BACKGROUND
colorectal cancer  is the third most common cancer in males and second most common in females world-wide
<cit> .
the majority of these cancers are considered preventable by appropriate diet and associated lifestyle factors
<cit> .
dietary patterns consisting of micronutrient dense, low-fat, high-fibre food patterns protect against colorectal cancer
<cit> .
conversely, specific sources of dietary protein have been linked to increased crc risk
<cit>  and animal studies have indicated that different dietary proteins can induce dna damage in the rats’ colon
<cit> .
consequently, the challenge is to translate this information into strategies that prevent crc.
one of the first steps to doing this is to understand the early molecular events involved in oncogenesis and develop hypotheses on the role played by environmental factors such as diet in this process.
the azoxymethane -treated rodent provides an important tool in the study of sporadic crc development and progression
<cit> .
it has been used extensively to study colon carcinogenesis and its prevention, in at least two formats that model different aspects of crc
<cit> .
one version of this model studies tumour development  to find the underlying signalling pathways of colon carcinogenesis.
for instance, it has been used to investigate mouse models of colorectal carcinogenesis using gene expression profiling and has provided significant insights into the role of reactivated embryonic signatures in colon tumours
<cit> .
the other main version of the aom model is the ‘cancer initiation’ model, which is used to study the early response to the carcinogen, where tissues are harvested shortly after treatment
<cit> .
using this latter acute aom model, we report here some of the early transcriptional events induced by this carcinogen in mucosal tissue along the length of the colon in rats.
RESULTS
the colonic epithelium is one of the largest epithelial barriers in the body and is in a constant state of self-renewal.
in order to understand the effects of a carcinogenic insult to this tissue, it is important to develop an understanding of the natural morphologic and molecular features of the normal rat colon.
it has been demonstrated that rat colonic stem cells are located in different positions and behave differently in crypts sampled from different points along the anterior-posterior length of the colon
<cit> .
in distal sections, stem cells are located in the crypt base from whence progeny differentiating cells then migrate up towards the lumen, ultimately undergoing anoikis and sloughing off into the digesta
<cit> .
in proximal sections, stem cells are located in the middle one-third of the crypt.
differentiating cells migrate bi-directionally from this source with some differentiating colonocytes migrating towards the lumen, while others migrate into the crypt base
<cit> .
our own data confirm the observations of others that the crypt height in the normal rat distal colon is greater than that for the proximal colon .
despite these morphological differences, no significant differences in rates of baseline apoptosis between the proximal and distal normal  colon  were observed.
it has been shown in mice that significant numbers of genes are differentially expressed along functionally distinct regions of the gastrointestinal tract
<cit>  and this is also true for the normal human colon
<cit> .
when gene expression in the normal colon of the rat was examined at the level of individual genes, the proximal expression profile differed markedly from that of the distal colonic epithelium with  <dig> genes differentially expressed   <dig> ) .
these genes are listed in additional file
1: table s <dig>  this microarray dataset was also validated by demonstrating that the top  <dig> genes most differentially expressed between the proximal and distal colon were also 100% consistently differentially expressed using real time rtpcr .
when the functional groupings of these genes were considered through pathway analysis, most of the top  <dig> pathways identified were broadly associated with intestinal metabolism functions .
the magnitude of these changes in expression can be very large .
we consider this association with metabolism most likely reflects the changing profile of digestive functions naturally occurring along the length of the colon.
consequently, these position-associated profiles provide the background against which changes in gene expression induced by colonic carcinogens need to be assessed.
the carcinogens aom and  <dig> -dimethylhydrazine are metabolised by cytochrome p <dig>  into methylazoxymethanol.
in turn this breaks down to form highly reactive alkylating species which can lead to the addition of methyl adducts at the o <dig> position of guanine residues in the dna to form the promutagenic modified base o <dig> methyl guanine .
if this modified base is not repaired, it can lead to g:c to a:t transition mutations during replication tan
<cit> .
these dna adduct-induced mutations are found commonly in colorectal cancers
<cit> .
so not surprisingly, aom induces substantial transcriptional changes in the mucosa of the rat colon six hours after subcutaneous administration .
the genes differentially expressed in response to aom are listed in additional file
1: table s <dig>  there were  <dig> and  <dig> genes differently expressed  in the proximal and distal colons respectively of aom-treated rats when compared with the same tissues from normal  animals.
the fold changes were up to  <dig>  in the proximal and  <dig>  in the distal colon.
at a whole genome level, principal component analysis  revealed that the magnitude of the site effect on gene expression  was equal to or greater than that of aom for the two highest principal components  .
further examination of the pca revealed that pc <dig> and pc <dig> best explained the effect of aom , and pc <dig> and pc <dig> best explained the effect of ‘site’ .
as it has been previously shown that the greatest effects of aom in the rat, in terms of tumours numbers are exhibited in the distal colon
<cit>  and human tumours predominately occur in the most distal colonic region, i.e.
sigmoid colon and the rectum
<cit> , it is not surprising that there almost 10-fold more genes expressed in the distal rat colon at  <dig> hours post treatment.
as a consequence, this report will concentrate predominantly on the effects this carcinogen in this colonic region with a particular focus on dna damage and repair.
in a previous study using the “cancer initiation” aom model in sprague dawley rats, tan et al.
measured levels of o6-mdgua accumulating in the dna from a number of tissues harvested  <dig> hours and  <dig> hours after subcutaneous injection of this carcinogen.
they observed that  <dig> hours after aom exposure, the highest levels o6-mdgua occurred in the following tissues : liver, distal colon, proximal colon, proximal small intestine , and kidney.
the stomach, distal si, bladder, spleen, blood and lung had relatively low levels o6-mdgua.
while levels of this highly mutagenic alkylation product had dropped in most tissues tested by  <dig> h post aom administration, o6-mdgua levels remained high at this time point in the proximal and distal colon, kidney and bladder.
this is a significant finding as the distal colon is more prone to aom induced tumours than any other tissue
<cit>  and tumours in the bladder and kidney have been observed in animals treated with high levels of dimethyl hydrazine, a precursor of aom
<cit> .
a key enzyme involved in the repair of o6-mdgua is o-6-methylguanine dna methyltransferase .
in a ‘suicide’ reaction the methyl adduct from one modified guanine base is transferred to a cysteine residue in the active site of one molecule of enzyme resulting in the inactivation of that molecule of enzyme and earmarking it for ubiquitination and degradation
<cit> .
interestingly, in the current study, in the normal colon, the level of expression of mgmt was greater in the distal section compared to the proximal section .
this would be consistent with an adaptation to a higher basal metabolic demand for dna adduct repair in the distal colonic mucosa relative to the proximal.
this could arise in response to dietary mutagens in the colonic digesta becoming more concentrated as more and more water is removed during its transit from proximal to distal colon.
whatever the drivers may be, however, this change in mgmt levels from proximal to distal colon is likely to form a part of an innate homeostatic process to maintain genomic integrity in a healthy colonic mucosa.
six hours after the administration of aom, mgmt expression was down-regulated in both the proximal and distal colonic epithelium .
as there are high levels of o6-mdgua present in the dna of the distal colon at this time
<cit>  and with mgmt being the primary enzyme for repair of dna methyl adducts, it appears that mgmt is rapidly depleted instead of being up-regulated in response to aom.
as the animals survive aom challenge well with no apparent significant loss of colonic function, this observation suggests that other repair mechanisms are brought into play to ensure the rapid return to normal colonic function.
further analysis revealed that the expression of a number of other dna repair and damage genes was also altered in response to aom, particularly in the distal colon .
expression of the damaged dna binding and sensing h2a histone family member x  gene was significantly up-regulated in response to aom  , confirming that repair mechanisms other than mgmt are deployed in response to the aom perturbation.
in terms of single strand break repair, there are a number of nucleotide-excision repair   genes differentially expressed in the distal colon in response to aom treatment and 80% of them were up-regulated.
this is important as ner is the most flexible of the dna repair pathways as it repairs bulky dna lesions
<cit> .
other base-excision repair associated genes also showing increased expression in response to treatment with aom include apex <dig>  had a  <dig>  fold change and polβ,  a  <dig>  fold change .
the mismatch repair  pathway is an important pathway involved in the dna damage response to carcinogen induced lesions resulting in cell cycle arrest and, at high lesion load, apoptosis
<cit> .
however, aom treatment led to the down-regulated response of mmr genes .
for instance, msh <dig> ), which recognises insertion/deletion mismatches containing two or more extra bases
<cit>  showed decreased expression  with aom .
these observations suggest that the mmr pathway in general may be down-regulated in response to aom and are consistent with aom’s major mode of action involving dna adduct formation and induction of point mutations rather than the formation of multi-base mismatches.
double-strand breaks , in which both strands in the dna double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements
<cit> .
dsb repair via homologous recombination  is an important process as it takes place late in the s- and g2-phases of the cell cycle to prevent unrepaired double strand breaks from causing down-stream problems in transcription, replication and chromosome segregation
<cit> .
in the distal colon there were nine genes from this pathway up-regulated in response to aom.
for instance, xrcc <dig>  which plays a central role in this pathway and encodes a member of the rad <dig> family of proteins, was up-regulated  <dig> -fold.
conversely, the dsb repair via non-homologous end-joining  pathway was down-regulated with aom, demonstrated by the decreased expression of xrcc <dig>  .
consequently, there is some evidence that single and double strand break repair functions may be compromised in response to aom treatment.
these data coupled with the accumulation of unrepaired o6-mdgua lesions in colonic epithelium in response to carcinogen, indicates that at six hours post treatment other cellular processes such as cell cycle arrest and apoptosis becomes more important in maintaining mucosal integrity in response to this genomic insult.
to investigate the biological consequences of unrepaired dna damage, such as the o6-mdgua lesions, the functionality of the top  <dig> genes differentially expressed in response to aom in the proximal and distal colon, was examined through pathways analysis.
the top  <dig> pathways in which the highest percentage of component genes displayed aom-associated differential expression relative to the saline treated control in the distal and proximal colonic mucosa are shown in figure
<dig>  there was a number of cell cycle regulation pathways differentially expressed in the distal colon  <dig> hours after the treatment with aom.
these included, “p <dig> signalling” and “cell cycle regulation by b cell translocation ” .
this is noteworthy as deregulated cell cycle processes are a prominent feature of oncogenesis
<cit> .
overall, the response suggests a trend towards cell cycle arrest in response to aom.
dna damage checkpoint control mechanisms tightly regulate progression through the cell cycle, ensuring the fidelity of cell division which is an important self-defence mechanism for the maintenance of genome stability
<cit> .
a number of observations support the involvement of the p <dig> signalling  and btg pathways in aom-induced cell cycle arrest in the distal colon: cyclin g <dig>  a protein involved in g2/m phase arrest and regulates p <dig>
<cit>  expression increased  <dig>  fold; cyclin dependent kinase inhibitor  <dig> , which inhibits the activity of cyclin-cdk <dig> or -cdk <dig> complexes
<cit> , is marginally up-regulated, and as a result retinoblastoma  <dig>  is down-regulated ; btg family member  <dig> , an important transcriptional regulator that impairs g1-s cell cycle progression
<cit>  increased  <dig>  fold and mdm <dig>   which is also a member of another expressed pathway, “cell cycle: g2/m dna damage checkpoint regulation” .
this latter cell cycle pathway has other genes differentially expressed.
for example, chek <dig>  which is a checkpoint regulator of cell cycle arrest and putative tumour suppressor in response to dna damage
<cit> , is up-regulated .
most cancer cells harbour mutations in tumour suppressors and/or oncogenes which would normally control cell cycle checkpoints
<cit> .
therefore, cell cycle regulation is important in the maintenance of genomic stability and to prevent cells that have undergone malignant transformation progressing through the cell cycle phases.
1: tables <dig>
the p <dig> signaling pathway  is significantly expressed  in response to aom.
loss of p <dig> function is thought to be a contributing factor in colorectal cancer, because the p53-dependent pathway shuts down damaged cells, either through apoptosis, cell-cycle arrest or cellular senescence
<cit> .
apoptosis was observed histologically to have significantly increased at six hours after the aom treatment .
as mgmt is depleted at this time point, we hypothesise that the main cellular response to aom involves the early depletion of mgmt then a switch to the induction of apoptosis and this most likely first occurs through the p <dig> signalling pathway.
this is demonstrated through closer examination of the p <dig> signalling pathway response to aom in the distal colon .
firstly, genes such as tp53inp <dig> , which is a key transcriptional regulator that responds to a variety of cellular stresses, including dna damage, oxidative stress and activated oncogenes, to regulate key cellular processes including the induction of apoptosis
<cit> , is up-regulated  <dig>  fold.
furthermore, caspases  <dig>  and casp <dig> are up-regulated in response to aom in the distal colon .
the activation of casp <dig> triggers an execution arm of the apoptosis response initiating dna fragmentation
<cit> .
the apoptotic function of caspases is regulated by the bcl- <dig> family of proteins
<cit> .
accordingly, in response to aom, bcl-associated x protein  which is critically important in the up-regulation of apoptosis, is increased two fold.
furthermore, there is also decreased expression of the caspase-activated inhibitor avon by  <dig> -fold.
therefore, these results indicate that at  <dig> hours after being treated with aom, one of the major effects of this carcinogen occurs through the p <dig> signalling pathway and the result is cell cycle arrest and a cellular switch towards apoptosis.
gene network analysis was used to further understand the function of genes expressed in response to aom in each colonic segment, in particular the early induction of apoptosis.
when the top three aom/proximal networks were merged  there were  <dig> genes with functional annotations  associated with colorectal cancer including the up-regulation  of transcriptional regulator c-jun  and the down-regulation of mgmt .
in terms of the aom/distal network there were eight genes associated with colon cancer , including the transcriptional regulator myc  and the previously mentioned c-jun .
when the p <dig> signalling pathway and apoptosis genes were cross-referenced in this network, c-jun kinase , which regulates cjun and is an important regulator of cell death
<cit> , was a gene common to both and is linked through the gene network to the down-regulated p <dig> signalling transcriptional regulator, retinoblastoma  <dig> .
taken together these results suggest that the early genomic damage effects of aom on the colonic mucosa may be mediated through the p <dig> pathway, favouring apoptosis through c-jun/jnk signalling and preventing cell cycle progression through reduced rb <dig> expression.
whilst this mechanism is hypothetical, it provides a framework for the further elucidation of the key mechanisms underpinning the cellular switch towards apoptosis in the gut mucosa in response to alkylating carcinogen challenge.
CONCLUSIONS
the healthy rat colonic mucosa exhibits extensive gene expression changes from its proximal to distal end reflecting regional changes in metabolic function.
the normal rat colon also has naturally occurring protective and genomic repair mechanisms expressed dynamically, albeit subtly, along the proximal/distal axis.
six hours after administration of aom, substantial changes in gene expression have occurred in the colonic mucosa and these also differ along the length of the colon.
the changes are greater in the distal colon and appear particularly associated with the sensing of genomic damage, associated cell cycle arrest and a cellular switch towards the induction of apoptosis.
consequently, the genomic homeostasis mechanisms that naturally exist to combat dietary and environmental insults in the colon of the normal rat appear to be dysregulated by aom resulting in a cellular switch through p <dig> signaling to more efficient genes associated with the apoptotic response, a genetic response that is also reflected histologically.

 colorectal cancer is the third most common cancer in males and second most common in females world-wide. the majority of these cancers are considered preventable by appropriate diet and associated lifestyle factors. dietary patterns consisting of micronutrient dense, low-fat, high-fibre food patterns protect against colorectal cancer. conversely, specific sources of dietary protein have been linked to increased crc risk and animal studies have indicated that different dietary proteins can induce dna damage in the rats’ colon. consequently, the challenge is to translate this information into strategies that prevent crc. one of the first steps to doing this is to understand the early molecular events involved in oncogenesis and develop hypotheses on the role played by environmental factors such as diet in this process. the azoxymethane -treated rodent provides an important tool in the study of sporadic crc development and progression. it has been used extensively to study colon carcinogenesis and its prevention, in at least two formats that model different aspects of crc. one version of this model studies tumour development to find the underlying signalling pathways of colon carcinogenesis. for instance, it has been used to investigate mouse models of colorectal carcinogenesis using gene expression profiling and has provided significant insights into the role of reactivated embryonic signatures in colon tumours. the other main version of the aom model is the ‘cancer initiation’ model, which is used to study the early response to the carcinogen, where tissues are harvested shortly after treatment. using this latter acute aom model, we report here some of the early transcriptional events induced by this carcinogen in mucosal tissue along the length of the colon in rats.  the colonic epithelium is one of the largest epithelial barriers in the body and is in a constant state of self-renewal. in order to understand the effects of a carcinogenic insult to this tissue, it is important to develop an understanding of the natural morphologic and molecular features of the normal rat colon. it has been demonstrated that rat colonic stem cells are located in different positions and behave differently in crypts sampled from different points along the anterior-posterior length of the colon. in distal sections, stem cells are located in the crypt base from whence progeny differentiating cells then migrate up towards the lumen, ultimately undergoing anoikis and sloughing off into the digesta. in proximal sections, stem cells are located in the middle one-third of the crypt. differentiating cells migrate bi-directionally from this source with some differentiating colonocytes migrating towards the lumen, while others migrate into the crypt base. our own data confirm the observations of others that the crypt height in the normal rat distal colon is greater than that for the proximal colon. despite these morphological differences, no significant differences in rates of baseline apoptosis between the proximal and distal normal colon were observed. it has been shown in mice that significant numbers of genes are differentially expressed along functionally distinct regions of the gastrointestinal tract and this is also true for the normal human colon. when gene expression in the normal colon of the rat was examined at the level of individual genes, the proximal expression profile differed markedly from that of the distal colonic epithelium with genes differentially expressed ). these genes are listed in additional file 1: table s this microarray dataset was also validated by demonstrating that the top genes most differentially expressed between the proximal and distal colon were also 100% consistently differentially expressed using real time rtpcr. when the functional groupings of these genes were considered through pathway analysis, most of the top pathways identified were broadly associated with intestinal metabolism functions. the magnitude of these changes in expression can be very large. we consider this association with metabolism most likely reflects the changing profile of digestive functions naturally occurring along the length of the colon. consequently, these position-associated profiles provide the background against which changes in gene expression induced by colonic carcinogens need to be assessed. the carcinogens aom and -dimethylhydrazine are metabolised by cytochrome p into methylazoxymethanol. in turn this breaks down to form highly reactive alkylating species which can lead to the addition of methyl adducts at the o position of guanine residues in the dna to form the promutagenic modified base o methyl guanine. if this modified base is not repaired, it can lead to g:c to a:t transition mutations during replication tan. these dna adduct-induced mutations are found commonly in colorectal cancers. so not surprisingly, aom induces substantial transcriptional changes in the mucosa of the rat colon six hours after subcutaneous administration. the genes differentially expressed in response to aom are listed in additional file 1: table s there were and genes differently expressed in the proximal and distal colons respectively of aom-treated rats when compared with the same tissues from normal animals. the fold changes were up to in the proximal and in the distal colon. at a whole genome level, principal component analysis revealed that the magnitude of the site effect on gene expression was equal to or greater than that of aom for the two highest principal components. further examination of the pca revealed that pc and pc best explained the effect of aom, and pc and pc best explained the effect of ‘site’. as it has been previously shown that the greatest effects of aom in the rat, in terms of tumours numbers are exhibited in the distal colon and human tumours predominately occur in the most distal colonic region, i.e. sigmoid colon and the rectum, it is not surprising that there almost 10-fold more genes expressed in the distal rat colon at hours post treatment. as a consequence, this report will concentrate predominantly on the effects this carcinogen in this colonic region with a particular focus on dna damage and repair. in a previous study using the “cancer initiation” aom model in sprague dawley rats, tan et al. measured levels of o6-mdgua accumulating in the dna from a number of tissues harvested hours and hours after subcutaneous injection of this carcinogen. they observed that hours after aom exposure, the highest levels o6-mdgua occurred in the following tissues: liver, distal colon, proximal colon, proximal small intestine, and kidney. the stomach, distal si, bladder, spleen, blood and lung had relatively low levels o6-mdgua. while levels of this highly mutagenic alkylation product had dropped in most tissues tested by h post aom administration, o6-mdgua levels remained high at this time point in the proximal and distal colon, kidney and bladder. this is a significant finding as the distal colon is more prone to aom induced tumours than any other tissue and tumours in the bladder and kidney have been observed in animals treated with high levels of dimethyl hydrazine, a precursor of aom. a key enzyme involved in the repair of o6-mdgua is o-6-methylguanine dna methyltransferase. in a ‘suicide’ reaction the methyl adduct from one modified guanine base is transferred to a cysteine residue in the active site of one molecule of enzyme resulting in the inactivation of that molecule of enzyme and earmarking it for ubiquitination and degradation. interestingly, in the current study, in the normal colon, the level of expression of mgmt was greater in the distal section compared to the proximal section. this would be consistent with an adaptation to a higher basal metabolic demand for dna adduct repair in the distal colonic mucosa relative to the proximal. this could arise in response to dietary mutagens in the colonic digesta becoming more concentrated as more and more water is removed during its transit from proximal to distal colon. whatever the drivers may be, however, this change in mgmt levels from proximal to distal colon is likely to form a part of an innate homeostatic process to maintain genomic integrity in a healthy colonic mucosa. six hours after the administration of aom, mgmt expression was down-regulated in both the proximal and distal colonic epithelium. as there are high levels of o6-mdgua present in the dna of the distal colon at this time and with mgmt being the primary enzyme for repair of dna methyl adducts, it appears that mgmt is rapidly depleted instead of being up-regulated in response to aom. as the animals survive aom challenge well with no apparent significant loss of colonic function, this observation suggests that other repair mechanisms are brought into play to ensure the rapid return to normal colonic function. further analysis revealed that the expression of a number of other dna repair and damage genes was also altered in response to aom, particularly in the distal colon. expression of the damaged dna binding and sensing h2a histone family member x gene was significantly up-regulated in response to aom, confirming that repair mechanisms other than mgmt are deployed in response to the aom perturbation. in terms of single strand break repair, there are a number of nucleotide-excision repair genes differentially expressed in the distal colon in response to aom treatment and 80% of them were up-regulated. this is important as ner is the most flexible of the dna repair pathways as it repairs bulky dna lesions. other base-excision repair associated genes also showing increased expression in response to treatment with aom include apex had a fold change and polβ, a fold change. the mismatch repair pathway is an important pathway involved in the dna damage response to carcinogen induced lesions resulting in cell cycle arrest and, at high lesion load, apoptosis. however, aom treatment led to the down-regulated response of mmr genes. for instance, msh ), which recognises insertion/deletion mismatches containing two or more extra bases showed decreased expression with aom. these observations suggest that the mmr pathway in general may be down-regulated in response to aom and are consistent with aom’s major mode of action involving dna adduct formation and induction of point mutations rather than the formation of multi-base mismatches. double-strand breaks, in which both strands in the dna double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. dsb repair via homologous recombination is an important process as it takes place late in the s- and g2-phases of the cell cycle to prevent unrepaired double strand breaks from causing down-stream problems in transcription, replication and chromosome segregation. in the distal colon there were nine genes from this pathway up-regulated in response to aom. for instance, xrcc which plays a central role in this pathway and encodes a member of the rad family of proteins, was up-regulated -fold. conversely, the dsb repair via non-homologous end-joining pathway was down-regulated with aom, demonstrated by the decreased expression of xrcc. consequently, there is some evidence that single and double strand break repair functions may be compromised in response to aom treatment. these data coupled with the accumulation of unrepaired o6-mdgua lesions in colonic epithelium in response to carcinogen, indicates that at six hours post treatment other cellular processes such as cell cycle arrest and apoptosis becomes more important in maintaining mucosal integrity in response to this genomic insult. to investigate the biological consequences of unrepaired dna damage, such as the o6-mdgua lesions, the functionality of the top genes differentially expressed in response to aom in the proximal and distal colon, was examined through pathways analysis. the top pathways in which the highest percentage of component genes displayed aom-associated differential expression relative to the saline treated control in the distal and proximal colonic mucosa are shown in figure there was a number of cell cycle regulation pathways differentially expressed in the distal colon hours after the treatment with aom. these included, “p signalling” and “cell cycle regulation by b cell translocation ”. this is noteworthy as deregulated cell cycle processes are a prominent feature of oncogenesis. overall, the response suggests a trend towards cell cycle arrest in response to aom. dna damage checkpoint control mechanisms tightly regulate progression through the cell cycle, ensuring the fidelity of cell division which is an important self-defence mechanism for the maintenance of genome stability. a number of observations support the involvement of the p signalling and btg pathways in aom-induced cell cycle arrest in the distal colon: cyclin g a protein involved in g2/m phase arrest and regulates p expression increased fold; cyclin dependent kinase inhibitor, which inhibits the activity of cyclin-cdk or -cdk complexes, is marginally up-regulated, and as a result retinoblastoma is down-regulated; btg family member, an important transcriptional regulator that impairs g1-s cell cycle progression increased fold and mdm which is also a member of another expressed pathway, “cell cycle: g2/m dna damage checkpoint regulation”. this latter cell cycle pathway has other genes differentially expressed. for example, chek which is a checkpoint regulator of cell cycle arrest and putative tumour suppressor in response to dna damage, is up-regulated. most cancer cells harbour mutations in tumour suppressors and/or oncogenes which would normally control cell cycle checkpoints. therefore, cell cycle regulation is important in the maintenance of genomic stability and to prevent cells that have undergone malignant transformation progressing through the cell cycle phases. 1: tables the p signaling pathway is significantly expressed in response to aom. loss of p function is thought to be a contributing factor in colorectal cancer, because the p53-dependent pathway shuts down damaged cells, either through apoptosis, cell-cycle arrest or cellular senescence. apoptosis was observed histologically to have significantly increased at six hours after the aom treatment. as mgmt is depleted at this time point, we hypothesise that the main cellular response to aom involves the early depletion of mgmt then a switch to the induction of apoptosis and this most likely first occurs through the p signalling pathway. this is demonstrated through closer examination of the p signalling pathway response to aom in the distal colon. firstly, genes such as tp53inp, which is a key transcriptional regulator that responds to a variety of cellular stresses, including dna damage, oxidative stress and activated oncogenes, to regulate key cellular processes including the induction of apoptosis, is up-regulated fold. furthermore, caspases and casp are up-regulated in response to aom in the distal colon. the activation of casp triggers an execution arm of the apoptosis response initiating dna fragmentation. the apoptotic function of caspases is regulated by the bcl- family of proteins. accordingly, in response to aom, bcl-associated x protein which is critically important in the up-regulation of apoptosis, is increased two fold. furthermore, there is also decreased expression of the caspase-activated inhibitor avon by -fold. therefore, these results indicate that at hours after being treated with aom, one of the major effects of this carcinogen occurs through the p signalling pathway and the result is cell cycle arrest and a cellular switch towards apoptosis. gene network analysis was used to further understand the function of genes expressed in response to aom in each colonic segment, in particular the early induction of apoptosis. when the top three aom/proximal networks were merged there were genes with functional annotations associated with colorectal cancer including the up-regulation of transcriptional regulator c-jun and the down-regulation of mgmt. in terms of the aom/distal network there were eight genes associated with colon cancer, including the transcriptional regulator myc and the previously mentioned c-jun. when the p signalling pathway and apoptosis genes were cross-referenced in this network, c-jun kinase, which regulates cjun and is an important regulator of cell death, was a gene common to both and is linked through the gene network to the down-regulated p signalling transcriptional regulator, retinoblastoma. taken together these results suggest that the early genomic damage effects of aom on the colonic mucosa may be mediated through the p pathway, favouring apoptosis through c-jun/jnk signalling and preventing cell cycle progression through reduced rb expression. whilst this mechanism is hypothetical, it provides a framework for the further elucidation of the key mechanisms underpinning the cellular switch towards apoptosis in the gut mucosa in response to alkylating carcinogen challenge.  the healthy rat colonic mucosa exhibits extensive gene expression changes from its proximal to distal end reflecting regional changes in metabolic function. the normal rat colon also has naturally occurring protective and genomic repair mechanisms expressed dynamically, albeit subtly, along the proximal/distal axis. six hours after administration of aom, substantial changes in gene expression have occurred in the colonic mucosa and these also differ along the length of the colon. the changes are greater in the distal colon and appear particularly associated with the sensing of genomic damage, associated cell cycle arrest and a cellular switch towards the induction of apoptosis. consequently, the genomic homeostasis mechanisms that naturally exist to combat dietary and environmental insults in the colon of the normal rat appear to be dysregulated by aom resulting in a cellular switch through p signaling to more efficient genes associated with the apoptotic response, a genetic response that is also reflected histologically. 
in particular, the pathways associated with cell cycle and dna damage and repair processes appear to be disrupted in favour of apoptosis. these latter changes presumably protect and maintain a healthy colonic epithelium against incidental dietary and environmental insults. aom induces substantial changes in gene expression, resulting in an early switch in the cell cycle process, involving p signalling, towards cell cycle arrest leading to the more effective process of apoptosis to counteract this genotoxic insult. aom administration superimposes substantial changes on these basal gene expression patterns in both the distal and proximal rat colonic epithelium. the healthy rats’ colon exhibits extensive gene expression changes between its proximal and distal ends. in this report we define the normal rat colonic epithelium by describing transcriptional events along the anterior-posterior axis and then investigate the acute effects of azoxymethane on gene expression, with a particular emphasis on pathways associated with the maintenance of genomic integrity in the proximal and distal compartments using whole genome expression microarrays. there are large transcriptional changes that occur in epithelial gene expression along the anterior-posterior axis of the normal healthy rat colon.
 the acute response to genotoxic carcinogens in rats is an important model for researching cancer initiation events. in this report we define the normal rat colonic epithelium by describing transcriptional events along the anterior-posterior axis and then investigate the acute effects of azoxymethane on gene expression, with a particular emphasis on pathways associated with the maintenance of genomic integrity in the proximal and distal compartments using whole genome expression microarrays.  there are large transcriptional changes that occur in epithelial gene expression along the anterior-posterior axis of the normal healthy rat colon. aom administration superimposes substantial changes on these basal gene expression patterns in both the distal and proximal rat colonic epithelium. in particular, the pathways associated with cell cycle and dna damage and repair processes appear to be disrupted in favour of apoptosis.  the healthy rats’ colon exhibits extensive gene expression changes between its proximal and distal ends. the most common changes are associated with metabolism, but more subtle expression changes in genes involved in genomic homeostasis are also evident. these latter changes presumably protect and maintain a healthy colonic epithelium against incidental dietary and environmental insults. aom induces substantial changes in gene expression, resulting in an early switch in the cell cycle process, involving p signalling, towards cell cycle arrest leading to the more effective process of apoptosis to counteract this genotoxic insult. colorectal cancerazoxymethaneratsgene expression 