Abstract
The multiple intestinal neoplasia (Min) mouse is a model for intestinal tumourigenesis. On the C57BL/6J (B6) background, with an incidence of 100%, the Min mouse develops numerous tumours throughout the intestine, particularly in the small intestine. The Min phenotype was backcrossed to the FVB/NJ (FVB) genetic background in order to reduce the number of tumours. Control FVB Min mice had an incidence of 7% tumours, both in the small intestine and in the colon. One or more flat aberrant crypt foci (ACF) were also observed in the colon of 20% of the control mice. Neonatal mice were given one dose of the food-processing contaminant 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in order to test the chemical induction of tumours and flat ACF. Treatment with PhIP significantly increased both the number and incidence of tumours in the small intestine, and gave a non-significant increase of tumours and flat ACF in the colon.
The multiple intestinal neoplasia (Min) mouse was discovered by Moser et al. in 1990 as a result of random mutagenesis with ethylnitrosourea (1). These mice have a heterozygous mutation in the tumour suppressor gene Adenomatous polyposis coli (Apc), which leads to spontaneous development of tumours, mainly in the small intestine, but also in the colon (2). On the C57BL/6J (B6) background, the Min mice develop approximately 30-60 tumours throughout the intestinal tract (1). They also develop several flat aberrant crypt foci (ACF) in the colon, which are believed to be precancerous lesions (3).
Concerning both the number of tumours and flat ACF, a large variation among both untreated and carcinogen-treated Min mice on the B6 background is experienced. In addition, we have observed that control Min mice have a relatively high level of tumours and flat ACF in the intestine (3). As a consequence of this, the sensitivity towards intestinal carcinogens in the B6 Min mouse model is possibly reduced since there is a limit to intestinal tumour development.
The genetic background has an effect on the number of tumours in Min mice. The Min mouse on the B6 background has been crossed with several different strains, such as AKR/J (3, 4) and FVB/NJ (FVB) (5, 6). In both of these cases, the subsequent hybrid Min F1 generation had a lower number of small intestinal tumours compared with the Min mice on a pure B6 background. Moser et al. backcrossed the Min phenotype from B6 to the AKR/J background for several generations and observed a further reduction in small intestinal tumours in comparison with the hybrid F1 mice (2), which indicated that the reduction in spontaneous tumour development on the AKR/J background was not caused by resistance to disease as is often observed in hybrid F1 mice. Untreated Min mice on a pure A/J background developed significantly more flat ACF in the colon, but fewer small intestinal tumours than the B6 Min mice. The A/J Min mice were also more susceptible towards the colon carcinogen azoxymethane (AOM) and developed four times more lesions in the colon than the AOM treated B6 Min mice (7).
Several different Modifier of Min (Mom) genes, which affect the tumour burden in the Min mice, have been identified to directly or indirectly interact with the Apc mutation (8). One of these genes, Mom1, is believed to encode for the secretory phospholipase A2 (Pla2s), and is thought to account for around 50% of the variation in tumour number in hybrid Min F1 mice (2). Mouse strains such as FVB and AKR/J, with high expression of Pla2s, are generally known to be relatively resistant to carcinogen-induced tumourigenesis, whereas susceptible strains, such as B6 and A/J, express low levels due to a mutation in the Mom1 gene (9).
The aim of this study was to explore both the spontaneous formation and chemical induction of tumours and flat ACF in the new Min mouse model on a pure FVB background. The food-processing contaminant, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was chosen to test the chemical induction of lesions in this new Min mouse model, because it has been previously shown that this compound increases the number of tumours both in the small intestine and the colon in the Min mouse on the B6 background (6, 10, 11). The FVB mice are widely used in experimental studies, in particular with transgens, because of their transgenic capabilities (12). Crossing transgenic FVB mice with Min mice on an FVB background would provide opportunities to study possible genes that are important for intestinal tumourigenesis on a pure background.
Materials and Methods
Animals, chemicals and treatment. FVB mice were bred at the Norwegian Institute of Public Health, Oslo, Norway, from inbred mice originally purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The Min phenotype was backcrossed to the FVB strain for eight generations from the B6 background at the institute. Each cage housed one female and one male mouse. The animals were housed in plastic cages with a 12 h light/dark cycle. Water and food were given ad libitum. The mice were fed the breeding diet Harlan Teklad 2018 (E) Global (Harlan Teklad U.K., Bicester, Oxfordshire, UK) during gestation and until four weeks of age, then the litter was separated from the mother. Thereafter they were given a standard maintenance diet, SDS RM1 (E), from Special Diets Services Ltd. (Witham, Essex, UK). The Min and wild-type (wt) pups were identified by an allele-specific PCR-assay, as described previously (13). Only the Min mice were used in this study.
PhIP of >98% purity (Wako, Japan) was dissolved in saline and adjusted to a pH of 3.5. Pups were given one subcutaneous injection of 50 mg/kg body weight (bw) PhIP (n=20, ten males and ten females) or 0.9% saline (n=15, nine males and six females) three to six days after birth. The mice were sacrificed by cervical dislocation at eight weeks of age. Scoring of flat ACF and tumours was carried out as reported previously (10).
Statistical analysis. Data were analysed using SigmaStat (SigmaStat software; Jandel Scientific, Erkrath, Germany). Data for the number of tumours and flat ACF were analysed by Mann-Whitney rank sum test. Differences in incidence between the groups were tested by Z-test. A p-value of <0.05 was considered significant.
Results
In the present study, the Min mutation in Apc was, through eight backcrossings from the original B6 background, established on the FVB background. This was done to study both the spontaneous and carcinogenic susceptibility to intestinal tumourigenesis of the Min trait on this genetic background.
Susceptibility to spontaneous intestinal tumourigenesis. The spontaneous tumour incidence in the small intestine was relatively low in the FVB Min mice. Only one of the fifteen control mice (incidence: 7%) had a tumour in the small intestine (Table I). Also in the colon, only one tumour was observed in one of the fifteen control FVB Min mice, again yielding a mean tumour number of 0.1 and an incidence of 7% (Table I). Twenty percent of the control FVB Min mice had one or more flat ACF, which gave a mean colonic flat ACF number of 0.2 (Table I).
Susceptibility to carcinogen-induced intestinal tumourigenesis. One dose of 50 mg/kg bw PhIP three to six days after birth significantly increased both the tumour number and incidence in the small intestine. The tumour number was increased from an average of 0.1 to 2.9 (p<0.001) (Table I). For the FVB Min mice in the present study, there was a large difference in small intestinal tumour incidence between the control and PhIP-treated mice, with an increase from 7 to 95% (p<0.001) (Table I).
After treatment with PhIP there was only a small non-significant increase both in colonic tumour number and incidence in comparison with the control mice (Table I). There was a strong tendency for the PhIP-treated FVB Min mice to have a higher number of flat ACF than the control mice (p=0.056) (Table I). Similarly, a non-significant increase in the incidence of flat ACF was observed in the PhIP-treated mice (Table I).
Breeding, litter size and lifespan. The FVB mice were experienced to be good breeders, and cannibalism was almost non-existent. The litter size was large with an average of 10.2 pups per litter. The FVB Min mice have an approximately normal lifespan. The mice in this experiment were sacrificed at eight weeks of age because this time point is commonly used for the B6 Min mouse model, but the current results indicate that the duration of the experiment can be increased in the FVB Min mouse model and tumour development can be studied for a much longer period.
Discussion
Intestinal tumour development in control FVB Min mice was low. Untreated B6 Min mice in a previous study given the same diet and sacrificed at the same age had a much higher tumour development, with a number of 43 (median) and incidence of tumours in the small intestine of 100%, and a colonic tumour number of 2 (median) and with 75% incidence. The observed number of flat ACF in the FVB Min mice was somewhat higher than the tumour number, with an incidence of 20% (submitted for publication by Svendsen, C., 2010). In comparison, the B6 Min mice had a median flat ACF number of 12, and all of the mice had one or more flat ACF in the colon (submitted for publication by Svendsen, C., 2010). This indicates that FVB mice in general are more resistant to intestinal tumour development than the B6 mice. In support of this view, it was found that the hybrid Min F1 generation resulting from a cross between B6 Min and FVB wt were less susceptible for spontaneous intestinal tumourigenesis and was also more resistant to non-steroidal anti-inflammatory drug suppression with significantly less reduction of tumour number compared to the B6 Min mice (7). The lower tumour development in the intestine can be considered as an advantage when studying relatively potent carcinogens, as it is time-saving during counting of tumours.
Number and incidence of tumours and flat ACF in eight-week-old FVB Min mice after one subcutaneous injection of 50 mg/kg bw PhIP or vehicle (control) 3-6 days after birth.
Even though the FVB Min mice seems to be relatively resistant to spontaneous induction of intestinal lesions, there was a considerably increase in the number and especially the incidence of small intestinal tumours after PhIP treatment. This again suggests that the FVB Min mice model can be useful for studying relatively potent carcinogens. In an earlier study, it was found that for B6 Min mice given the same treatment and sacrificed at the same age (but were given a different breeding diet, RM3 (SDS) instead of Harlan Teklad), treatment with PhIP also increased the tumour number, but only 2.5-fold (submitted for publication by Svendsen, C., 2011). In another previous study, it was shown that PhIP exposure increased the relative small intestinal tumour number in AKR/J × B6 hybrid Min mice by 3.8-fold and in A/J × B6 hybrid Min mice by 3.7-fold compared with only a 3-fold increase in the B6 Min mice (4). No significant increase in tumour development or the number of flat ACF in the colon was observed in the FVB Min mice. This is in contrast to findings in B6 Min mice; here PhIP significantly increased the number of colonic tumours (4, 11).
The FVB mice are considered to be much better breeders than the B6 mice, where normally 30% of the litters are eaten by the mothers (14). Cannibalism is almost non-existent for the FVB mice (Authors' personal experience), and also the litter size is bigger, with an average of 10.2 pups per litter compared to B6 mice where the average is 5. Others have observed similar litter sizes, with 9.5 and 6.7 for the FVB and B6 mice, respectively (12). The lifespan for the B6 Min mice is only around 120 days (1), whereas the FVB Min mice have an approximately normal lifespan. The short lifespan for the B6 Min mice is due to intestinal bleeding, anaemia, and rectal prolapse caused by the high tumour burden (15). The breeding efficiency of the FVB Min mice makes this a useful cost- and time-efficient model for studying intestinal carcinogenesis.
In conclusion, the spontaneous intestinal tumour development of the FVB Min mouse is low, and therefore this model is probably not suitable for testing chemopreventives or dietary compounds that are believed to reduce the number of intestinal lesions; for this purpose the B6 model is better suited. However, the FVB Min mouse might be a relevant model for testing possible human carcinogens since in the present study it was shown that the mutagen PhIP increased the tumourigenesis in the intestines of these mice.
Acknowledgements
We thank Andre Larsen, Hege Hjertholm, Tone Rasmussen and Victor Labay Ong for excellent technical assistance. This work was supported by the Norwegian Research Council (175157/V40).
- Received December 20, 2010.
- Revision received February 28, 2011.
- Accepted February 28, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
