Risk factors for colorectal cancer in man induce aberrant crypt foci in rats: Preliminary findings

Figures & data

Table 1. Ingredients of control Diet A and presumed high-risk Diet B, both based on AIN-76A.

Ingredients	AIN-76A diet (g)		Diet A (g)	Diet B (g)
	Grams per 1000 grams of diet
Sucrose	500		0	0
Dextrose (glucose)	0		560	0
Fructose	0		0	560
Corn starch	150		0	0
Maltodextrin	0		90	90
	As in AIN-76A except as noted here
Iron (as ferric chloride)	0.05		0.05	0.6
Calcium	5.2		5.2	0.4
Phosphate	5.4		5.3	5.3
Vitamin D3, IU	1000		1000	100
Sodium	1		2	2

Diet A = control diet based on AIN-76 with glucose, maltodextrin; Diet B = as Diet A with low calcium, low vitamin D3, and high iron, with fructose rather than glucose (high oxidative and carbonyl stress); The Research Diets products codes for the diets are: D10001, D11011301, and D10102604, respectively.

Figure 1. An example of the very few discrete (or well defined) aberrant crypt foci (ACF) observed in animals on Diet B in Study 3. Note the slit-like lumens likely indicative of a dysplastic histology. (The mucosal field is 1.7 × 0.8 mm.)

Figure 2. Two examples of heterogeneous (or ill-defined) aberrant crypt focus (ACF) as observed on the colonic mucosal surface after Bouin's fixation in Study 2. The ACF are the foci of large, darkly staining enlarged crypts bordering an infolding pattern over what appears to be a lymphoid follicle. Faint outlines of the openings of small crypts are seen at the center of both ACF. (The mucosal fields are 1.7 × 0.8 mm and 2.7 × 1.3 mm.)

Figure 3. Two examples of heterogeneous (or ill-defined) aberrant crypt focus (ACF) as observed on the colonic mucosal surface following formalin fixation and staining with Methylene Blue in Study 3. Again, a faint outline of the openings of small crypts can be seen at the center of the ACF. We suggest that these changes follow the loss of clusters of crypts and are an early response to the loss of adjacent crypts (The mucosal fields are 1.7 × 0.8 mm.).

Figure 4. An example of the histological appearance at low and high power of an inflammatory/hyperproliferative focus observed in Study 3. On histologic examination of the colons that had been “Swiss rolled,” the foci were noted by their narrow caliber, mucin, and Goblet cell-depleted crypts (yellow arrow in Fig. 4A), and then by the frequent mitotic figures (red arrows in Fig. 4B), the damaged luminal surface of the aberrant crypt focus, and the many acute and chronic inflammatory cells. The large crypts, so evident on the topologic en face preparation, were not as evident in this histological section.

Table 2. Metabolic, biochemical and clinical measures for rats after 14 weeks on Diet A and B (Study 1).

Dietary groups	Diet A (Control)	Diet B (Carbonyl stress and oxidative stress)
Number of animals	6	6
Metabolic measures
Weight gain (g)	140 ± 7	101 ± 5
Fasting blood glucose (mmol/L)	8.0 ± 0.3	9.2 ± 0.2
Fasting insulin (picoM)	35.2 ± 4.4	95.0 ± 33.6
Biochemistry measures
Iron concentration (μmol/L)
Serum	0.1 ± 0.1	13.4 ± 4.6
Colon	52.4 ± 6.5	216.2 ± 23.8
MDA concentration (μmol/L)
Serum	3.0 ± 1.0	1.3 ± 0.6
Colon	0.2 ± 0.2	2.2 ± 1.1
MG-derived AGEs (μg/L)
Serum	207.7 ± 17.2	270.8 ± 20.0
Colon	71.6 ± 14.7	94.9 ± 13.5
Clinical chemistry measures
Total protein (g/L)	64 ± 1	63 ± 1
Albumin (g/L)	42 ± 1	42 ± 1
ALP (IU/L)	77 ± 3	160 ± 12
ALT (IU/L)	75 ± 6	91 ± 4
CK (IU/L)	774 ± 425	435 ± 71
Amylase (IU/L)	2577 ± 445	2040 ± 152
Cholesterol (mmol/L)	1.9 ± 0.1	3.1 ± 0.1
Glucose (mmol/L)	9.5 ± 0.9	10.0 ± 0.5
Creatinine (mmol/L)	30 ± 3	28 ± 2
Sodium (mmol/L)	144 ± 1	143 ± 1
Potassium (mmol/L)	3.9 ± 0.3	3.6 ± 0.2
Na/K Ratio (mmol/L)	37 ± 3	40 ± 2
Chloride (mmol/L)	97 ± 1	97 ± 0
Calcium (mmol/L)	2.63 ± 0.03	2.13 ± 0.17
Phosphorus (mmol/L)	2.0 ± 0.2	2.8 ± 0.2

MDA = malondialdehyde; MG-derived AGEs = methylglyoxal derived advanced glycation end products.

¹ Diets A, and B as defined in Table 1.

Values are shown as means ± SEM.

^a Significant as compared to animals on diet A based on nonparametric 2-tailed t-tests (P ≤ 0.05).

^b Significant as compared to animals on diet A based on nonparametric 2 tailed t-tests (P ≤ 0.01).

^c Significant as compared to animals on diet A based on nonparametric 2-tailed t-tests (P ≤ 0.001).

Table 3. Colonic mucosal surface measures for rats after 40 weeks on Diet A and B (Study 2).

Dietary groups	Diet A (control)	Diet B (carbonyl stress and oxidative stress)
Number of animals	20	41
Weight gain (g)	266 ± 6	224 ± 4
Discrete ACF (AOM-like)	0.30 ± 0.13	0.20 ± 0.06
Heterogeneous ACF/colon	4.15 ± 1.12	9.05 ± 1.79
ILF/colon	1.40 ± 0.45	3.80 ± 0.98
MDF	0.06 ± 0.05	0.10 ± 0.05

¹ Diets A and B as defined in Table 1. ILF = isolated lymphoid follicles; ACF = aberrant crypt foci; AOM = azoxymethane; MDF = mucin-depleted foci. Values are shown as means ± SEM per animal.

² The ACFs per colon per animal as scored for exogenous carcinogens such as azoxymethane.

³ The ACFs per colon per animal as scored by criteria of Whiteley et al. (22).

⁴ Fraction of rats with MDF.

^a Significant as compared to animals on diet A based on nonparametric 2-tailed t-tests (P ≤ 0.05).

^b Significant as compared to animals on diet A based on nonparametric 2-tailed t-tests (P ≤ 0.01).

Table 4. Metabolic, biochemical, clinical, and colonic mucosal surface measures for rats after 14 weeks on Diet A and B with decreased fluorescent light (UV) (Study 3).

Dietary groups	Diet A (control)	Diet B (carbonyl stress and oxidative stress)
Number of animals	18	18
Metabolic measures
Weight gain (g)	180 ± 4	118 ± 4
Biochemistry measures
MDA concentration (mmol/L)
Serum	0.1 ± 0.0	0.1 ± 0.0
MG-derived AGEs (μg /L)
Serum	217 ± 16	241 ± 15
Clinical chemistry measures*
Total protein (g/L)	66 ± 0	68 ± 1
Albumin (g/L)	43 ± 0	43 ± 1
ALP (IU/L)	104 ± 3	203 ± 11
ALT (IU/L)	52 ± 1	57 ± 2
CK (IU/L)	389 ± 52	644 ± 68
Amylase (IU/L)	2723 ± 21	2468 ± 54
Cholesterol (mmol/L)	2.2 ± 0.0	3.2 ± 0.1
Glucose (mmol/L)	8.3 ± 0.1	8.8 ± 0.1
Creatinine (mmol/L)	24.7 ± 0.5	31.0 ± 1.3
Sodium (mmol/L)	142.0 ± 0.5	142.7 ± 0.7
Potassium (mmol/L)	4.7 ± 0.1	4.3 ± 0.0
Na/K Ratio (mmol/L)	30.7 ± 0.4	33.3 ± 0.4
Chlorine (mmol/L)	97.3 ± 0.3	93.7 ± 0.6
Calcium (mmol/L) *	2.85 ± 0.09	1.69 ± 0.03
Phosphorus (mmol/L)	2.1 ± 0.0	4.2 ± 0.2
Bicarbonate (mmol/L)	23.8 ± 0.6	17.7 ± 0.8
Magnesium (mmol/L)	0.7 ± 0.0	0.9 ± 0.0
25-hydroxyvitamin D * (nmol/L)	42.5 ± 3.2	12.0 ± 0.7
Colon mucosal surface measures
Heterogeneous ACF/colon	0.7 ± 0.4	13.6 ± 3.5
Isolated lymphoid follicles/colon	0.6 ± 0.4	7.4 ± 1.4

¹ Diets A and B as defined in Table 1. ILF = isolated lymphoid follicles; ACF = aberrant crypt foci. Values are shown as means ± SEM. Two sample 2-tailed test was performed. Heterogeneous ACF and isolated lymphoid follicles are scored per colon per animal.

* Sample size 6 + 6 for all Clinical chemistry, except calcium (12 + 12) and 25-hydroxyvitamin D (4 + 4).

^a Significant as compared to animals on diet A based on nonparametric 2-tailed t-tests (p ≤ 0.05).

^b Significant as compared to animals on diet A based on nonparametric 2-tailed t-tests (P ≤ 0.01).

^c Significant as compared to animals on diet A based on nonparametric 2-tailed t-tests (P ≤ 0.001).

Whiteley LO, Hudson L, Jr., and Pretlow TP: Aberrant crypt foci in the colonic mucosa of rats treated with a genotoxic and nongenotoxic colon carcinogen. Toxicol Pathol 24, 681–689, 1996.

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