Neonatal exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine via breast milk or directly induces intestinal tumors in multiple intestinal neoplasia mice
Jan Erik Paulsen*,
Inger-Lise Steffensen*,
Åshild Andreassen,
Rose Vikse and
Jan Alexander1
Department of Environmental Medicine, National Institute of Public Health, PO Box 4404 Torshov, N-0403 Oslo, Norway
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Abstract
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We examined whether the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) could increase intestinal tumorigenesis in neonatal C57BL/6J-Min/+ mice, a murine model for familial adenomatous polyposis. Min/+ mice are heterozygous for a nonsense mutation in the adenomatous polyposis coli gene and spontaneously develop multiple intestinal adenomas, primarily in the small intestine. Neonatal Min/+ mice (36 days old) were exposed to PhIP via breast milk from lactating dams given 8 s.c. injections of 50 mg/kg PhIP three times a week or to 8 s.c. injections of 25 or 50 mg/kg PhIP directly, over the same period. At the age of 11 weeks, the number, diameter and location of the intestinal tumors were scored. Remarkably, a 2- to 4-fold increase in the number of small intestinal tumors was seen in Min/+ mice exposed to PhIP via breast milk (P < 0.001). To our knowledge, this is the first time PhIP has been reported to induce tumors following exposure via breast milk from PhIP-exposed dams. Upon direct exposure to 50 mg/kg PhIP, a 6- to 9-fold increase in the number of small intestinal tumors was observed (P < 0.001). The diameter of the PhIP-induced small intestinal tumors was slightly increased (P < 0.001). In the colon, a 3- to 4-fold increase in the number of tumors was seen in Min/+ mice exposed to PhIP via breast milk (P = 0.004). Direct exposure to 50 mg/kg PhIP caused a 2- to 6-fold increase in the number of colonic tumors (P = 0.014). The PhIP-induced colonic tumors were located more distally and displayed a smaller diameter than the tumors from the controls (P < 0.05). In contrast to a previous study, where PhIP showed only a moderate tumorigenic effect in adult Min/+ mice, the present study demonstrates a strong tumorigenic effect of PhIP in neonatally exposed Min/+ mice, even after exposure via breast milk from PhIP-exposed dams.
Abbreviations: ACF, aberrant crypt foci; APC/Apc, adenomatous polyposis coli gene (human/murine); FAP, familial adenomatous polyposis; Min, multiple intestinal neoplasia; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
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Introduction
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Colorectal cancer appears to be associated with a high consumption of red meat (1), especially cooked well-done (2). Highly mutagenic and carcinogenic heterocyclic amines isolated from the crust of fried meat have therefore been suggested as candidate human colorectal carcinogens (3). The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was first isolated from fried ground beef (4) and is the most abundant heterocyclic amine in various cooked fish and meats (3,4). In cultured mammalian cells, PhIP is more genotoxic than the other heterocyclic amines (5,6). In rats, PhIP induces tumors in the colon, small intestine and cecum, as well as in the mammary glands in females (79), and also colonic aberrant crypt foci (ACF) (9,10). In mice, however, PhIP induces lymphomas and no intestinal tumors (11), although PhIP-induced ACF have been reported (12).
Familial adenomatous polyposis (FAP) is a dominantly inherited autosomal disorder, which leads to an early development of multiple colorectal adenomas (13). Some of these lesions inevitably progress to carcinomas if not removed. FAP is caused by germline mutations in the tumor suppressor gene adenomatous polyposis coli (APC) (1416). As in human FAP, various germline mutations in the murine Apc, which is homologous to the human APC gene, induce multiple intestinal adenomas, of which some progress to adenocarcinomas in older mice (1720). However, unlike FAP, most of the tumors develop in the small intestine. One of these mouse strains, C57BL/6J-Min (multiple intestinal neoplasia), is heterozygous for a germline nonsense mutation in codon 850 of the Apc gene, changing a leucine (TTG) to a stop (TAG) codon, thereby producing a truncated non-functional Apc protein (17,18).
Inactivation of the tumor suppressor gene APC seems to be an early and important event in the development of colorectal cancer, whether it is caused by a somatic or an inherited mutation (13). The Min mouse and analogous strains provide good carcinogenesis models for both sporadic and inherited forms of colorectal cancer and they represent an opportunity to study the pathogenesis of a neoplasm in which the initial molecular defects are the same in both human and mouse. It appears that intestinal tumor development both in humans (13,21) and mice (2023) is associated with loss of function of both APC/Apc alleles. Accordingly, it seems likely that the murine FAP models are particularly susceptible to intestinal carcinogens that affect the Apc gene. In PhIP-induced tumors in rats, a specific 5'-GGGA-3'
5'-GGA-3' mutation in the Apc gene has been found (24).
Recently, we reported that adult Min/+ mice were more susceptible to PhIP than wild-type mice (25). However, this effect was restricted to a slight increase in the number of nascent tumors in the proximal small intestine and in the number of ACF in the colon. According to this, in other murine FAP models with one mutated Apc allele, PhIP exposure after 4 weeks of age caused no (26) or only a moderate (27) tumorigenic effect. The results from our previous study (25) indicated that spontaneous tumor initiation in Min/+ mice took place much earlier than the PhIP exposure at 47 weeks of age. Furthermore, mice seem to be more susceptible to PhIP in the early neonatal period than later in life (28). PhIP given to lactating rodent dams is transferred through the breast milk to the pups (2931). The main objective of the present study was therefore to examine the effect of PhIP in neonatal Min/+ mice. We exposed Min/+ pups to PhIP either via breast milk from PhIP-injected lactating dams or by injections directly.
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Materials and methods
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Mouse breeding
The mice were bred at the National Institute of Public Health (Oslo, Norway) from inbred mice originally purchased from The Jackson Laboratory (Bar Harbor, ME). The Min pedigree was maintained by mating C57BL/6J-+/+ (wild-type) females with C57BL/6J-Min/+ males, and procedures to secure inbreeding were followed. The Min mutation was propagated through the males, since anemia and intestinal adenomas interfere with pregnancy (17). All mice used in the experiment were related within the number of generations (<12) necessary for securing their status as inbred. The Min/+ mice used in the present study were identified by allele-specific PCR, as described in detail previously (25), using DNA isolated from blood drawn after the lactation period. The mice were housed in plastic cages in a room with a 12 h light/dark cycle and controlled humidity (55 ± 5%) and temperature (2024°C). Water and diet were given ad libitum. The mice were given a breeding diet, SDS RM3 (E), from Special Diets Services (Witham, UK) during gestation and until 5 weeks of age; thereafter they were given a standard maintenance diet from B&K Universal (Grimston, UK). The mice were killed by cervical dislocation at 11 weeks of age.
PhIP treatment
PhIP of >98% purity purchased from Toronto Research Chemicals (North York, Ontario, Canada) was dissolved in concentrated HCl, which thereafter was evaporated. The PhIPHCl was dissolved in 0.9% saline and adjusted to pH 3.5. From 3 to 6 days after birth the pups were injected s.c. on Mondays, Wednesdays and Fridays, in total eight times, with either 25 or 50 mg/kg body wt PhIP, or the lactating dams (n = 5) were given eight s.c. injections of 50 mg/kg PhIP.
Scoring of tumors
The large and small intestines were removed separately, rinsed in ice-cold phosphate-buffered saline and slit open along the longitudinal axis. The small intestines were divided into proximal, middle and distal sections. The intestinal tissues were then spread flat between sheets of filter paper and fixed in absolute ethanol. The number, diameter and location of adenomas in the large and small intestines were scored by transillumination in an inverse light microscope at a magnification of x20. The diameters of the adenomas were scored with an eyepiece graticule. The smallest adenomas (<0.1 mm in diameter) observed were monocryptal as confirmed by histology (not shown). The tumor position along the intestines is given in centimeters from the ventricle. For each experimental group, the incidence of tumors, defined as the number of mice with tumors/number of mice in the group, the mean number of tumors/mouse ± SEM, the mean tumor diameter (mm)/mouse ± SEM and the mean number of tumors/cm intestine/mouse were calculated for the small intestine and colon separately.
Statistical analysis
We used a two-way ANOVA (SigmaStat software; Jandel Scientific, Erkrath, Germany) after rank transformation of the data due to their non-normal distribution, to test the following three null hypotheses: (i) there is no difference between the various experimental groups; (ii) there is no difference between males and females; (iii) the effect of treatment does not depend on gender. When a significant difference was found by two-way ANOVA, the Bonferroni all pairwise multiple comparison procedure was used to isolate the groups that were different and the sizes of the differences. The statistical difference in cumulative frequency distribution of tumor sizes between groups and difference in tumor incidence were calculated by
2 analysis or the Fischer exact probability test (two-tailed probability), as found appropriate. Correlation between tumor size and location along the intestine was calculated by the non-parametric Spearman rank order correlation coefficient rs. A P value of <0.05 was considered significant.
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Results
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Effects of PhIP on terminal body weight
None of the PhIP treatments affected the terminal body weight. However, males generally had a greater mean terminal body weight than the females (25 ± 0.5 versus 21 ± 0.4 g, P < 0.001).
Effects of PhIP on tumor formation
Remarkably, the Min/+ mice exposed through breast milk from dams injected with 50 mg/kg PhIP showed a 2- to 4-fold increase in the number of small intestinal tumors compared with untreated Min/+ control mice (Table I
). This effect was highly significant both in males and females (P < 0.001). An even greater increase (P
0.001) was seen in pups directly exposed to 25 (5- to 7-fold) or to 50 mg/kg PhIP (6- to 9-fold). However, the small additional increase in the number of small intestinal tumors after direct exposure to 50 compared with 25 mg/kg did not reach statistical significance. Female controls had significantly more tumors than the corresponding male controls (P = 0.028). However, there was no gender difference in the various PhIP-treated groups. The incidence of small intestinal tumors was 100% in all groups, including controls (Table I
).
In the colon, Min/+ mice exposed via breast milk showed a 3- to 4-fold statistically significant increase (P = 0.014) in the number of tumors compared with controls (Table II
). A 3- to 4-fold increase in tumor number compared with the controls was seen in the pups directly exposed to 25 (P = 0.02) and a 2- to 6-fold increase was seen after 50 mg/kg PhIP (P = 0.004). However, when the various PhIP treatments were compared no significant differences between them were found. In females alone, there were no significant effects on colonic tumor number of any of the PhIP treatments compared with controls. In males alone, 50 mg/kg PhIP directly to the pups increased the number of colonic tumors significantly (P = 0.004), whereas the other two PhIP treatments did not reach statistical significance. Although a general statistically significant difference was found between males and females by two-way ANOVA (P = 0.006), the multiple comparison procedure only isolated a gender difference in one group, i.e. male pups treated with 50 mg/kg PhIP directly had more tumors than females (P = 0.03). The incidence of colonic tumors seemed to increase in the treatment groups compared with the controls (Table II
), but this increase was statistically significant only in pups exposed via breast milk (P = 0.03) and pups exposed directly to 50 mg/kg PhIP (P = 0.041).
The distribution of tumors along the intestine was plotted separately for each experimental group (mean number of tumors/cm intestine/mouse). The curves clearly show an accumulation of tumors in the distal part of the small intestine in both the PhIP-treated Min/+ mice and in the controls (Figure 1
). In this area, PhIP seemed to induce small intestinal tumors in a dose-dependent manner. In the colon, the distribution curves show an increased accumulation of tumors in the distal part in pups exposed to 50 mg/kg PhIP directly and in the mid part in pups exposed to 25 mg/kg PhIP directly or exposed via breast milk (Figure 1
). In the controls, the colonic tumors were apparently more proximally distributed.

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Fig. 1. Distribution of tumors along the small intestine and colon in Min/+ mice. Tumors from males and females are pooled. The tumor position is given as distance from the ventricle measured in cm. The mean number of tumors/cm intestine/mouse was scored. , untreated controls; ········, 50 mg/kg PhIP to dams; - - -, 25 mg/kg PhIP to pups; - ·· - ·· -, 50 mg/kg PhIP to pups.
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Effects of PhIP on tumor growth
Within each treatment group all the tumors were pooled for statistical analysis. In the small intestine, PhIP treatment induced a moderate, dose-dependent and statistically significant (P < 0.001) increase in tumor diameter (Table III
). The tumor diameter after 50 mg/kg PhIP directly was larger than after 25 mg/kg directly (P < 0.001). In females alone, the increased tumor size was statistically significant after all the PhIP treatments compared with controls (P < 0.001). In males alone, a significant increase was seen only in the pups exposed directly to 50 mg/kg PhIP (P < 0.001). Although the growth-enhancing effect of PhIP on small intestinal tumors was significantly more pronounced in females than in males (two-way ANOVA, P = 0.031), the multiple comparison procedure found only a statistically significant gender difference in mice exposed via breast milk (P < 0.001).
In the colon, PhIP treatment tended to decrease the tumor diameter (Table IV
). However, this effect was only statistically significant in male pups treated with 50 mg/kg PhIP directly (P = 0.02), where the colonic tumor diameter was almost half the size of control tumors. No gender difference was found in colonic tumor diameter.
In the small intestine, the tumor diameter tended to decrease along the cranio-caudal axis in all the treatment groups (0.66 < rs < 0.47, 0.001 < P < 0.02). In the colon, the tumor diameter tended to increase along the same axis (0.55 < rs < 0.93), although this effect was only statistically significant in the pups exposed to 50 mg/kg PhIP directly (P = 0.02) and via breast milk after 50 mg/kg PhIP to lactating dams (P = 0.04).
Tumor populations induced by PhIP
It appears that PhIP may induce tumor populations that are different from those formed spontaneously in the untreated controls (Tables III and IV
and Figure 1
). To illustrate these PhIP-specific tumor populations, we subtracted the tumor size distribution of control mice from the tumor size distribution of PhIP-treated mice (Figure 2
). Tumors from males and females were pooled.

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Fig. 2. Tumor size distributions of PhIP-induced tumors pooled from male and female Min/+ mice. The PhIP-induced tumor populations were calculated by subtracting the spontaneous tumors formed in the controls from the tumors formed in the PhIP-treated mice for each tumor size class. (a) Distribution of tumor diameter (mm)/mouse in the small intestine. The interval between tumor size classes is 0.25 mm. (b) Distribution of tumor diameter (mm)/mouse in the colon. The interval between tumor size classes is 0.5 mm. , untreated controls; ········, 50 mg/kg PhIP to dams controls; - - -, 25 mg/kg PhIP to pups controls; - ·· - ·· -, 50 mg/kg PhIP to pups controls. In (a) the ······· group represents only females. Males did not differ from control.
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In the small intestine, PhIP apparently induced a tumor population consisting of larger tumors than those formed spontaneously (Figure 2a
). This was confirmed by statistical analysis of the cumulative frequency distribution of tumor sizes, where the PhIP-induced tumor populations in all the treatment groups were found to be significantly shifted towards higher tumor size classes compared with the controls (P < 0.001, data not shown). However, for animals exposed to PhIP via breast milk, this effect was only apparent in females.
An opposite effect of PhIP on tumor size was observed in the colon. The size distribution curves for PhIP-induced colonic tumors appeared to be shifted in a dose-dependent manner towards lower tumor size classes in comparison with the controls (Figure 2b
). Upon statistical analysis of the cumulative frequency distributions of tumor sizes we found a significant increase in the proportion of small tumors compared with controls in pups directly exposed to 25 and 50 mg/kg PhIP (P < 0.05, data not shown).
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Discussion
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The most striking finding in the present work was the 4-fold increase in the number of small intestinal tumors in neonatal Min/+ mice exposed to PhIP via breast milk. It has been shown previously that PhIP is transferred in the milk of mice and rats to the offspring (2931) and formed adducts in the livers of rat offspring (32). Neonatal rats have also been reported to metabolize PhIP (33). Transfer of PhIP is even seen at doses comparable to those occurring in the human diet (31). However, to our knowledge, this is the first time PhIP has been reported to induce tumors after exposure solely via breast milk. The importance of PhIP exposure via breast milk is supported by the findings of Hasegawa et al. (34). They reported an increased risk of mammary carcinoma development following transplacental and trans-breast milk exposure to PhIP, but only when the rat pups were also continuously exposed to dietary PhIP after weaning.
Given a linear doseresponse effect up to 25 mg/kg, calculations based on the tumor numbers in the small intestine suggest an exposure via breast milk as large as 712 mg/kg, or 1425% of the maternal exposure. This accords well with previous studies of PhIP transfer in breast milk (29,32). However, this PhIP effect was apparently not merely dependent on maternal metabolism of PhIP, since direct s.c. injections of neonatal Min/+ mice in the same developmental period resulted in even higher increases in tumor numbers, i.e. up to 9- and 6-fold in the small intestine and colon, respectively.
The great increase in the number of intestinal tumors, especially in the small intestine, in neonatal Min/+ mice after PhIP exposure either directly or through breast milk clearly demonstrates that neonatal Min/+ mice are highly susceptible to PhIP. A similar high susceptibility of neonatal Min/+ mice has also been reported with N-ethyl-N-nitrosourea (35). It has been known for a long time that neonatal animals or humans have an increased susceptibility to carcinogens compared with adults, probably because of differences in age-dependent factors (3638). These could be cell proliferation, differentiation or maturity, a different ratio between body weight and target organ weight and the ability to absorb, metabolize or excrete xenobiotic compounds. In theory, antigenic neoplastic cells may also be protected by immune tolerance as a consequence of emerging prior to functional maturation of the immune system.
We visualized putative PhIP-specific tumor populations by subtracting the tumor size distribution of the controls from the tumor size distribution of the PhIP-exposed Min/+ mice. In the small intestine, these PhIP-induced tumors seemed to be larger than the spontaneous ones, probably because of earlier tumor induction, increased tumor growth, or both. It could be speculated whether PhIP has a general increasing effect on cell proliferation in this organ, as shown in male rat colon (39). However, PhIP is also known to be a potent mutagen in the stem cells of the murine small intestinal epithelium (40). In the colon, the PhIP-induced tumors appeared to be smaller than the spontaneous ones. Hence, the mechanisms by which PhIP induces tumors might be different in the small intestine and colon.
PhIP affected both the number and growth of tumors to a greater extent in the small intestine than in the colon, as was also shown previously in adult PhIP-exposed Min/+ mice (25) and in the Apc1638N transgenic mouse model (27). Whereas PhIP did not change the location of tumors in the small intestine, PhIP tended to induce tumors more distally in the colon. A distal location of colonic tumors thought to be caused by dietary and other environmental factors is also seen in humans (41).
At present, the effect of PhIP exposure on the Apc gene in the Min/+ mice is unknown. Inactivation of the remaining wild-type allele of the Apc gene is found in 100% of the spontaneously formed intestinal adenomas from Min/+ mice studied so far (21,22). In theory, the PhIP-induced tumors may also have lost the function of the remaining normal Apc allele, e.g. by loss of heterozygosity, mutation or deletion. However, it cannot be excluded that PhIP affects other genes than Apc relevant for intestinal carcinogenesis. This study forms a solid basis for further studies of the mechanism(s) of the tumorigenic effects of PhIP in the Min/+ mouse, which we will address in the near future.
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Acknowledgments
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We thank Marit Hindrum for excellent technical assistance with the PCR genotype analysis of the Min/+ mice. I.-L.S. is a post-doctoral fellow of the Norwegian Research Council and Å.A. is a post-doctoral fellow of the Norwegian Cancer Society.
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Notes
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1 To whom correspondence should be addressed Email: jan.alexander{at}folkehelsa.no 
* These authors have contributed equally to this work 
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Received November 13, 1998;
revised January 29, 1999;
accepted March 12, 1999.