Ranjana P. Bird

Role of aberrant crypt foci in understanding the pathogenesis of colon cancer

Aberrant crypt foci (ACF) are present in carcinogen treated rodent colons and in the colons of humans with a high risk for developing the disease. It is proposed that ACF are preneoplastic lesions. Quantification of the number and growth features of ACF has been employed to study modulators of colon carcinogenesis. In this review, examples are presented to support the concept that ACF are preneoplastic lesions and that sequential quantification of their number and growth features (crypt multiplicity) in animal colons may provide further insight into the pathogenesis of colon cancer. It is proposed that cellular and molecular heterogeneity among ACF with different growth and morphologic features will be invaluable in the identification of events critically associated with cancer development.

The significance of aberrant crypt foci in understanding the pathogenesis of colon cancer.

Animal models provide a unique opportunity to study the biology of the disease process, and to test hypotheses linking environmental factors in the etiology and prevention of colon cancer. The concept of cancer prevention is to retard, regress or eliminate precancerous lesions. To actuate this concept, it is important to identify and enumerate preneoplastic lesions of various growth dimensions. The study of the precancerous stages in the colon is possible by the identification of aberrant crypt foci (ACF) in rodent colons treated with a carcinogen. The growth, morphological and molecular features of ACF support the contention that ACF are putative preneoplastic lesions. The ACF system is used extensively to identify modulators of colon carcinogenesis. Among the various endpoints being used in cancer research, ACF are the only endpoints which provides a quantitative approach to assess the disease process and the underlying cellular and molecular events as affected by cancer preventive or promoting agents. Many dietary components have been classified as tumor promoters or inhibitors based on their ability to change the tumor outcome. Whether they affect the growth of very early or advanced preneoplastic lesions is not known and can be explored by using ACF system. This information will provide a better understanding of cancer pathogenesis and will lead to the development of different cancer preventive strategies for high-risk individuals and the general population.

The effect of chenodeoxycholic acid on the development of aberrant crypt foci in the rat colon

Bile acids are reported to enhance experimentally-induced colonic tumorigenesis. Previously we have reported that cholic acid, a known tumor promoter, actually reduced the number of aberrant crypt foci (ACF), purported preneoplastic lesions (B.A. Magnuson and R.P. Bird, Cancer Lett., 68 (1993), 15-23). This observation was unexpected and has prompted us to explore the effect of other bile acids on the development of ACF. The primary objective of this investigation was to evaluate the effect of feeding varying dosages of chenodeoxycholic acid (CDC) on the induction and growth of ACF and on the proliferative indices of the colonic epithelium. Sprague-Dawley male rats were injected with azoxymethane (+AOM, 20 mg/kg) or saline (-AOM). One week later they were randomly allocated to five groups and were fed diets containing CDC at varying levels (0.0, 0.025, 0.05, 0.1 and 0.2% by weight) for 2 weeks. After completion of the feeding period the number and crypt multiplicity of ACF were quantified, and three different proliferative indices, including mitotic index, BUDR labelling index (percentage S-phase cells) and proliferating cell nuclear antigen labelling index (percentage cycling cells) were determined. CDC at all dosages increased the number of ACF having the maximum effect at the 0.1% CDC level. A significant dose-related increase in crypt height was noted in CDC-fed+AOM groups when compared with the +AOM control groups. The mitotic indices of colonic crypts were higher (P < or = 0.05) only in the 0.025% CDC -AOM group when compared with the 0% CDC -AOM group (5.97 +/- 0.63 vs. 3.92 +/- 0.79). The BUDR labelling indices were not altered by CDC feeding (P > or = 0.05). PCNA labelling indices increased consistently among the CDC-fed groups. Among the -AOM group the 0.05% CDC group had the maximum value, which was significantly different from the control value (19.21 +/- 1.92 vs. 10.93 +/- 0.56, respectively). Among the +AOM groups the PCNA labelling indices increased with increasing levels of CDC. It was concluded that CDC stimulated the development of ACF and altered cell cycle associated events in colonic crypts undergoing neoplastic changes.

Reduction of aberrant crypt foci induced in rat colon with azoxymethane or methylnitrosourea by feeding cholic acid

Recent studies in our laboratory have demonstrated that feeding cholic acid (CHA) to rats treated with a single dose of azoxymethane (AOM) reduces the growth of putative preneoplastic lesions, aberrant crypt foci (ACF), in a dose-dependent manner. This finding was unexpected since CHA has been reported to promote colon cancer in rats receiving multiple treatments of the colon carcinogen, methylnitrosourea (MNU). The main objective of the present investigation was to evaluate the effect of the type of carcinogen and treatment protocol on the induction and growth of ACF in conjunction with CHA treatment. Male Sprague-Dawley rats received 0, 1 or 2 treatments with AOM or MNU and were fed either the AIN-76A or AIN-76A plus 0.2% CHA diet for 4 weeks. The total number and average size of ACF were significantly reduced in CHA-fed animals regardless of the type or number of treatments of carcinogen. The greatest reduction of ACF due to CHA-feeding was seen in the distal colon. The average crypt multiplicity (number of crypts in each ACF) was not altered by diet or carcinogen treatment. Colonic cell proliferation (crypt height and number of mitotic figures) was significantly increased in CHA-fed animals compared to control diet animals. Therefore, feeding CHA for 4 weeks reduced the number and size of ACF in rats induced by 1 or 2 injections of AOM or MNU, despite stimulation of colonic cell proliferation. These findings suggest further investigation is needed to understand the mechanism of promotion by cholic acid and the value of number and growth characteristics of ACF as a biological endpoint in the pathogenesis of colon cancer.

Further investigation of the effect of cholic acid on the induction, growth characteristics and stability of aberrant crypt foci in rat colon

We previously reported that the colons of animals injected with azoxymethane (AOM) and fed a diet containing cholic acid (CHA) had lower numbers of aberrant crypt foci (ACF) than those in animals fed a control diet. To follow up on this observation, a series of studies was conducted to determine whether CHA affects the development of ACF in a dose- and time-dependent manner, and the possible mechanism(s) involved. Sprague Dawley male rats were injected with AOM (20 mg/kg s.c.), and one week later randomly allocated to groups fed diets containing 0, 0.05, 0.1 or 0.2% CHA by weight, for 4 weeks. Their colons were scored for the number size and location of ACF, number of crypts per ACF, and mitotic activity. It was observed that the number and size of ACF decreased with increasing levels of CHA. Mitotic activity was higher (P < 0.05) in the 0.2% CHA diet (CHA-diet) group compared to the 0% CHA group. To determine if timing of intervention with the CHA-diet was critical, rats were allocated to the CHA-diet before or after AOM injection. The ACF-reducing effect of 0.2% CHA diet was evident (P < or = 0.05) only after AOM injection. Intervention with the CHA-diet 4 weeks after AOM injection demonstrated that the diet eliminated and/or remodelled a large proportion (50%) of ACF which had developed within 4 weeks and inhibited the growth of those ACF that persisted. This effect was also associated with higher (P < or = 0.05) mitotic activity in the colon. Discontinuing the treatment of rats with the CHA-diet resulted in a rapid increase in the number of ACF in their colons, establishing that the growth inhibitory effect of the CHA-diet on ACF was reversible. In conclusion, it was demonstrated that the CHA-diet modulated the number of ACF by inhibiting their development and growth and by eliminating or remodelling a selected population of ACF.

Comparative effects of secondary bile acids, deoxycholic and lithocholic acids, on aberrant crypt foci growth in the postinitiation phases of colon carcinogenesis

The objective of this study was to investigate the effect of deoxycholic (DCA) and lithocholic (LCA) acids on the postinitiation phases of colon cancer. Male Sprague-Dawley rats (n = 170) were injected with azoxymethane (2 injections at 15 mg/kg body wt sc given 1 wk apart) and fed a control (CON) AIN-93 diet. Two weeks after the second azoxymethane injection, 10 animals were killed and aberrant crypt foci (ACF) were enumerated. The remaining animals were randomly assigned to four diet groups: 1) CON, 2) DCA, 3) LCA, and 4) high fat (HF, a positive control group). Bile acid diets consisted of 0.2% by weight DCA or LCA; HF diets consisted of 20% fat (5% soybean oil + 15% beef tallow by weight). Animals were killed at Weeks 3, 12, and 20 (from 1st carcinogen injection), and number and growth features of ACF and adenomatous lesions were enumerated in the colon. At Week 12, ACF number and small, medium, and large (1-3, 4-6, and > or = 7 crypts/focus, respectively) ACF were higher in the HF group than in the DCA, LCA, and CON groups (p < or = 0.05). By Week 20, ACF number and small, medium, and large ACF were similar in the LCA and HF groups, whereas the response was similar in the DCA and CON groups. Average crypt multiplicity was higher in the HF and LCA groups than in the DCA and CON groups (p < or = 0.05). Microadenoma (MA) incidence was higher in the HF group than in the CON and LCA groups (p < or = 0.05). Regional distribution patterns for ACF number were similar to MA and tumor distribution patterns within the CON, DCA, and HF groups. In the LCA group, ACF number and MA showed a proximal predominance in regional distribution, whereas tumors showed a distal predominance. HF diets provided the most stimulatory environment, immediately enhancing the number and growth of ACF and MA incidence. In conclusion, HF and LCA diets exerted distinct effects on postinitiation phases of colon cancer, whereas the DCA diet did not.

A NAD(P)H:Quinone Oxidoreductase 1 Polymorphism Is a Risk Factor for Human Colon Cancer

Colon cancer is one of the most common cancers in North America and generally develops from colonic epithelial cells following initiation by carcinogens. We have shown that the phase II detoxifying enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1) contributes to the inhibition of carcinogen-induced colon cancer in rats at both the initiation and postinitiation stages. An inactivating polymorphism at base 609 of the NQO1 gene, 609C (NQO1 *1) → 609T (NQO1 *2), occurs at high frequency in the human population. Thus, we carried out a case-control study to determine if this polymorphism is associated with an increased risk of developing colon cancer. A total of 298 patients with colon cancer and 349 healthy controls matched for age, gender, and ethnic origin were enrolled in the study. There was an increased incidence of the NQO1 *2/*2 genotype in patients with colon cancer, with a gender and age-adjusted odds ratio of 2.68 (95% confidence intervals, 1.14-6.28). However, the incidence of the NQO1 *1/*2 genotype was not increased in patients with colon cancer compared with controls. When the patient and control groups were stratified by tobacco and alcohol use, the incidences of the NQO1 *2/*2 genotype were increased in patients with colon cancer for tobacco and alcohol users and nonusers, suggesting that there is no interaction between the NQO1 base 609 polymorphism and tobacco or alcohol use. These results strongly suggest that NQO1 plays a significant role in preventing the development of colon cancer, and individuals with an NQO1 *2/*2 genotype are at an increased risk of developing this disease. (Cancer Epidemiol Biomarkers Prev 2006;15(12):2422–6)

Effect of dietary fat on colonic protein kinase C and induction of aberrant crypt foci

A major objective of the present study was to determine whether a high-fat diet affects early events during colon carcinogenesis. Female Sprague-Dawley rats were injected with saline or azoxymethane (20 mg/kg) and fed either a normal (5% corn oil w/w) or a high (5% corn oil and 15% beef tallow w/w) fat diet. To assess the effect of a known tumor-promoting diet on the early events of neoplastic transformation, Study 1 examined the induction and growth of aberrant crypt foci (ACF) as well as of proliferative indices. The total number of ACF were similar in both groups even after 8 wk of dietary treatment; however, ACF with accelerated growth characteristics (≥4 crypts/focal lesions) were more prevalent (P≤0.05) in the colons of animals fed the high-fat diet. Metaphase arrest cells and 5′-bromo-2′-deoxyuridine labelled cells showed no appreciable response to dietary changes. To determine whether changes in colonic signal transduction pathways represent an early response to dietary modification, Study 2 evaluated the activity of protein kinase C (PKC), proliferative indices and changes in phospholipid fatty acid profiles. In comparison to the normal fat group, the colons of high-fat fed animals exhibited higher (P≤0.05) membranes and lower soluble PKC activity; however, proliferation patterns of these colons were not altered. Changes in the membrane lipid composition were minor; however, an increase in the phosphatidylcholine/phosphatidylethanolamine ratio and in 20∶4n−6 was noted. Our results demonstrate that in comparison to a normal-fat diet, a high-fat diet stimulated the growth of a population of ACF, i.e., preneoplastic lesions leading to advanced growth characteristics. In addition, a high-fat diet exerted a marked influence on total, cytosolic and membrane associated PKC activities. The findings suggest that modulation of PKC may play a critical role at the early stages of colon carcinogenesis.

Measurement of the proliferative status of colonic epithelium as a risk marker for colon carcinogenesis: Effect of bile acid and dietary fiber

The proliferative status of mouse colonic epithelium, as affected by dietary fibers with or without cholic acid (CA), was studied by autoradiography and the metaphase arrest technique. In the first study, groups of mice were fed natural ingredient (laboratory chow) or semisynthetic diets containing 0% (control) or 0.2% (test) CA. After the mice were fed two weeks, the effect of CA was significantly more pronounced in the semisynthetic diet group than in the natural ingredient diet group with respect to labeled cells/crypt section (7.8 +/- 0.8 vs. 2.9 +/- 0.4) and mitotic figure (MF)/crypt section (3.0 +/- 0.5 vs. 1.8 +/- 0.2). In the second study, diets formulated to contain 5 or 10% cellulose (C), pectin (P), or wheat bran (WB) with or without CA (0.2%) were fed to animals for two weeks and colonic proliferative indices were measured. When compared with 5% C group, the 10% WB group exhibited lower labeling index (LI) values (4.2 +/- 0.5 vs. 6.4 +/- 1.0) and the 10% P group exhibited higher LI values (10.0 +/- 1.1 vs. 6.4 +/- 1.0). CA-induced increases in the LI and MF values responded independently in some cases to dietary fiber. Among the CA-treated groups, only the 10% P diet resulted in lower LI when compared with the 5% C group (p less than 0.05) (7.4 +/- 0.8 vs. 12.5 +/- 2.8) but had no effect on MF/crypt section. However, the 5 or 10% WB diet resulted in lower MF values (1.7 +/- 0.2 and 1.8 +/- 0.6 vs. 2.6 +/- 0.3). A long-term feeding study comparing 10% P with 10% C diets also demonstrated that the LI was elevated in the 10% P group without any effect on the mitotic activity of the colonic epithelium. This paradoxical finding suggests that the value of the LI and/or mitotic index as a risk marker of colon carcinogenesis should be further investigated.

Modulation of colonic xenobiotic metabolizing enzymes by feeding bile acids: comparative effects of cholic, deoxycholic, lithocholic and ursodeoxycholic acids.

Primary and secondary bile acids such as cholic (CHA), deoxycholic (DCA) and lithocholic (LCA) acids have been shown to increase colon tumorigenesis. It has been suggested that inhibition of xenobiotic metabolizing enzymes such as glutathione S-transferase (GST) and UDP-glucuronyltransferase (UGT) by bile acids may be a factor in the development of colon cancer. While enzyme inhibition has been demonstrated in vitro, it is unclear whether feeding bile acids modulates colonic GST and UGT in vivo. To test this notion, male, Sprague-Dawley rats (n = 100) were assigned to a control (CON) or test diets containing 0.2% CHA, DCA, LCA or ursodeoxycholic acid (UDCA). After 5 weeks, colonic tissue was harvested and used for enzyme and cell proliferation measurements. The response to bile acids varied with the enzyme measured and appeared isoenzyme specific. GST-alpha activity was lower in the bile acid fed groups compared with CON. While GST-mu was lower in the LCA-fed group, GST-pi was lower in the DCA-, CHA- and UDCA-fed groups. Unlike GST, both UGT and NADPH-cytochrome P-450 reductase (CYC) activities were increased by bile acids. The proliferative response of the colonic epithelium varied with the bile acids and was regionally specific. These data demonstrate that feeding bile acids alters the activity of colonic phase I and II enzymes; however, the physiological effect of these enzymatic perturbations is yet to be determined.