14.4.1 Carcinogenesis induced by chemicals
Among the various experimental models used to study colon carcinogenesis, those using azoxymethane (AOM) or 1,2-dimethylhydrazine (DMH) to induce colonic cancer in rodents are very important, since these two carcinogens induce tumors through the sequential formation of histopathological lesions similar to those observed in spontaneous carcinogenesis in humans (Chang, 1984). Accordingly, these methods have been widely used to study the biology of the various phases of colon cancer but also to study the correlation between diet and cancer, by comparing cancer incidence in DMH/AOM initiated rodents fed with different dietary regimens (Fig. 14.3). The DMH/AOM model is also very popular for study of the effect on colon carcinogenesis of putative chemopreventive chemicals such as non steroidal anti-inflammatory drugs (Corpet and Taché, 2002).
Other carcinogens more related to food, such as heterocyclic amines have also been used to induce intestinal tumors in rodents (Ochiai et al., 2003). However, AOM or DMH are less expensive than these carcinogens (e.g., 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine: PhIP) and can more
Early lesions (ACF, MDF)
Early lesions (ACF, MDF)
Standard diet + chemopreventive agent
Fig. 14.3 Scheme of an experimental model to study the effects of nutrients on colon carcinogenesis. In both treated and control rats, colon carcinogenesis is induced with injections of azoxymethane (AOM) or 1,2-dimethylhydrazine (DMH). The putative chemopreventive agent, for instance a dietary compound, can be given through the experiment mixed in the diet. Animals are sacrificed after 2-3 months to observe early prenoplastic lesions (ACF or MDF) or later, to observe mascroscopic tumors. The effect of the putative chemopreventive agent is evaluated comparing the frequency of early lesions or tumors in the controls and treated rats.
conveniently be administered via subcutaneous or intraperitoneal injections. On the contrary, PhIP has to be administered mixed in the diet for a long period of time (Ochiai et al., 2003).
Rats are the animals used most frequently in the AOM/DMH models, since cancer can be easily induced with just two injections of the carcinogen in these animals, whereas multiple injections are required in mice. However, mice have a smaller body weight than rats so smaller amounts of the chemicals are required. Various dose regimens can be used. We used two subcutaneous injections, one week apart, of DMH, 150 mg/kg each (total dose 300 mg/kg), or, alternatively, two subcutaneous injections, one week apart, of AOM, 15 mg/kg each (total dose 30 mg/kg) in rats. With these dosages and schedule we obtained a good yield of preneoplastic lesions (ACF and MDF) and tumors (Femia et al., 2005). Tumor induction with doses either higher or lower than those we use has been reported (for the websitehttp://www.inra.fr/ reseau-nacre/sci-memb/corpet/indexan.html for a review).
Rats treated with two injections of AOM or DMH (total dose 30 mg/kg or 300 mg/kg, respectively) develop tumors about eight months later. Both carcinogens induce the majority of cancers in the colon (about one/two tumors per rat with the dosages reported above), but tumors in the small intestine and in the inner ear are also induced, though at a lower frequency. Animals showing rectal bleeding are sacrificed before time.
Typically, groups of 20-30 animals are treated. Treatment with the putative dietary regimen can be administered during the various phases of carcinogenesis, before induction with the carcinogen, during induction or after, during the promotion-progression phase of carcinogenesis.
At sacrifice using CO2 asphyxiation, all organs are macroscopically examined for the presence of tumors or other pathological lesions. Tissues showing a deviation from normal morphology are fixed in 10% buffered formalin and embedded in paraffin blocks. If possible (due to the dimension of the tumor), part of the lesion is also kept frozen at -80°C for further analysis. Paraffin blocks are then sectioned and stained with hematoxylin-eosin to confirm the presence and type of tumors by histopathological examination, which is performed by a pathologist unaware of the codes of the specimens. Before being fixed in formalin, suspected macroscopic lesions are measured with a caliper and their dimensions calculated by multiplying the two main diameters of each lesion. Cancer histological types are evaluated on the basis of the histotype, grading and pattern of growth (Morson et al., 2003). Adenomas are classified on the basis of their microscopic architecture as tubular, tubulovillous and villous according to Morson et al. ( 2003). To circumvent the necessity of waiting a long time to observe the growth of tumors, purported preneoplastic lesions such as ACF have also been used as endpoint related to carcinogenesis. As reported above, ACF have characteristics of preneoplastic lesions, they are easily quantified and are visible as early as one month from carcinogen induction. Moreover, with a standard dose of carcinogen (f.i. 30 mg/kg of AOM) each rat develops about one hundred
ACF, so that the number of animals/group needed to see a significant effect is relatively smaller with ACF experiments than with long-term carcinogenesis studies (12-15 animals compared with 20-30 animals/group). For these reasons and also because of the simplicity of their quantification, ACF determination has been widely used as a short-term assay to predict carcinogenesis outcome (Corpet and Taché, 2002, see also the website: http://www.inra.fr/reseau-nacre/sci-memb/corpet/indexan.html). According to PubMed records of the National Library of Medicine, almost 500 papers have been published on ACF as colorectal cancer biomarkers.
ACF are clearly visible two months after induction with the carcinogen. At sacrifice, the colon is carefully removed from the abdomen, washed with saline, longitudinally opened and pinned flat on a polystyrene board or on two filter papers. After fixation in buffered formalin (at least 4 h), the colon can be cut into three segments: proximal (closer to the cecum and characterized by 'herring-bone' folding of the mucosa), mid and distal (obtained by cutting the remaining colon into two equally long segments). ACF are determined according to Bird (1987) using methylene blue (MB) staining. ACF appear as crypt aggregates, morphologically altered (larger, intensely stained and with a higher peri-cryptic area) (Bird, 1987).
Determination of ACF is performed by counting the number of lesions present in the entire colon and also the number of aberrant crypts (AC) forming each ACF (multiplicity of ACF). Another ACF parameter that has been calculated to predict carcinogenesis outcome is the number of 'large' ACF, which have been defined with different criteria (Corpet and Taché, 2002, Corpet, et al., 1990, Pretlow, et al., 1992). For instance, Pretlow and colleagues (1992) defined 'large' ACF as lesions with a multiplicity equal to or higher than four crypts/ACF. Corpet et al. defined large ACF (1990) as ACF being of such a multiplicity that at least one large ACF/rat is present in the control group. It is obvious that using different definitions, different results can be obtained.
The number of total ACF, the multiplicity of ACF and 'large' ACF are all considered parameters correlated to cancer outcome. In the last few years some observers have questioned the use of ACF as a parameter correlated with carcinogenesis. For instance, compounds such as genistein, selected as possible chemopreventive agents based on ACF results, have later been shown to increase tumor development (Magnusson et al., 1993, Zheng et al.,
1999, Papanikolaou et al., 2000). Different studies have tried to explain the discrepancy between ACF and cancer (Jen et al., 1994, Papanikolau et al.,
2000, Paulsen et al., 2001), suggesting that only a small portion of ACF, probably constituted by the most dysplastic ones, will become tumors.
Other preneoplastic lesions such as MDF, which are easily detectable in the unsectioned colon of rats 10-15 weeks after induction with the carcinogen, have been proposed as endpoint related to colon carcinogenesis. Accordingly, in AOM or DMH-induced rats the number of MDF increases with promoters of carcinogenesis such as cholic acid, high-fat or heme-rich diets (Femia et al., 2004, Pierre et al., 2004) whereas it decreases with chemopreventive agents such as polyethylene glycole, or synbiotics (Femia et al., 2005, Caderni et al., 2003).
MDF determination can be performed in colons which have been processed for the determination of ACF with methylene blue (MB). MB-stained colons can be kept in formalin and then processed with the high-iron diamine Alcian blue staining (HID-AB). Briefly, the entire colon is rinsed in distilled water for 5 min and transferred into a Petri dish containing a freshly prepared solution (referred to as diamine solution), obtained by dissolving simultaneously 120 mg of N-N'-dimethyl-m-phenylene diamine and 20 mg of N-N'-dimethyl-p-phenylene diamine in 50 ml of distilled water and then adding 1.4 ml of 60% ferric chloride. The Petri dish is covered with aluminum foil to protect it from the light and the colon allowed to be stained with the diamine solution for 18 h at room temperature. The colon is then rinsed three times in distilled water and stained for 30 min with 1% Alcian Blue in 3% acetic acid. The colon is then rinsed three times with 80% ethanol followed by distilled water and then observed under the microscope (mucosa side up) for the determination of MDF. To score the colon we use Optiphot-2, NIKON. With HID-AB staining, normal crypts in the distal part of rat colon are stained brown (indicative of a predominant production of sulphomucins) while in the more proximal part of the colon the crypts are blue (indicative of a predominant production of sialomucins). In HID-AB stained colons, ACF appear as brown or blue depending on their multiplicity and dysplasia (Caderni et al., 1995). MDF are identified as focal lesions characterized by the absence or very limited production of mucins (Caderni et al., 2003). Besides this defect in mucin production, MDF can be recognized since they are focal (with a very clear cut distinction between the lesions and the normal adjacent crypts) and are formed by crypts with a lumen which is often, not always, distorted when compared with normal surrounding crypts. Elevation of the lesion above the surface of the colon, and a multiplicity (i.e., the number of crypts forming each focus) of more than three crypts, are also frequent features of MDF. The colon is then coded and the scoring is performed blindly by two observers. For each colon we determine the number of MDF/colon and their multiplicity (i.e., the number of crypts forming each focus).
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