[CANCER RESEARCH 63, 2388 –2392, May 15, 2003] Identification of Mucin-depleted Foci in the Unsectioned Colon of Azoxymethane- treated Rats: Correlation with Carcinogenesis 1 Giovanna Caderni, 2 Angelo Pietro Femia, Augusto Giannini, Alessandro Favuzza, Cristina Luceri, Maddalena Salvadori, and Piero Dolara Department of Pharmacology, University of Florence, 50139 Florence, Italy [G. C., A. P. F., A. F., C. L., M. S., P. D.], and Department of Pathology, S. M. Annunziata Hospital, Florence [A. G.], Italy ABSTRACT We tested the association between aberrant crypt foci (ACF) and tumor induction by feeding azoxymethane-induced rats (15 mg/kg  2, s.c.) with synbiotics (Raftilose Synergy 1, a derivative of inulin, 10% of the diet, along with lactobacilli and bifidobacteria). After 16 weeks of feeding, synbiotics significantly increased ACF multiplicity. On the contrary, after 32 weeks, synbiotics significantly decreased intestinal tumors. When the same unsectioned colon used for ACF determination was stained with high-iron diamine Alcian blue, foci of crypts with scarce or absent mucins were identified. We defined these lesions as mucin-depleted foci (MDF), and they were visible in all azoxymethane-treated rats and correlated with tumor induction (MDF/colon: 8.2  0.9 and 3.8  0.9 in controls and synbiotic-fed rats, respectively, P < 0.01; crypts/MDF: 12.2  2 and 6.4  1 in controls and synbiotic-fed rats, respectively, P < 0.05, means  SE, n  7). There were fewer MDF/colon than ACF, and they were histologically more dysplastic than mucinous lesions identified as ACF in high-iron diamine Alcian blue-stained colon. In conclusion, MDF may be premalignant lesions that predict colon carcinogenesis. INTRODUCTION Colon carcinogens such as AOM 3 or 1,2-dimethylhydrazine induce colon cancer in rodents through a multistep process characterized by the sequential formation of histopathological lesions similar to those observed in spontaneous carcinogenesis in humans (1). As part of this process, ACF have been identified as preneoplastic lesions visible in the unbedded colon of rodents as early as 2 weeks after carcinogen administration (2, 3). ACF have also been identified in humans (4, 5), are easy to quantify in the entire colon, and have a preneoplastic nature (6 –10). Therefore, ACF determination has been widely used as a short-term test for predicting colon carcinogenesis (11), although some studies reported conflicting results on the association between ACF and tumor development (12, 13). In fact, in humans and rodents, ACF have variable levels of dysplasia, and it has been suggested that only a few dysplastic ACF will develop into cancers (7, 8, 14). Recently, “-catenin accumulated crypts” have been described as possible premalignant lesions in AOM-treated rodents (15). Although these lesions and dysplastic ACF may represent true preneoplastic lesions, their identification in the unsectioned colon is problematic because there is no practical way to look at the entire colon of a rat histologically. Given these considerations, we were interested in correlating ACF and tumors in a study in which AOM-treated rats were treated with synbiotics, compounds with potential chemopreventive activity (16), comprising a prebiotic, Raftilose Synergy 1 (a derivative of inulin), and the probiotics, lactobacilli and bifidobacteria. Moreover, in the unsectioned colon of the treated animals, we searched for crypt foci with altered mucin production, one of the most prominent features of dysplasia in the colon (17), hoping to find alternative biomarkers for cancer. This led us to the identification of MDF, which may be precursor lesions of colon tumors. MATERIALS AND METHODS AOM was purchased from Sigma (Milan, Italy). Raftilose Synergy 1, a derivative of inulin, was provided by Orafti (Tienen, Belgium). Lactobacillus GG, L. delbrueckii subsp. rhamnosus, and Bifidobacterium lactis Bb12 were provided by Valio (Helsinki, Finland) and purchased from Chr. Hansen (Hor- sholm, Denmark), respectively. Animals and Treatments. We used 4 –5-week-old, male F344 rats (Nos- san, Correzzana, Milan, Italy). The animals were housed according to the European Union Regulations on the Care and Use of Laboratory Animals, as reported previously (18). Rats (n = 92) were randomly allocated to two groups with the following diets. The control group (n = 46) was fed a high-fat diet, based on the AIN76 diet (16), modified to contain a high amount of fat [230 g/kg corn oil (w/w)], a low level of cellulose [20 g/kg (w/w)] and maltodextrins [100 g/kg (w/w)], and sucrose [360 g/kg (w/w)] as sources of carbohydrates. The synbiotic group (n = 46) was fed the same diet as controls, but malto- dextrins were replaced by 100 g/kg (w/w) Raftilose Synergy 1; this diet also contained Lactobacillus GG, L. delbrueckii subsp. rhamnosus, and Bifidobac- terium lactis Bb12 strains (5  10 8 colony-forming units of each strain/g diet). Ten days after beginning the feeding of the experimental diets, rats were injected with AOM (1 week apart; 15 mg/kg  2, s.c). In each dietary group, some animals were treated with saline instead of AOM (4 animals in the control group and 5 animals in the synbiotic group). Either 7 or 15 weeks after the first AOM injection, two groups of seven rats (control and synbiotic groups) were sacrificed by CO 2 inhalation, and ACF were determined according to Bird (2). The colons were coded and scored independently by two observers. The correlation coefficient between the scores of the two observers on a set of 60 colon samples was 0.78 (P  0.0001) for the number of ACF/colon and 0.87 (P  0.0001) for the multiplicity of the ACF (ACs/ACF). Thirty-one weeks after the first AOM injection, tumors were determined in the two experimental groups (synbiotic and control groups, 28 animals/group) using methods previously described in detail (16, 18). Determination of Mucin Production in the Unbedded Colon and Iden- tification of MDF. Mucin production was analyzed by restaining with the HID-AB procedure formalin-fixed colons that were previously stained with methylene blue to visualize ACF, as described previously (18). The HID-AB- stained, unbedded colons were then scored at the microscope (40 magnifi- cation), mucosa side up. MDF were identified as focal lesions by the following criteria: (a) absence or very small production of mucins; (b) distortion of the opening of the lumen compared with normal surrounding crypts; (c) elevation of the lesion above the surface of the colon; and (d) multiplicity (i.e., the number of crypts forming each focus) higher than 3 crypts. To be defined as MDF, a focus had to fulfill the first criterion (absence or very low production of mucins) and at least two of the other criteria listed above. The colons were coded and scored independently by two observers. The correlation coefficient between the scores of the two observers on a set of 14 colon samples was 0.86 (P  0.001) for the number of MDF/colon and 0.91 (P  0.001) for the multiplicity of MDF (number of crypts/focus). Dissection of MDF and ACF and Evaluation of Dysplasia. MDF and ACF were identified at the microscope as described above, marked with permanent ink (The Davidson Marking System; Bradley Products, Blooming- Received 7/29/02; accepted 3/13/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by EU Projects QLKI-1999-00346 and QLRT 1999-00505 and by Min- istero Ricerca Scientifica Tecnologica (MURST), Italy. 2 To whom requests for reprints should be addressed, to e-mail: giovanna. caderni@unifi.it. 3 The abbreviations used are: AOM, azoxymethane; AC, aberrant crypt; ACF, aberrant crypt foci; MDF, mucin-depleted foci; HID-AB, high iron diamine–alcian blue. 2388 Research. on February 28, 2016. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from