Ubagai et al
. studied the combined effects of PhIP and a HF diet on colon carcinogenesis in the F344 rat.(11
) Animals were given 400 p.p.m. PhIP in the basal diet for 2 weeks, followed by HF diet for up to 110 weeks, and this resulted in 3 of 19 rats with large intestinal tumors (19% incidence). Alternatively, 400 p.p.m. dietary PhIP was given for 2 weeks, followed by 4 weeks on HF diet, and the PhIP/HF cycling was repeated three times, ending with continuous HF diet for up to 60 weeks. Large intestinal tumors were seen in 9 out of 20 rats (45% incidence). The latter result compares favorably with the 43% incidence observed in rats treated continuously with 100 p.p.m. dietary PhIP for 104 weeks,(3
) in which a ~10-fold greater total amount of PhIP was administered per animal. Thus, PhIP/HF cycling represents an efficient means of inducing colon tumors, using a fraction of the amount of total carcinogen. We further modified the experimental protocol by giving PhIP via oral gavage, at a dose (50 mg/kg body wt per day) that was matched to the daily carcinogen intake from 400 p.p.m. PhIP in the diet,(3
) and after three cycles of PhIP/HF treatment, standard AIN93M diet rather than HF diet was administered. Using this modified protocol, the colon tumor incidence after 1 year was 41.6%.(12
In the current investigation, rats were euthanized immediately after completing the third and final cycle of PhIP dosing, so as to examine changes in colonic crypt homeostasis before the onset of frank tumors. Under these conditions, there was a striking increase in BrdU-positive cells throughout the entire length of the colon, with particularly high staining in the lower two-thirds of the crypt column. Moreover, there was an increase in cleaved caspase-3-positive cells in the proliferative zone of each crypt (central and basal regions), coupled with a reduction of apoptotic cells in the luminal region. The present study was not designed to specifically compare intermittent PhIP/HF dosing with continuous dietary PhIP treatment, but this might be interesting in a follow up investigation. Previously, a modest 1.5-fold increase in colonic BrdU labeling was seen at 8 weeks in male (but not female) rats fed continuously with 400 p.p.m. PhIP in the diet, and no significant changes were seen in cell proliferation rates for male or female rats at 4 or 12 weeks.(13
) However, the latter study did not assess changes in apoptosis or β-catenin expression. In general, we observed that cleaved caspase-3 did not show as good a concordance as did BrdU labeling for the corresponding β-catenin positive cells.
The high cytoplasmic β-catenin expression seen in the present study is noteworthy, because Ubagai et al
) reported that all of the large intestinal tumors obtained following PhIP/HF treatment also had high accumulation of cytoplasmic and nuclear β-catenin. Interestingly, only 55% of the tumors harbored a mutation in the Apc
(β-catenin) genes, and it was suggested that PhIP/HF cycling produced ‘unknown genetic alterations’ in the Wnt–Apc–β-catenin signaling pathway.(11
) Using the identical exposure protocol described here, colon tumors obtained at 1 year had a 36% frequency of β-catenin mutations, although this was increased to 79% when rats were subjected to postinitiation treatment with caffeine, which resulted in tumor promotion.(12
) This clearly supports the notion that cells harboring specific genetic changes within a population can be influenced to progress (or not to progress) to neoplasia by external factors, such as phytochemicals and a HF diet. As discussed before,(11
) intermittent exposure to PhIP might allow populations of cells to survive and progress to tumors, when continuous carcinogen treatment might otherwise lead to removal of those cells via apoptosis. In the present study, there was increased apoptosis in the lower half of the colonic crypt following PhIP cycling, but this appeared to be a compensatory mechanism triggered by the greatly enhanced rate of cell proliferation, resulting in expansion of the crypt-wide proliferative zone ().
Despite the high levels of β-catenin detected in the present study following PhIP/HF treatment (), no β-catenin mutations were observed in the colonic mucosa scrapings, using PCR-based single strand conformation polymorphism (PCR-SSCP) screening (data not presented). One interpretation is that β-catenin mutations were indeed present in the colonic scrapings, but below the limit of detection of the PCR-SSCP methodology, and that only after clonal expansion and tumor formation were the mutations readily detected owing to their enrichment relative to the wild type allele. Interestingly, among the three reported β-catenin/Tcf target genes examined, only c-myc
was increased in the colonic mucosa of rats given PhIP/HF diet, as noted in the colon tumors obtained at 1 year.(12
) Interestingly, Ctnnb1
mRNA also was elevated in the colonic mucosa of rats given PhIP/HF diet (). Although this increase was modest, it is consistent with prior reports showing elevated levels of Ctnnb1
mRNA expression in PhIP- and 1,2-dimethylhydrazine-induced rat colon tumors,(5
) as well as in primary human colon carcinomas and their liver metastases.(14
) In some of the vehicle controls, low or undetectable levels of β-catenin were seen via immunoblotting (), despite the presence of Ctnnb1
mRNA in most samples () and membrane-associated β-catenin being detected in immunohistochemical analyses (). It is unclear whether this reflects the efficiency of the membrane extraction and/or immunoblot procedures used here, and further work is needed to clarify this question, perhaps on a larger subset of tissue samples from vehicle and PhIP-treated animals. However, for the vehicle control samples shown here (), repeated immunoblotting confirmed the low or undetectable expression of β-catenin, suggesting the data in this case were reproducible.
In summary, we have shown that in rats given three cycles of PhIP alternating with exposure to HF diet, there was an increase in colonic cell proliferation, elevated apoptosis in the proliferative zone of the colonic crypt and augmented expression of cytoplasmic β-catenin. These results from the early stages of colon carcinogenesis suggested a possible independent dysregulation of β-catenin and c-myc expression, separate from the classical β-catenin/Tcf pathway,(15
) in accordance with data obtained from the corresponding colon tumors at 1 year.(12
) Thus, early changes affecting colonic crypt homeostasis at the time of exposure to PhIP and HF diet may have persisted into much later stages of colon tumor formation.