A New Medium-term Rat Colorectal Bioassay Applying Neoplastic Lesions as End Points for Detection of Carcinogenesis Modifiers Effects with Weak or Controversial Modifiers

Abstract

We have established a two-stage, medium-term rat colorectal carcinogenesis model featuring induction of neoplastic lesions within ten weeks. In the present study, we examined the ability of this model to detect weak modifiers. F344 male rats were given three subcutaneous (sc) injections of 1,2-dimethyl-hydrazine (DMH, 40 mg/kg b.w.) in one week followed by drinking water containing 1% dextran sodium sulfate (DSS) for a second week. One week after this regimen, basal diet alone, or diets containing 10% perilla oil, 10% corn oil, 10% dextrin, or 0.1% indole-3-carbinol (I3C) were supplied. The perilla oil and corn oil groups did not show significant differences in the numbers of aberrant crypt foci (ACF) and incidences or multiplicity of proliferative lesions as compared to the controls at either time point. In the dextrin group, the total number of ACF at week ten was significantly increased. With I3C, the total number of ACF and incidence and multiplicities of adenocarcinomas at week ten and the incidence of invasive tumors at week twenty were significantly increased. These data essentially correspond with earlier reported results, except in the vegetable oil cases. Thus, the system is suitable for detection of colorectal carcinogenesis modifiers with advantages over previous models using ACF alone as end points.

Introduction

Colorectal cancer is the fourth and third most common malignant neoplasm in men and women, respectively, in the world, with the highest incidence rates in North America, Australia/New Zealand, Western Europe, and, for men especially, Japan (Parkin et al. 2005). Epidemiological studies suggest a strong correlation with dietary factors, such as heterocyclic amines (Sinha and Rothman 1999) and a high-fat diet (Ahmed 2004). In Japan the incidence has increased with the shift to westernized dietary habits (Kono 2004). However, it remains unclear which components of the diet are of most importance in this regard, and it is also necessary to determine protective factors. For this purpose, animal models are needed. Several two-stage colorectal carcinogenesis models have been developed using 1,2-dimethyl-hydrazine (DMH) or its metabolite, azoxymethane (AOM), as initiators (Nigro 1985), but these models require long experimental periods. For short-term screening, aberrant crypt foci (ACF) of the colon have been used. Stained with methylene blue in animals treated with colon-specific carcinogens, first described by Bird (1987), they have long been presumed to be preneoplastic lesions (Bird 1995), and ACF assays have been widely used in rats for detection of colorectal carcinogenesis modifiers within short periods initiated with the colon carcinogen DMH (Maziere et al. 1998) or AOM (Onogi et al. 1996). However, evidence has documented a lack of any direct correlation with tumor development. A number of compounds with the ability to reduce the occurrence of ACF, for example, 2-(carboxyphenyl)retinamide (Zheng et al. 1999) and genistein (Rao et al. 1997), actually enhanced the development of colorectal cancers after initiation with AOM, whereas some tumor promoters like cholic acid decreased numbers of ACF in rats treated with AOM (Magnuson and Bird 1993). Recently, other types of histological alteration, termed β-catenin-accumulated crypts (BCAC) and dysplastic ACF, which feature nuclear accumulation of β-catenin protein and frequently demonstrate gene mutations, have been identified on histological evaluation of rat colon mucosa treated with AOM (Yamada et al. 2000) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) (Ochiai et al. 2003), respectively. It has been argued that BCAC, rather than ACF, should be applied as biomarkers for identifying modulators of colorectal carcinogenesis within a short period (Hirose et al. 2003). However, the necessity for a technique requiring a high level of skill in making cross-sections of colorectum for detection of BCAC argues against use for routine screening.

We have recently established a medium-term colorectal carcinogenesis model in rats initiated with DMH followed by one week of dextran sodium sulfate (DSS) treatment, in which neoplastic lesions including adenocarcinomas can be induced within ten weeks (Onose et al. 2003). A previous study showed that this system offers a useful tool for detection of potent colorectal carcinogenesis modifiers within ten to twenty weeks (Onose et al. 2006).

Numerous substances have already been shown to inhibit or enhance development of colon ACF and/or tumors in short- and long-term bioassay systems. For example, in rats, perilla oil, high in the n-3 polyunsaturated fatty acid (PUFA) α-linolenic acid (C18:3 n-3), weakly inhibited ACF and/or colorectal cancer development induced with different carcinogens, AOM (when given in a 10–12% medium-fat diet supplement before, during, and after initiation) (Komaki et al. 1996; Onogi et al. 1996), N-methyl-N-nitrosourea (MNU; during and after initiation) (Narisawa et al. 1991), and DMH (after initiation) (Hirose et al. 1990). Treatment during and after the initiation period with 10% corn oil, high in the n-6 PUFA α-linoleic acid (C18:2 n-6), is reported to increase formation of ACF or colorectal carcinomas induced by AOM in rats (Wu et al. 2004). Results for a high-carbohydrate diet containing dextrin, a starch hydrolysis product, which physiologically acts as an indigestible dietary fiber (Kishimoto et al. 1995), are controversial. For example, administration of a diet containing 67–68% carbohydrate mix (45% dextrin, 45% sucrose, 5% corn starch, 5% potato starch) during and after initiation with DMH (Kristiansen et al. 1995), AOM (Poulsen et al. 2001) or 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) (Molck et al. 2001), has been found to increase development of rat colon ACF as compared to the case with a reversed carbohydrate mix (5% dextrin, 5% sucrose, 45% corn starch, 45% potato starch). However, the incidence of tumors did not significantly correlate. Indole-3-carbinol (I3C), a glucobrassican derivative abundant in cruciferous vegetables such as broccoli, cabbage, brussels sprouts, and cauliflower (Jongen 1996), has both anticarcinogenic and tumor-promoting activities in animal models, depending on the initiator exposure protocol and species. For example, when male F344 rats were given 0.1% I3C before and during treatment with IQ, the number of ACF was significantly reduced (Xu et al. 1996). Post-initiational treatment of 0.1% I3C decreased the multiplicity of IQ-induced rat colorectal tumors and had no effect on the colorectal tumors induced by DMH (Xu et al. 2001). However, administration of 0.1% I3C to rats for three weeks before, sixteen weeks during, and twelve weeks after treatment with DMH enhanced tumor development in the colon (Pence et al. 1986).

In the present study, we used these agents as typical weak or controversial modifiers to test the validity of our recently established DMH-DSS rat model to detect influence on colorectal carcinogenesis modification within a ten- or twenty-week experimental period.

Materials and Methods

Chemicals and Animals

DMH was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan); DSS (MW 36,000–50,000) from ICN Biomedicals, Inc. (Aurora, OH, USA); perilla oil from Ohta Oil Mill Co., Ltd. (Aichi, Japan); corn oil from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan); dextrin (dietary fiber 89%, sugar 7.5%, moisture 3.5%) from Matsutani Chemical Industry Co., Ltd. (Hyogo, Japan); and 13C from Sigma Chemical Co. (St. Louis, MO, USA). A total of 150 male F344 rats, five weeks of age, were purchased from Charles River Japan Inc. (Kanagawa, Japan) and housed in polycarbonate cages with white wood chips for bedding under standard conditions (room temperature: 24 ± 1°C; relative humidity: 55 ± 5%; twelve-hour light and dark cycle), with free access to basal diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and drinking water. They were used in the experiment after one week of acclimatization.

Experimental Protocol

The experimental design is shown in Figure 1. All rats were given three sc injections of DMH (40 mg/kg body weight dissolved in saline) in the first week and allowed free access to deionized water containing 1% DSS for one week thereafter. All animals were then given tap water and basal diet ad libitum for one week and divided at week three into five treatment subgroups, supplied with basal diet alone as a control or containing the following compounds for seven or seventeen weeks: 10% perilla oil, 10% corn oil, 10% dextrin, and 0.1% I3C. The dose levels of perilla oil, corn oil, and I3C were determined based on the effective levels described in the previous literature (Hirose et al. 1990; Pence et al. 1986; Wu et al. 2004). The dose level for dextrin was modified to a lower level (10% corresponding to 8.9% dietary fiber) than that used in earlier studies (Kristiansen et al. 1995; Molck et al. 2001; Poulsen et al. 2001), because 14% dietary fiber in the diet significantly reduced body weight gain in rats treated for forty-eight weeks (Thorup et al. 1992). In each group, fifteen rats were euthanized under ether anesthesia at ten weeks, and the remaining animals at week twenty. The two different sacrifice time points were applied to detect sensitively the effect of modifiers. At autopsy, all animals were exsanguinated under deep ether anesthesia, and each entire colon was removed and opened longitudinally, and the number of grossly visible colorectal lesions at weeks ten and twenty, and their sizes at week twenty, were recorded. For size, dimensions were determined with a caliper to allow calculation of volume (length × depth × height × 0.52). After macroscopic observation, the entire colons at weeks ten and twenty were stretched flat on filter paper and fixed in 10% neutral buffered formalin for a day. At week ten, after staining with 0.2% methylene blue, numbers of ACF were counted under a light microscope at low-power magnification. For this analysis, each colon was divided into three segments, proximal, middle, and distal, as previously described (Onose et al. 2003). After counting the ACF, the fixed entire colons were cut longitudinally into three strips, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for microscopic examination. Each induced nodule in entire colon at week twenty was trimmed for microscopic examination. Proliferative lesions in the colon mucosa were classified as dysplastic foci, adenomas, and adenocarcinomas. Diagnostic criteria for proliferative lesions were based on our previous report (Onose et al. 2006). The experiments were carried out in accordance with the Guide for Animal Experimentation of the National Institute of Health Sciences of Japan.

Statistical Analysis

All quantitative data are presented as mean ± SD values. Numbers of ACF were analyzed by Student t test following the F test for homogeneity of variance in comparison with the DMH + DSS-alone control group. Incidences and multiplicity of histopathological findings were analyzed by Fisher’s exact probability test and the Mann-Whitney U test, respectively.

Results

One rat in the perilla oil group at two weeks died, probably as a result of colorectal hemorrhage before test chemical treatment, and its data were excluded from the present study.

The final body weights of rats in the corn oil group at week ten were significantly (p < .05) higher than in the control group. Data for ACF and proliferative lesions in each group at week ten are summarized in Tables 1 and 2, respectively. In the DMH + DSS-alone control group, most ACF were small, consisting of no more than four crypts. ACF and colorectal dysplastic foci, adenomas, and adenocarcinomas were observed in all groups (Figure 2), limited to the distal colon as described previously (Onose et al. 2003). In the perilla oil and corn oil groups, there were no significant differences in the numbers of ACF and incidence and multiplicity of proliferative lesions as compared to the controls. In the dextrin group, the total number of ACF was significantly (p < .001) increased, whereas the incidence and multiplicity of adenomas tended to decrease, and those for adenocarcinomas did not differ from the control values. In the I3C group, the total number of ACF was significantly (p < .05) increased, particularly because of large lesions consisting of four crypts or more (p < .01). The incidence and the multiplicity of adenocarcinomas were also significantly (p < .01 and .05, respectively) increased, whereas adenomas showed a tendency to decrease.

At week twenty, the final body weights of rats in the perilla oil and the corn oil group were significantly (p < .01) higher than in the control group (Table 3). Data for ACF and incidences and multiplicities of adenomas and adenocarcinomas at week twenty did not differ from the control values with any of the treatments (Tables 3 and 4, respectively). However, in the corn oil and I3C groups, invasion of adenocarcinomas beyond the lamina muscularis mucosae was observed for one tumor in one rat and eleven tumors in five rats, respectively, and the difference was statistically significant (p < .05) for the I3C group. Tumor volume in the I3C group was apparently elevated, but this elevation was not statistically significant.

Discussion

DSS, a synthetic sulfated polysaccharide composed of dextran with sulfate glucose, is known to induce colitis in rodents (Okayasu et al. 1990). Although DSS is a nongenotoxic compound, its long-term treatment has been reported to result in colon cancer development in rats (Hirono et al. 1981). We have recently succeeded in inducing a high incidence of colorectal adenocarcinomas in rats initiated with DMH followed by DSS treatment within ten weeks (Onose et al. 2003), and we demonstrated that the effects of typical known colon carcinogenesis modifiers, nimesulide and lactoferrin as inhibitors, deoxycholic acid as a promoter, and 1-hydroxyanthraquinone as a carcinogen, could be detected within ten or twenty weeks with this animal model (Onose et al. 2006). In the present study, known colorectal carcinogenesis modifiers—perilla oil, corn oil, dextrin, and I3C—with subtle effects were examined to see whether this system could achieve sensitivity expected for detection of colorectal carcinogenesis modifiers.

In the control group, colorectal tumors, including adenomas and adenocarcinomas, were observed in eight of fifteen and thirteen of fifteen animals at weeks ten and twenty, respectively, consistent with our previous experiments (Onose et al. 2003; Onose et al. 2006). Studies in animal models have shown that in rats, 10% or 12% perilla-oil–rich diets suppress ACF and/or colorectal cancer development induced with different carcinogens—AOM (before, during, and after initiation) (Komaki et al. 1996; Onogi et al. 1996), MNU (during and after initiation) (Narisawa et al. 1991), and DMH (after initiation) (Hirose et al. 1990)—but no such influence was evident in the present study. Treatment with perilla oil during the initiation period with AOM or MNU (Komaki et al. 1996; Narisawa et al. 1991; Onogi et al. 1996) might be partially responsible for the preventive effects on colorectal carcinogenesis. With the inhibition after DMH initiation (Hirose et al. 1990), defatting of basal diet CRF-1 was performed before use, and it is noteworthy that the 5.7% crude fat in basal diet CRF-1 contains 47.8% α-linoleic acid (C18:2 n-6) from soybeans (manufacturer’s data), which might interfere with the preventive effects of α-linolenic acid on colorectal carcinogenesis. Treatment with a 10% corn oil diet rich in α-linoleic acid as soybean oil during and after the initiation period with AOM has been shown to enhance colorectal cancer development in rats (Wu et al. 2004), but again, in the present study this result could not be confirmed, which might partly be explained by the absence of treatment of corn oil during the initiation period.

The observed modulation by dextrin of the development of ACF and tumor occurrence in the present experiment is generally in accordance with results for diets high in sucrose and dextrin (Kristiansen et al. 1995; Molck et al. 2001; Poulsen et al. 2001), although the timing and the duration, as well as the doses, did not completely coincide. Similarly, our finding of the effects of I3C on the progression stage add to the earlier evidence of promotion (Pence et al. 1986). The report that I3C treatment for forty-five weeks after initiation by DMH had no effect on the colorectal tumors does not necessarily coincide with the present study; however, the data did not describe tumor volume and invasion. In other experiments, colorectal carcino-genesis was enhanced when I3C was given three weeks before, sixteen weeks during, and twelve weeks after DMH initiation (Pence et al. 1986). Taken together, it can be interpreted that I3C enhances DMH-initiated colorectal carcinogenesis.

ACF have been widely used as biomarkers for colorectal carcinogenesis (Maziere et al. 1998; Onogi et al. 1996), despite the doubts as to their relevance to the occurrence of cancers (Magnuson and Bird 1993; Rao et al. 1997; Zheng et al. 1999). Usual two-stage rat colorectal carcinogenesis models require approximately thirty weeks to detect modifiers with neoplasms as the end points (Hirose et al. 1990; Reddy et al. 1985). Therefore, our model has distinct advantages, and the present results for weak or controversial modifiers point to its application as a useful tool for the detection of agents that impact colorectal carcinogenesis. Further studies for verification employing other model chemicals are now necessary.