow and these were similar to those fed WB: in faeces, pH 6.4 ± 0.1, acetate 28.8 ± 3.2 mmol/g, propionate 3.2 ± 1.0 mmol/g and butyrate 10.6 ± 1.7 mmol/g.

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Fig. 9. Effects of fermentable fibre WB and non-fermentable fibre MC on fecal SCFA concentrations. Vertical bars represent SEM.
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Discussion
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The intestinal response to DNA damage has been most studied in the small intestine, using radiation as the damaging agent. Irradiation of the small intestine results in markedly increased apoptosis in the proliferative compartment with the maximum at 3 h (2,18). The DNA damage-recognition protein p53 is critical as radiation-induced apoptosis is attenuated in p53 null mice; the cell cycle arrest cyclin-dependent kinase p21Waf1 is also involved (18). A second wave of apoptosis follows at
48 h, that is not dependent on p53 (15).
Information on the colonic acute apoptotic response to DNA insult is more limited. The response to irradiation is similar in magnitude along the length of the colon and the maximum is reached at 45 h (2). Apoptosis occurs in the proliferative compartment of the crypt, predominantly in the basal cell positions of 16, implying that stem cells are affected (2). Apoptosis in proliferative cells is much more frequent than the occasional, senescence-related, apoptosis occurring in mature cells on the surface epithelium (2,19), although some investigators have claimed fairly frequent apoptosis in the differentiated compartment on the basis of the TUNEL technique (3,20).
In response to DNA insult by the alkylating agent AOM in the colon, we show a maximal apoptotic response at 8 h of greatest magnitude in distal colon. While AARGC in the colon is confined to the proliferative compartment, as is the case with irradiation, the maximal effect is at position 9, well above that of the stem cells and implying that daughter cells are more susceptible to damage by carcinogen. Expression of the anti-apoptotic protein bcl-2 has been shown to be largely restricted in the colon to just the most basal cells (positions 13) (21). This could explain why AARGC is more common in cell positions above this level.
Thus, the colonic apoptotic response to DNA damage by genotoxic carcinogen (AARGC) is clearly different from that occurring in response to irradiation. The maximum time-point and peak position in the crypt are different. Furthermore, there is no `second wave' apparent out as far as 72 h, as occurs in small intestine after irradiation (15).
Our observations extend those of Hirose et al. (12) and agree with their observations that AARGC is more frequent in the distal colon and maximal at 8 h; however, they did not study sufficient time-points to exclude a second wave at
4872 h. In an earlier study, Ijiri (14) has observed a circadian rhythm for AARGC in the mouse colon following administration of DMH (a pro-drug for AOM). They also examined the effect of irradiation in the colon and confirmed an earlier maximal effect at 3 h.
Various methods for measuring apoptosis are available that vary in utility and biological significance (22). Characteristic morphological changes in nuclei are considered to be the most accurate (22) but careful training in their recognition is important as they can be difficult to see and easily confused with intra-epithelial lymphocytes and mitotic figures. So, we compared morphological changes observed by H&E with TUNEL. TUNEL is an in situ direct immuno-peroxidase method for detecting DNA strand breaks. Our results indicated that both methods correlated well and provided identical distributions of apoptosis along the colonic crypt axis. We saw very little DNA fragmentation by TUNEL at the surface, agreeing with some (2,19) but in contrast to others, where staining might have been non-specific (3,20). The regression equation showed that counts were higher by TUNEL than H&E, an observation that corresponds with that of others (23). It is probable that TUNEL is more sensitive, detecting DNA strand breaks before nuclear changes become advanced and obvious. Indeed, some studies have used TUNEL as confirmation of H&E morphology and only count those TUNEL-positive cells also positive by H&E (23). We considered morphologic analysis by H&E to be adequate to identify apoptosis in the remaining experiments.
As many epidemiological studies indicate that environmental factors, largely dietary, influence CRC (24), it is conceivable that dietary factors might regulate AARGC in the colon. Protective factors might improve the efficiency of AARGC in deleting mutated cells with the potential to progress to cancer. Dietary factors might act systemically, or locally via changes in the colonic lumenal environment. Thus, we chose to test the regulatory effect of fermentable dietary fibre, using a non-fermentable fibre as control, to see if any regulation could be related to changes in the lumenal environment. Colonic fermentation generates SCFAs including butyrate; WB in particular, elevates distal colonic butyrate levelsthe site at which CRC is most common (8). The distal colon is also the site at which AARGC is at maximum. Because butyrate has been shown to facilitate apoptosis in vitro in CRC cell lines, by inhibition of histone deacetylase (10), we hypothesized that any regulatory effect of WB in vivo might be mediated in part by generation of butyrate.
In the present study, supplementation of diets with WB for 4 weeks altered the lumenal environment relative to MC, as a result of active fermentation. This was associated with a significantly higher rate of AARGC in the distal colon. At the same time, WB did not significantly affect proliferation, indicating that increased AARGC was not simply due to increased DNA damage subsequent upon increased exposure of DNA to the carcinogen in S-phase. AARGC is obviously subject to exogenous regulation.
The protective value of dietary fibre, at least in the rat carcinogen model, has been linked to the production of butyrate and especially the concentration of butyrate in the distal colon (8,9), although the association has not always been seen (25). WB, in contrast to other fibres, generates a greater concentration of butyrate in the distal colon (8). Apart from being the preferred metabolic fuel for the colonocyte (26), butyrate has been shown to increase differentiation and induce apoptosis (10,27). In the present study, WB had a range of lumenal effects, increasing the concentration of all three major SCFAs and lowering pH relative to MC. Thus, it is not possible to be certain that butyrate is responsible, but given its in vitro effects of apoptosis induction, the findings are consistent with the proposition that in vivo generation of butyrate does regulate AARGC.
Based on epidemiological evidence and some experimental models, it is possible that lowered pH is protective for CRC and could thus possibly modulate AARGC. But a number of other studies have failed to show that merely acidifying the colon had a protective effect (28).
The effects of other exogenous factors on apoptosis, has been examined at various stages in the carcinogen model. Chicory-derived ß-fructans increase apoptosis 24 h after DMH (23) but the timing has been criticized because it does not coincide with the maximum apoptotic response (14). The tumour promoter lithocholic acid decreases apoptosis in the DMH model, but 4 weeks after administration of the carcinogen (29); no acute response was measured here. Sinigrin, found in brassica vegetables, also increases apoptosis with the same carcinogen but after a complex dosing schedule of AOM started 5 days prior to measurement (30). The relevance of these experiments to regulation of AARGC is thus uncertain. Standardization of methods so that interventions are evaluated for their impact at the point of maximal AARGC is important, if we are to determine biological relevance of regulation of AARGC to subsequent events in tumourigenesis.
In conclusion, AARGC in the colon is maximal at 8 h in response to AOM, involves daughter cells in the proliferative compartment, not just stem cells, and is greatest in the distal colon. There is no `second wave' as is the case for the apoptotic response to irradiation in small intestine. AARGC is regulated by fermentable fibre; lumenal generation of butyrate may be the mediator of this effect. Certainly, dietary agents can regulate AARGC. Whether regulation of AARGC protects by more efficient removal of mutated cells that might progress to cancer, remains to be demonstrated by measurement of post-initiation DNA damage and/or events in tumourigenesis occurring soon after initiation. Validation of this model in such a manner should also clarify the tumour-protective contribution of regulation of AARGC relative to regulation of postinitiation events.
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Notes
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1 To whom correspondence should be addressed Email: graeme.young{at}flinders.edu.au 
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Acknowledgments
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This study was supported by a project grant from the National Health and Medical Research Council of Australia.
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Received October 23, 2001;
revised March 28, 2002;
accepted April 5, 2002.