A chemoprotective fish oil- and pectin-containing diet temporally alters gene expression profiles in exfoliated rat colonocytes throughout oncogenesis.

We have demonstrated that fish oil- and pectin-containing (FO/P) diets protect against colon cancer compared with corn oil and cellulose (CO/C) by upregulating apoptosis and suppressing proliferation. To elucidate the mechanisms whereby FO/P diets induce apoptosis and suppress proliferation during the tumorigenic process, we analyzed the temporal gene expression profiles from exfoliated rat colonocytes. Rats consumed diets containing FO/P or CO/C and were injected with azoxymethane (AOM; 2 times, 15 mg/kg body weight, subcutaneously). Feces collected at initiation (24 h after AOM injection) and at aberrant crypt foci (ACF) (7 wk postinjection) and tumor (28 wk postinjection) stages of colon cancer were used for poly (A)+ RNA extraction. Gene expression signatures were determined using Codelink arrays. Changes in phenotypes (ACF, apoptosis, proliferation, and tumor incidence) were measured to establish the regulatory controls contributing to the chemoprotective effects of FO/P. At initiation, FO/P downregulated the expression of 3 genes involved with cell adhesion and enhanced apoptosis compared with CO/C. At the ACF stage, the expression of genes involved in cell cycle regulation was modulated by FO/P and the zone of proliferation was reduced in FO/P rats compared with CO/C rats. FO/P also increased apoptosis and the expression of genes that promote apoptosis at the tumor endpoint compared with CO/C. We conclude that the effects of chemotherapeutic diets on epithelial cell gene expression can be monitored noninvasively throughout the tumorigenic process and that a FO/P diet is chemoprotective in part due to its ability to affect expression of genes involved in apoptosis and cell cycle regulation throughout all stages of tumorigenesis.

Upregulation of p21Waf1/Cip1 expression in vivo by butyrate administration can be chemoprotective or chemopromotive depending on the lipid component of the diet.

The overall goal of this research was to separate out the effects of butyrate from its fiber source and determine in vivo if it upregulates colonic histone acetylation, p21(Waf1/Cip1) expression (p21) and apoptosis and if this sequela of events is protective against aberrant crypt foci (ACF) formation. Eighty Sprague-Dawley rats were provided defined diets with either corn oil or fish oil as the lipid source, +/- butyrate-containing capsules targeted for release in the colon and +/- azoxymethane (AOM) (10 rats per group). Diets were provided for 11 weeks and at termination colonocyte nuclear histone H4 and p21 expression were determined by immunohistochemistry, apoptosis was measured by the terminal deoxynucleotide transferase biotin-dUTP nick end labeling assay and aberrant crypt numbers and multiplicity were enumerated. Luminal butyrate levels were also quantified. AOM injection repressed p21 expression, which was reversed by butyrate supplementation. Although butyrate enhanced p21 expression with both dietary lipid sources, the increase in p21 resulted in an increase in apoptosis and decrease in ACF with fish oil, but had no effect on apoptosis and increased ACF with corn oil. This significant interaction between fat, butyrate (fiber) and p21 expression with one combination being protective and the other promotive of colon carcinogenesis reinforces the importance of considering diet as a key factor in chemoprevention.

Suppression of colon carcinogenesis by bioactive compounds in grapefruit.

This study evaluated the hypothesis that untreated and irradiated grapefruit as well as the isolated citrus compounds naringin and limonin would protect against azoxymethane (AOM)-induced aberrant crypt foci (ACF) by suppressing proliferation and elevating apoptosis through anti-inflammatory activities. Male Sprague-Dawley rats (n = 100) were provided one of five diets: control (without added grapefruit components), untreated or irradiated (300 Gy, 137Cs) grapefruit pulp powder (13.7 g/kg), naringin (200 mg/kg) or limonin (200 mg/kg). Rats were injected with saline or AOM (15 mg/kg) during the third and fourth week and colons were resected (6 weeks post second injection) for evaluation of ACF, proliferation, apoptosis, and cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) protein levels. Experimental diets had no effect on the variables measured in saline-injected rats. However, in AOM-injected rats, the experimental diets suppressed (P < or = 0.02) aberrant crypt and high multiplicity ACF (HMACF; P < or = 0.01) formation and the proliferative index (P < or = 0.02) compared with the control diet. Only untreated grapefruit and limonin suppressed (P < or = 0.04) HMACF/cm and expansion (P < or = 0.008) of the proliferative zone that occurred in the AOM-injected rats consuming the control diet. All diets elevated (P < or = 0.05) the apoptotic index in AOM-injected rats, compared with the control diet; however, the greatest enhancement was seen with untreated grapefruit and limonin. Untreated grapefruit and limonin diets suppressed elevation of both iNOS (P < or = 0.003) and COX-2 (P < or = 0.032) levels observed in AOM-injected rats consuming the control diet. Although irradiated grapefruit and naringin suppressed iNOS levels in AOM-injected rats, no effect was observed with respect to COX-2 levels. Thus, lower levels of iNOS and COX-2 are associated with suppression of proliferation and upregulation of apoptosis, which may have contributed to a decrease in the number of HMACF in rats provided with untreated grapefruit and limonin. These results suggest that consumption of grapefruit or limonin may help to suppress colon cancer development.

Dietary fish oil reduces oxidative DNA damage in rat colonocytes.

Prolonged generation of reactive oxygen species by inflammatory mediators can induce oxidative DNA damage (8-oxodG formation), potentially resulting in intestinal tumorigenesis. Fish oil (FO), compared to corn oil (CO), has been shown to downregulate inflammation and upregulate apoptosis targeted at damaged cells. We hypothesized FO could protect the intestine against 8-oxodG formation during dextran sodium sulfate- (DSS-) induced inflammation. We provided 60 rats with FO- or CO-supplemented diets for 2 weeks with or without 3% DSS in drinking water for 48 h. Half the treated rats received 48 additional h of untreated water before termination. Due to DSS treatment, the intestinal epithelium had higher levels of 8-oxodG (p =.04), induction of repair enzyme OGG1 mRNA (p =.02), and higher levels of apoptosis at the top of colonic crypts (p =.01) and in surface cells (p <.0001). FO-fed rats, compared to CO, had lower levels of 8-oxodG (p =.05) and increased apoptosis (p =.04) in the upper crypt region; however, FO had no significant effect on OGG1 mRNA. We conclude that FO protects intestinal cells against oxidative DNA damage in part via deletion mechanisms.