The food mutagen 2-amino-9H-pyrido[2,3-b]indole (A{alpha}C) but not its methylated form (MeA{alpha}C) increases intestinal tumorigenesis in neonatally exposed multiple intestinal neoplasia mice

Inger-Lise Steffensen,1, Jan Erik Paulsen and Jan Alexander,1

Department of Food Toxicology, Division of Environmental Medicine, Norwegian Institute of Public Health, PO Box 4404 Nydalen, N-0403, Oslo, Norway


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The heterocyclic amines 2-amino-9H-pyrido[2,3-b]indole (A{alpha}C) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA{alpha}C) are carcinogenic in several organs in rodents, but not in the intestinal tract. However, A{alpha}C induces DNA adducts, mutations and preneoplastic aberrant crypt foci (ACF) in rodent colons. The purpose of this study was to examine whether A{alpha}C and MeA{alpha}C could affect intestinal tumorigenesis in C57BL/6J-Min/+ (multiple intestinal neoplasia) mice. These mice are heterozygous for a germline nonsense mutation in codon 850 of the tumor suppressor gene adenomatous polyposis coli (Apc), producing a truncated non-functional Apc protein. They develop multiple intestinal adenomas, and are particularly susceptible to intestinal carcinogens that affect the Apc gene, especially when exposed neonatally. Whole litters consisting of Min/+ and +/+ (wild-type) mice of both sexes were given a single s.c. injection of 0.22 mmol/kg A{alpha}C (40.3 mg/kg) or MeA{alpha}C (43.4 mg/kg) or the vehicle 1:1 dimethylsulfoxide:0.9% NaCl on days 3–6 after birth, and were terminated at 11 weeks. A{alpha}C increased the number and diameter of small intestinal tumors, but not the number of colonic tumors or dysplastic ACF, in female and male Min/+ mice separately. In pooled data from females and males, colonic tumors and ACF found after A{alpha}C exposure appeared to be smaller than the spontaneous lesions, indicating later induction, slower growth or both. In contrast to A{alpha}C, MeA{alpha}C did not affect intestinal tumorigenesis in Min/+ mice. No effects were found by any of the amino-{alpha}-carbolines in the +/+ mice. A{alpha}C was less potent than the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Abbreviations: A{alpha}C, 2-amino-9H-pyrido[2,3-b]indole; AC, aberrant crypt; ACF, aberrant crypt foci; APC/Apc, adenomatous polyposis coli gene, human/murine; DMSO, dimethylsulfoxide; FAP, familial adenomatous polyposis; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MeA{alpha}C, 2-amino-3-methyl-9H-pyrido[2,3-b]indole; Min, multiple intestinal neoplasia; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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). We have shown previously that the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) could increase intestinal tumorigenesis in a sensitive mouse model. In the present study, we examined whether the amino-{alpha}-carbolines 2-amino-9H-pyrido[2,3-b]indole (A{alpha}C) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA{alpha}C) could also increase intestinal tumorigenesis.

A{alpha}C and MeA{alpha}C are mutagenic and carcinogenic (3) heterocyclic amines that were first isolated from the pyrolysis products of soybean globulin (4). They were later found to be present in various foods, such as fried meats, fish, mushroom and bouillon concentrates (5–8). These heterocyclic amines are also found in the environment, i.e. in cigarette smoke condensates (5,9,10), diesel exhaust particles (11) and A{alpha}C also in river water (12). A{alpha}C is the second most mass-abundant heterocyclic amine in foods, while MeA{alpha}C is less abundant (3,6).

A{alpha}C and MeA{alpha}C induced preneoplastic foci in the liver of F344 rats (13). Carcinogenicity of A{alpha}C has not been reported in rats, while MeA{alpha}C induced tumors in the liver, pancreas, skin, salivary gland and urinary bladder in F344 rats (14). A{alpha}C induced liver nodules in neonatal B6C3F1 mice (15). In CDF1 mice, A{alpha}C and MeA{alpha}C induced tumors in the liver and blood vessels (16). However, in contrast to the lack of significant tumor induction in the colon or small intestine by these heterocyclic amines, preneoplastic colonic aberrant crypt foci (ACF) were induced by A{alpha}C in C57BL/6N mice (17), DNA adducts were induced by A{alpha}C in the colon and small intestine of Sprague–Dawley rats (18), and mutations were induced by A{alpha}C specifically in the colon, but not in the small intestine, of the Big BlueTM mouse (19).

C57BL/6J-Min/+ (multiple intestinal neoplasia) mice are heterozygous for a germline nonsense mutation in codon 850 of the tumor suppressor gene adenomatous polyposis coli (Apc), changing a leucine (TTG) to a stop (TAG) codon, thereby producing a truncated non-functional Apc protein (20,21). These mice spontaneously develop multiple intestinal adenomas, of which some progress to adenocarcinomas in older mice (20,21). The Min/+ mouse is an excellent model for the dominantly inherited autosomal disorder familial adenomatous polyposis (FAP), characterized by an early development of multiple colorectal adenomas, as well as for sporadic colorectal cancer, both conditions being caused by various mutations in the human APC gene (22–25). It appears that intestinal tumor development both in mice (26–29) and humans (25,27) is associated with loss of function of both Apc/APC alleles. Accordingly, the murine FAP models would be particularly susceptible to intestinal carcinogens that affect the Apc gene.

Previously, we showed that PhIP had a much stronger effect on intestinal tumorigenesis in Min/+ mice exposed neonatally (days 3–6 after birth) compared with in adults (30,31), and that a single s.c. injection of PhIP neonatally was enough to cause an increase in tumorigenesis (32). We also showed that PhIP affected intestinal tumor induction in the Min/+ mice both by causing loss of heterozygosity (LOH) and truncation mutations in the wild-type Apc+ allele (33). In the present study, we examined whether a single s.c. injection of A{alpha}C and MeA{alpha}C in a dose equimolar to the previously used dose of PhIP could affect intestinal tumorigenesis in neonatal Min/+ mice, and found that A{alpha}C, but not MeA{alpha}C, could do so.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mouse breeding
The C57BL/6J-Min/+ male mice were bred at the Norwegian Institute of Public Health, Oslo, Norway, from mice originally purchased from The Jackson Laboratory (Bar Harbor, ME), and mated with C57BL/6JBom-+/+ (wild-type) females (M&B A/S, Ry, Denmark). The Min mutation was propagated through the males, as anemia and intestinal adenomas interfere with pregnancy (20). The Min/+ and +/+ offspring used in the present study were genotyped by allele-specific polymerase chain reaction (PCR), as described in detail previously (30), using DNA isolated from blood drawn after the lactation period. The mice were housed in plastic cages in a room with 12 h light/dark cycle, and controlled humidity (55 ± 5%) and temperature (20–24°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 3 weeks of age, thereafter they were given a standard maintenance diet from B&K Universal (Grimston, UK).

Exposure to A{alpha}C and MeA{alpha}C
A{alpha}C and MeA{alpha}C purchased from Toronto Research Chemicals (North York, Ontario, Canada) were dissolved in 1:1 dimethylsulfoxide (DMSO):0.9% saline. Whole litters consisting of Min/+ and +/+ mice of both sexes were randomly chosen to receive a single s.c. injection of either 0.22 mmol/kg bw A{alpha}C (40.3 mg/kg bw), MeA{alpha}C (43.3 mg/kg bw) or the vehicle 1:1 DMSO:0.9% NaCl on days 3–6 after birth. Eleven, nine and ten litters received A{alpha}C, MeA{alpha}C and vehicle, respectively. The mice were terminated by cervical dislocation at 11 weeks of age.

Scoring of intestinal tumors and dysplastic ACF
The colon and small intestine were removed separately, rinsed in ice-cold phosphate-buffered saline and slit open along the longitudinal axis. The intestinal tissues were then spread flat between sheets of filter paper, and fixed at least 48 h in 10% neutral-buffered formalin prior to staining with 0.2% methylene blue (Georg T. Gurr, UK). The number, diameter and localization of adenomas in the colon and small intestine 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 tumor position along the intestines was registered in centimeters from the stomach. For each experimental group, the incidence of tumors, defined as the number of mice with tumors/number of mice in the group, the number of tumors (group mean ± SD) and the tumor diameter in millimeters (group mean ± SD) were calculated, for the small intestine and colon separately. In addition, to illustrate the effects of the carcinogens above the spontaneous level of tumors, we subtracted the number of spontaneous tumors formed in the vehicle-treated mice from the number of tumors formed in the A{alpha}C- and MeA{alpha}C-treated mice, for each size class of tumors, and presented this graphically.

We have described recently a specific type of dysplastic ACF in the colons of Min/+ mice (34,35). In contrast to the classical ACF, these dysplastic ACF are not elevated above the surrounding mucosa and are dependent on transillumination after methylene blue staining to be identified. For each experimental group, the incidence of dysplastic ACF, defined as the number of mice with ACF/number of mice in the group, the number of dysplastic ACF (group mean ± SD) and the focal crypt multiplicity, AC/ACF (group mean ± SD), were calculated.

Statistical analysis
The tumor number and diameter and number of dysplastic ACF and AC/ACF were analyzed with one-way and two-way ANOVA followed by Student–Newman–Keuls all pairwise multiple comparison procedure, or with Kruskal–Wallis ANOVA on ranks followed by Dunn’s all pairwise multiple comparison procedure for non parametric data (SigmaStat Software, Jandel Scientific, Germany). In addition, Student’s t-test, or Mann–Whitney rank sum test for non parametric data, was sometimes used to compare two groups that did not reach statistical significance with ANOVA. Fischer exact probability test (two-tailed probability) was used to evaluate incidence data. A P value of <0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
None of the treatments affected the survival of the newborn mice after injection, or had any observable toxic effects. No effects of either A{alpha}C or MeA{alpha}C were found in the +/+ mice. The following results are therefore from Min/+ mice only. The mice were injected on days 3, 4, 5 or 6 after birth. When mice subjected to the same treatment at a different day after birth were compared, there were no significant differences (data not shown).

Tumors
A{alpha}C induced 2–3 fold higher number of small intestinal tumors compared with the vehicle in Min/+ mice of both sexes (P < 0.05), whereas MeA{alpha}C did not affect the tumor number (Table IGo). There was no difference in number of small intestinal tumors between female and male mice (Table IGo).


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Table I. Effects of A{alpha}C and MeA{alpha}C on incidence, number and diameter of small intestinal tumors in Min/+ mice
 
A{alpha}C, but not MeA{alpha}C, increased the diameter of the small intestinal tumors in both female and male Min/+ mice significantly (P < 0.05, Table IGo). The diameters of the small intestinal tumors were larger in male mice than in female mice in all treatment groups (P < 0.05, Table IGo).

To illustrate the small intestinal tumor populations induced in addition to the spontaneous tumors after exposure to A{alpha}C and MeA{alpha}C, we subtracted the tumors formed in the vehicle-treated mice from the tumors formed in the A{alpha}C- and MeA{alpha}C-treated mice for each size class of tumors (Figure 1aGo). These figures were essentially similar for female and male mice, and a figure of pooled tumor data from females and males is shown. The tumor size distribution curve after A{alpha}C exposure is shifted to the right of the curve for the spontaneous tumors, indicating larger tumors after A{alpha}C exposure, whereas there are no additional increase in tumor number after MeA{alpha}C exposure (Figure 1aGo).



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Fig. 1. Tumor size distributions based on data pooled from female and male Min/+ mice exposed to A{alpha}C and MeA{alpha}C. The mice were given a single s.c. injection of vehicle (1:1 DMSO:0.9% NaCl), 0.22 mmol/kg (40.3 mg/kg) A{alpha}C or 0.22 mmol/kg (43.3 mg/kg) MeA{alpha}C on days 3–6 after birth, and were terminated 11 weeks after birth. The additional tumor populations formed after A{alpha}C and MeA{alpha}C exposure were calculated by subtracting the spontaneous tumors formed in the vehicle-treated mice from the tumors formed in the A{alpha}C- and MeA{alpha}C-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. (Solid line) vehicle, (dashed line) A{alpha}C-vehicle, (dotted line) MeA{alpha}C-vehicle.

 
In the colon, much fewer tumors than in the small intestine were found. Neither A{alpha}C nor MeA{alpha}C increased the incidence or number of colonic tumors significantly compared with the vehicle in female or male mice separately (Table IIGo). More colonic tumors were found in males than in females independent of treatment (P < 0.05, two-way ANOVA), and this difference was significant for MeA{alpha}C (P = 0.005, t-test) (Table IIGo).


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Table II. Effects of A{alpha}C and MeA{alpha}C on incidence, number and diameter of colonic tumors in Min/+ mice
 
None of the treatments affected the diameter of the colonic tumors in each gender separate, and no differences in tumor size were found between the genders (Table IIGo). If tumor data from female and male mice were pooled, both apparent A{alpha}C- and MeA{alpha}C-induced tumors were smaller than the spontaneous tumors from the vehicle-exposed mice (P = 0.015 and P = 0.048, respectively, t-test). However, after subtractions of the tumors formed in the vehicle-treated mice from the tumors in the A{alpha}C- and MeA{alpha}C-exposed mice, it is clearly shown that only A{alpha}C induced additional tumors, which were smaller than the spontaneous tumors in the vehicle-treated mice (Figure 1bGo).

The distribution of tumors along the intestines was similar for all treatments and for both gender. Most of the tumors are located in the distal two-thirds of the small intestine (data not shown).

Dysplastic ACF
Neither A{alpha}C nor MeA{alpha}C affected the incidence or number of dysplastic ACF or AC/ACF in female or male mice separately (Table IIIGo). However, if data from females and males were pooled, A{alpha}C appeared to decrease the AC/ACF compared with the vehicle (P = 0.039, t-test). No significant differences between females or males were found with one-way ANOVA or Student’s t-test/Mann–Whitney rank sum test, but when analyzed by two-way ANOVA the AC/ACF was generally higher in male mice than in female mice (P = 0.011).


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Table III. Effects of A{alpha}C and MeA{alpha}C on incidence, number of dysplastic ACF and AC/ACF in the colon of Min/+ mice
 
Comparison with PhIP
We have previously treated Min/+ mice with the same concentration of PhIP (0.22 mmol/kg bw, 50.0 mg/kg bw) (32), using the same protocol as in the present work, but using untreated mice as the control group. The effects of A{alpha}C were compared with the effects of PhIP in percent after normalizing to the mean of the respective control groups using pooled data from female and male mice (Figure 2Go). Both A{alpha}C and PhIP increased the number and diameter of small intestinal tumors compared with their respective controls, and PhIP increased both parameters significantly more than A{alpha}C (P < 0.001 and P < 0.05, respectively). PhIP increased the number of colonic tumors more than A{alpha}C (P < 0.05), and PhIP increased the number of colonic tumors compared with the controls, whereas A{alpha}C did not. A{alpha}C-induced colonic tumors were smaller than the spontaneous tumors, whereas the same tendency did not reach statistical significance with PhIP. There was no significant difference between A{alpha}C and PhIP on the size of colonic tumors. The number of PhIP-induced ACF was lower than in the control group, whereas A{alpha}C did not affect the number of ACF significantly, and the effects of PhIP and A{alpha}C was not significantly different on this parameter. The effect of PhIP on AC/ACF was not significant, whereas A{alpha}C-induced ACF were smaller. A{alpha}C decreased the size of ACF more than PhIP (P < 0.05). The distribution of tumors along the intestines was similar for A{alpha}C and PhIP.



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Fig. 2. Effects of PhIP and A{alpha}C in percent after normalizing to the mean of the respective control groups, i.e. vehicle for A{alpha}C, untreated for PhIP, using pooled data from female and male Min/+ mice. The mice were left untreated or given a single s.c. injection of vehicle (1:1 DMSO:0.9% NaCl), 0.22 mmol/kg (40.3 mg/kg) A{alpha}C or 0.22 mmol/kg (50.0 mg/kg) PhIP on days 3–6 after birth. All mice were terminated 11 weeks after birth. (a) Number of tumors in the small intestine and colon and ACF in the colon. Treatments with the same letter are significantly different (aP < 0.001, bP <0.05, one-way ANOVA). (b) Diameter of tumors in the small intestine and colon and AC/ACF. Treatments with the same letter are significantly different (c,dP < 0.05, one-way ANOVA). The PhIP data are taken from reference (32), with permission.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The main results in this study were that a single s.c. injection of 0.22 mmol/kg A{alpha}C given to neonatal female or male Min/+ mice increased both the number and diameter of small intestinal tumors, but not the number of colonic tumors or ACF. In addition, colonic tumors and ACF pooled from A{alpha}C-exposed female and male mice appeared to be smaller than the spontaneous lesions found in the control group. In contrast to A{alpha}C, MeA{alpha}C in equimolar dose did not affect intestinal tumorigenesis.

A{alpha}C is clearly able to affect intestinal tumorigenesis in the sensitive Min/+ mouse, in contrast to no effects found by A{alpha}C on the intestines of CDF1 mice (16). It was shown previously that DNA adducts were induced by a single dose of A{alpha}C in higher level in the colon than in the small intestine of Sprague–Dawley rats (18), and mutations were induced by A{alpha}C in the colon, but not in the small intestine, of the Big BlueTM mouse (19), indicating that A{alpha}C is a more potent inducer of DNA adducts and mutations in the colon than the small intestine. However, A{alpha}C could clearly increase tumorigenesis also in the small intestine in the present study.

Compared with the spontaneous tumors found in the vehicle-treated group, the A{alpha}C-induced tumors in the small intestine were larger. This was illustrated in the figure of the tumor size distribution curves where the curve after A{alpha}C exposure is shifted to the right of the curve for the spontaneous tumors (Figure 1aGo). The reason for this could be earlier tumor induction, increased tumor growth, or both, after A{alpha}C exposure. Based on pooled data from the colons of female and male mice, both tumors and ACF appeared to be smaller after A{alpha}C exposure compared with the spontaneous lesions, indicating that the additional lesions formed after A{alpha}C exposure in the colon were specific populations induced somewhat later or were growing slower than the spontaneous lesions. These results are similar to results previously found with PhIP (32), and indicate that the mechanisms for induction of intestinal lesions may be different in the small intestine and colon. Although it is not possible to rule out A{alpha}C-induced increased and decreased growth rate in the small intestine and colon, respectively, it is probable that the tumor size distributions rather reflect time of tumor induction by A{alpha}C relative to the spontaneously formed tumors.

When the spontaneous tumors in the small intestine were subtracted from the tumors found after MeA{alpha}C exposure, the size distribution curves show that no additional tumors were formed (Figure 1aGo). The only statistically significant effect of MeA{alpha}C found was that based on pooled data from female and male mice colonic tumors after MeA{alpha}C exposure appeared to be smaller than the spontaneous tumors. However, the size distribution curves show that no additional tumors were formed after MeA{alpha}C exposure in the colon either (Figure 1bGo).

Both A{alpha}C and MeA{alpha}C form the same reactive metabolites, N2-OH-A{alpha}C (36) and N2-OH-MeA{alpha}C (37), and induce similar major DNA adducts, N2-(deoxyguanosin-8-yl)-A{alpha}C (38) and N2-(deoxyguanosin-8-yl)-MeA{alpha}C (39), respectively. In CDF1 mice, whether A{alpha}C or MeA{alpha}C were the most potent carcinogen varied with tissue, stage of tumor and gender (16). When A{alpha}C and MeA{alpha}C were compared in various in vitro tests, i.e. morphological transformation, bacterial mutagenicity and several genotoxicity assays, the ranking of potency was higher for MeA{alpha}C than for A{alpha}C in most assays (40). The only difference in chemical structure between A{alpha}C and MeA{alpha}C is that MeA{alpha}C has a methyl group, ortho to the exocyclic amino group. It was reported that the heterocyclic amine Trp-P-1 having an ortho-methyl group was a more potent genotoxicant in hepatocytes than Trp-P-2 lacking this group, and stated that for reasons not fully understood ortho-methyl amines are stronger carcinogens than the corresponding amines (41). Also, the presence of a methyl group in the 1- or 3-imidazole position in the 2-aminoimidazole-azaarenes favors high mutagenic potency, probably by stabilizing the ultimate reactive nitrenium ion (42). This is in contrast to the present study, where A{alpha}C lacking the ortho-methyl group was more potent that MeA{alpha}C having such a group. However, many other chemical and biological factors besides chemical structure determine the ultimate mutagenic and carcinogenic potency of a compound (43).

The diameter of small intestinal tumors and number of colonic tumors and AC/ACF were generally higher in male Min/+ mice than in female mice independent of treatment in this study. A higher sensitivity of males compared with females have also often, but not always, been observed in previous studies (30–32). Whether this apparent gender difference is due to differences in DNA adduct level, mutation rate, cell proliferation or other factors, is not yet known.

There are two broad structural categories of mutagenic and carcinogenic nitrogen-heterocyclic aromatic compounds containing an exocyclic amino group found in cooked food (44). A{alpha}C and MeA{alpha}C belong to the category aminopyridoindoles, whereas PhIP belongs to the aminoimidazoazaarenes. We have studied previously the effects of PhIP on intestinal tumorigenesis in the Min/+ mice using the same protocol of a single s.c. injection of 0.22 mmol/kg (50.0 mg/kg) neonatally (32), and wanted, therefore, to compare the effects of A{alpha}C and PhIP, representing these two categories of food mutagens (Figure 3).

Both PhIP (32) and A{alpha}C clearly affected tumorigenesis in the small intestine, by increasing both number and size of the tumors in this organ. PhIP was more potent than A{alpha}C. In the colon, PhIP increased the number of tumors, while decreasing the number of dysplastic ACF, indicating that the ACF had become tumors at the time point studied, i.e. at 11 weeks. PhIP induced more tumors in the colon than A{alpha}C. A{alpha}C did not affect either the number of tumors or dysplastic ACF in the colon, but A{alpha}C-induced tumors and ACF were smaller than the spontaneous lesions. Although not statistically significant, colonic tumors induced by PhIP were also smaller than the spontaneous tumors, but the size of ACF was not affected by PhIP. Therefore, both types of food mutagens, PhIP and A{alpha}C, are able to affect intestinal tumorigenesis both in the small intestine and colon, and PhIP is more potent than A{alpha}C.

When A{alpha}C, MeA{alpha}C and PhIP were compared in various in vitro tests, i.e. morphological transformation, bacterial mutagenicity and different genotoxicity assays, PhIP was more potent than both amino-{alpha}-carbolines in all assays except in a micronucleus assay where MeA{alpha}C was more potent than PhIP (40).

We have also done an experiment where 0.25 mmol/kg (43.6 mg/kg) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a quinoline-type aminoimidazoazaarene (44), was given to Min/+ mice using the same protocol as in the present study (45). However, the intestines were fixed in absolute ethanol instead of formalin in this study, making the smallest tumors harder to notice. Anyway, although the numbers of tumors are not precisely comparable, the numbers of tumors after IQ exposure, i.e. 97.3 ± 15.3 (mean ± SD) and 1.2 ± 1.1, in the small intestine and colon, respectively, suggest that IQ is less potent than both A{alpha}C and PhIP, in the Min/+ mice.

We have shown previously that the mechanisms for PhIP-induced intestinal tumorigenesis in Min/+ mice include both LOH and truncation mutations in the wild-type Apc+ allele (33). Whether A{alpha}C affects intestinal tumorigenesis in the Min/+ mice by the same mechanisms as PhIP is not yet known. However, it could be of interest to note that although both PhIP and A{alpha}C predominantly induced G:C -> T:A transversions in the lacI gene in the Big BlueTM mouse, there were striking differences in the sequence context of their respective mutations. PhIP induced G:C -> T:A transversions preferentially at G:C runs, in addition to G:C base pair deletions in 5'-GGGA-3' sequences, whereas A{alpha}C induced G:C -> T:A transversions in 5'-CGT-3' sequences (46).

A{alpha}C is the second most mass-abundant heterocyclic amine in foods after PhIP, while MeA{alpha}C is of less abundance (6). The average daily intakes of PhIP and A{alpha}C in cooked meats and fish in a Western diet are estimated to be 16.64 and 5.17 ng/kg/day, respectively (6). Even if the effects on intestinal tumorigenesis in this animal model are observed after a much higher dose than the estimated human intake, the results still point to the importance of these substances as etiological agents for human cancers after lifelong exposure.

In conclusion, we have shown that A{alpha}C given in a single s.c. injection of 22 mmol/kg to neonatal Min/+ mice increased intestinal tumorigenesis, whereas MeA{alpha}C in the same dose did not.


    Notes
 
1 To whom correspondence should be addressed Email: inger-lise.steffensen{at}fhi.no Back


    Acknowledgments
 
We thank Marit Hindrum and Lars Pettersen for excellent technical assistance with the PCR genotype analysis of the Min/+ mice.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 13, 2002; revised April 29, 2002; accepted April 30, 2002.





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