Tumorigenesis in the GI tract
A total of 99 intestinal tumors were found from the mice studied over 24 months with total tumor numbers of 13, 32 and 54 at the three time points. They were all studied for histological type. Among 99 tumors, 11% were adenocarcinomas and 89% were adenomas. Adenocarcinomas were only 3% (2 of 58) in the small intestine and 22% (9 of 41) in the right side of colon. Among adenocarcinomas, only three were fully invasive carcinomas including two in NWD/Methn and one NWD/Fb group. The other nine were early invasive carcinomas involving the submucosa. Seventy-three percentage of the adenocarcinomas occurred after 18 months of feeding. Tubular adenomas were often seen, especially in the small intestine. Flat adenomas were also observed in the cecum. In this report, we cannot compare the effects of dietary nutritional components on tumor progression due to the limited number of each histological type of tumors in each diet group and at each time point. Actually, both adenomas and carcinomas were seen in each diet group. This restriction allowed us to focus primarily on the tumor incidence and multiplicity in the small intestine and colon and to analyze the effects of dietary nutritional components on tumor development. Only one microadenoma was found in the stomach of a mouse fed NWD for 18 months; for convenience in analysis, this was included in the total of small intestine. Therefore, the data reported here were mainly tumor incidence and multiplicity in the small intestine and colon of seven diet groups.
After 6 months of feeding, a few mice from each group were killed and examined for visible lesions in the intestinal tract. None were found. These early, apparently normal colons were examined for early genetic alterations, by gene expression analysis, of the flat colonic mucosa induced by diet at 6 months (4
). The results indicated that feeding the NWD generated significantly altered profiles in gene expression in the flat mucosa, which also inhibited heterogenicity among the mice similar to that initiated in the mucosa by inheritance of mutant Apc allele (4
After 12 months of feeding, there were no intestinal tumors in mice fed AIN-76A and NWD. The remaining groups had tumors in the small intestine and/or in colon. The overall tumor incidence was 15% in NWD/CaD, 7% in NWD/FA, 20% in NWD/Methn, 22% in NWD/Chln and 6% in NWD/Fb. There were no tumors in the colon of three diet groups, NWD/CaD, NWD/FA and NWD/Fb. For the other two groups, NWD/Methn and NWD/Chln, the tumor incidence was similar in both small intestine and colon with 10% in NWD/Methn and 17% in NWD/Chln. There was no statistical significant difference of tumor incidence either in small intestine or in colon between groups of AIN-76A and NWD or between NWD and NWD with any of the added supplements at 12 months of feeding. The mean tumor multiplicity showed no significant difference in all five groups found with intestinal tumors, compared with NWD, except for NWD/Methn group. At 12 months, the tumor multiplicity increase was significant in the overall intestine (0.39, P
0.01), the colon (0.22, P
0.05) and not fully significant in the small intestine (0.17, P
0.08) compared with NWD (0.00) ( and ).
Tumor incidence in small and large intestine of C57Bl/6 mice at three time points during 24 months of feeding
Tumor multiplicity in the small and large intestine of C57/Bl6 mice at three time points during 24 months of feeding
After 18 months of feeding, intestinal tumors developed in both small intestine and colon of mice in every diet group of the seven diet groups studied including AIN-76A, NWD and NWD with supplements. In the colon, the tumor incidence and tumor multiplicity was low in the seven diet groups. But it showed a trend suggesting that the tumor multiplicity increased in NWD compared with AIN-76A (0.23 versus 0.06, P
0.05) and decreased in NWD/CaD (0.05 versus 0.23, P
0.05). There was no significant difference of tumor incidence and multiplicity overall in the total intestine, or in the small intestine and colon individually between groups of NWD and AIN-76A, and between groups of NWD and NWD with supplements ( and ).
At 18 months of feeding, this study failed to show significant increase in the intestinal tumors in the NWD as demonstrated in the pilot study (1
), but did show this effect at 24 months. Other than normal biological and perhaps seasonal and experimental variability, we have no real explanation for the difference in the 18 months time period.
After 24 months of feeding, a statistically borderline difference was seen in tumor multiplicity overall in the intestine (P
0.08) and in the colon (P
0.08) among the seven diet groups. NWD increased intestine tumor incidence overall (53 versus 27%, P
0.05) and in the colon by 2.9-fold (27 versus 7%, P
0.05) compared with AIN-76A controls. The total tumor multiplicity increase was also observed in the intestine by 1.5-fold (0.67 versus 0.27, P
0.08) and in the colon by 3.7-fold (0.33 versus 0.07, P
0.05). However, the NWD with increased calcium and vitamin D significantly inhibited tumor development in the intestine. The tumor incidence decreased by 89% (6 versus 53%, P
0.01) overall in the intestine and no tumors in the colon (0 versus 27%, P
0.05) of mice in NWD/CaD compared with NWD group. Calcium and vitamin D supplement also decreased the tumor multiplicity by 91% overall in the intestine (0.06 versus 0.67, P
0.01) and by 82% in the small intestine (0.06 versus 0.33, P
0.02) and in the colon (0.00 versus 0.33, P
0.02) compared with NWD. Tumor development in the intestine was not inhibited by any of the other supplements modified in NWD, including folic acid, methionine, choline and fiber ( and ).
These results indicate that a NWD with increased lipid and decreased nutrients of calcium, vitamin D, methyl donors and fiber produced intestinal tumor development in wild-type C57Bl/6 mice after long-term feeding. The colonic tumors caused by the NWD were significantly inhibited by increased dietary calcium and vitamin D content, but not by any individually increased nutrient of folic acid, methyl donors (methionine and choline) or fiber. In this study, increased folic acid, methionine, choline or dietary fiber did not inhibit the increased development of colon tumors induced by the NWD, a mimic of the human NWD. However, increased dietary calcium and vitamin D completely inhibited such colon tumor development.
Tumorigenesis outside the GI tract
Besides tumors in the intestine, extra-GI tract tumors were observed in C57Bl/6 mice after long-term feeding of the diets studied described above. In seven diet groups, a total of 48 (14%) mice had extra-GI tumors including three (1%) in AIN-76A group and 45 (13%) in NWD groups among 356 mice over the 24 months studied. Most of the extra-GI tumors were observed after 18 months of feeding, except for one mouse in AIN-76A group. Four major types of extra-GI tumors were found: 4.2% with lung tumors (alveolar/bronchiolar adenomas/carcinomas), 3.9% with non-Hodgkin’s lymphomas, 2.8% soft tissue tumors (hemangioma and leiomyosarcoma) and 2.8% with liver tumors (hepatoblastoma and Kupffer cell sarcoma). The incidence of lung tumors, non-Hodgkin’s lymphoma and soft tumors, but not liver tumors, was higher in NWD than AIN-76A (all P
0.05). It was interesting to note that the incidence of extra-GI tumors was also higher in NWD with supplementation of methyl donors than in NWD alone. This is similar to the effect of long-term effect of increased folic acid over 7 years observed in a human study by Cole et al.
). There were no soft tissue tumors in the NWD/Fb group and no liver tumors in the NWD/Methn group. The extra-GI tumors showed no statistical significant difference between NWD and AIN-76A.
The data of 24 months as indicated in illustrates the potential relation between GI tumors and diet in this study group. A suggested effect in the test is that increased calcium and vitamin D reduced the increased tumors of NWD addition by itself (paragraph starting with ‘after 24 months of feeding…’).