Aberrant crypt foci and colon cancer: comparison between a short- and medium-term bioassay for colon carcinogenesis using dimethylhydrazine in Wistar rats

! " #$% & ' ( ) * #$% ! + , ! ! ./ " 0 0 * #$% 1 2. 3/ 4 3/ ! 5 2/ 0 ) ! 6 .! ! & ) 6 ' 0 #$% ) ! Correspondence M.A.M. Rodrigues Departamento de Patologia, Box 564 Faculdade de Medicina, UNESP 18618-000 Botucatu, SP Brasil Fax: +55-14-6821-2348 E-mail: mariar@fmb.unesp.br Research supported by FAPESP


Introduction
Colorectal cancer is an increasingly important cause of cancer death in Brazil (1,2). To establish better methods for the clinical management of patients with colon cancer it is necessary to understand the mechanisms of carcinogenesis. Development of colon cancer is a multistep process involving a series of pathological alterations ranging from discrete microscopic mucosal lesions, like aberrant crypt foci (ACF), to malignant tumors (3,4).
ACF are focal lesions of the colonic mucosa composed of one to several enlarged crypts, which are specifically induced by colon carcinogens (5,6). Easily identified in methylene blue-stained whole-mount preparations under a dissecting microscope, they are usually quantified as number per animal or per colon and used as early indicators of colon carcinogenesis (3,7,8). However, no correlation between the number of ACF and the incidence of adenocarcinomas was found in some studies (9,10). Evaluation of the predictive value of such precursor lesions as surrogates for neoplasia is increasingly needed in the context of carcinogen or anticancer drug evaluation (11,12).
The genotoxic chemical dimethylhydrazine (DMH) is one of the agents most frequently used in experimental models of colon carcinogenesis in rodents (13)(14)(15). It is a complete carcinogen that induces the initiation and promotion steps of carcinogenesis yielding macroscopically visible neoplasms in a dose-dependent manner (13,15). Thus, the DMH-induced colon cancer model in rats is a good tool to investigate the relationship of ACF to colon cancer. To clarify whether ACF are directly related to colon carcinogenesis, this study was carried out to evaluate the relationship between a shortterm (4 weeks) and medium-term (30 weeks) assay in a model of colon carcinogenesis induced by DMH in the rat.

Animals and chemicals
Male Wistar rats aged 6 weeks and weighing about 160 g were obtained from the Paraná Institute of Technology (TECPAR, Curitiba, PR, Brazil). The animals were housed five per polypropylene plastic cage covered with metal grids in a temperature-controlled room (21-25ºC) with a 12-h light/ 12-h dark cycle. They were maintained on a Nuvilab-CR1 basic diet (Nuvital, Curitiba, PR, Brazil) and tap water ad libitum. 1,2-Dimethylhydrazine was purchased from Sigma (St. Louis, MO, USA). DMH (40 mg/ kg body weight) was dissolved immediately in PBS before use in 1 mM EDTA and the pH of the solution brought to pH 6.5.

Experimental design
After 10 days of acclimation the rats were randomly assigned to four groups of 10 animals each. Two groups received four subcutaneous injections of DMH (40 mg/kg body weight, twice a week for 2 weeks). Control groups received injections of PBS plus the vehicle (EDTA). One DMH-treated group and one control group were killed at the end of the 4th week (short-term assay). The other DMH-treated group and its respective control were maintained without any further treatment until the 30th week (medium-term assay). All groups were sacrificed by diethyl ether inhalation. This protocol was approved by the Ethics Committee on Care and Use of Laboratory Animals of the National Research Council.

Necropsy and tissue preparation for aberrant crypt foci counting
At autopsy the entire colon was removed, opened longitudinally and rinsed in saline. Cecum was excised and the remaining colon was divided into three segments of 8 cm each, referred to as proximal colon (adjacent to the cecum), middle colon and distal colon (adjacent to the rectum). Each segment was fixed flat on filter paper in 10% buffered formalin for 24 h and then stained with 0.1% methylene blue in PBS for 20 min for ACF counting. Specimens were carefully examined, mucosal side uppermost, by two pathologists at 40X magnification under a compound light microscope. The number of ACF was determined, and their distribution pattern along the colon and crypt multiplicity were scored. Samples for histological analysis were collected and histological sections were cut perpendicular to the muscularis mucosa and stained with hematoxylin and eosin. The material was analyzed for the presence and degree of dysplasia according to Siu et al. (16).

Tumor analysis
The presence of tumors was carefully noted in terms of number, size and location along the colon. Samples for histological study were processed, sectioned at 4-5 µm and stained with hematoxylin and eosin. The neoplasms were classified according to the histopathological classification proposed by Sunter et al. (13).

Statistical analysis
Comparisons of ACF number and crypt multiplicity between the short-term (4 weeks) and medium-term (30 weeks) assays were evaluated for significance by the Fisher exact method. The differences were considered significant when P<0.05.

Results and Discussion
ACF were stereoscopically distinguished from normal crypts by their darker staining and larger size, elliptical shape, thicker epithelial lining, and larger pericryptal zone ( Figure 1A). No ACF were detected in control rats. They were observed only in DMHtreated rats.
The distribution and temporal evolution of ACF in the colon are presented in Figure  2. The majority of ACF were present in the middle and distal colon both in the shortterm (4 weeks) and medium-term (30 weeks) assays, demonstrating that ACF are not random lesions of the rodent colon treated with genotoxic colon carcinogens (12).   No significant differences in the number of ACF/cm 2 were observed between the shortterm (4 weeks) and medium-term (30 weeks) assays, especially in the middle and distal colon (Figure 2A). These findings support the view that ACF represent focal morphological alterations induced by DMH in the colonic mucosa and this may reflect the initiation step of carcinogenesis (3,8).
The number of crypts per ACF increased significantly from the 4th to the 30th week in the middle and distal colon ( Figure 2B). At the 4th week 91.5% ACF had one or two crypts and only 8.5% ACF had three or more crypts, while at the 30th week 46.9% ACF had three or more crypts (Table 1). This progression of ACF morphology consisting of multiple crypts observed from the 4th to the 30th week may correspond to the promotion step of colon carcinogenesis (17,18).
Histologically, ACF also presented progressive changes. At the 4th week they exhibited hyperplastic features, while mild to severe dysplasia was observed at the 30th week ( Figure 1B).
Nine tumors were found in 8 of 10 DMHtreated rats examined at the 30th week. Seven tumors were located in the distal colon and two in the middle colon, which were the sites most populated by ACF. Thus, ACF and colon cancer developed at the same sites in the colonic mucosa of DMH-treated rats, meaning that they may be sequentially related biological events (3,7,8). Interestingly, all tumors were group I well-differentiated adenocarcinomas according to Sunters classification (13). These findings indicate that ACF may be regarded as early morphological markers in the pathway for well-differentiated tumors in colon carcinogenesis (14). In contrast to other studies (13-15), we did not find tumors in the proximal colon and no tumor was of the poorly differentiated, mucin-secreting histological type. These results may be related to the lower dose of DMH used in the present study compared to previous reports (13)(14)(15).
Overall, the results of the present study demonstrate that induction of ACF by DMH in the short-term (4 weeks) assay was correlated with development of well-differentiated adenocarcinomas in the medium-term (30 weeks) assay. The observed relationship supports the view that ACF are precursor lesions for colon tumors and can be used in short-term assays as surrogate end-points to assess carcinogenic or anticancer activity in chemically induced models of colon carcinogenesis. Table 1. Number of aberrant crypt foci (ACF) and crypt multiplicity (%) in the short-term (4 weeks) and medium-term (30 weeks) assays from rats treated with DMH.

Groups
Total