Mutational analysis of the liver, colon and kidney of Big Blue® rats treated with 2-amino-3-methylimidazo[4,5-f]quinoline
Sandra A.M. Bol,
Janine Horlbeck,
Jovanka Markovic,
Johan G. de Boer1,
Robert J. Turesky and
Anne Constable2
Nestlé Research Centre, Nestec Ltd, PO Box 44, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland and
1 Centre for Environmental Health, Department of Biology, University of Victoria, PO Box 3020, Victoria, BC, Canada V8W 3N5
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Abstract
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The heterocyclic aromatic amine (HAA) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induces intestinal tumours and hepatocellular carcinomas in rats, but no tumourigenic effects have been identified in the kidney. The tissue-specific mutagenicity of IQ was studied at the lacI locus in the liver, colon and kidney of Big Blue® transgenic rats. At the highest dosing regime of IQ (20 mg/kg for 5 consecutive days) the mean mutant frequencies were significantly increased above background (P < 0.05) and were highest in the liver (12.9 ± 6.2x105), followed by colon (7.4 ± 1.4x105) and kidney (5.9 ± 0.8x105). The mutational spectra from the livers of IQ-treated rats was statistically significantly different to that from the livers of control rats (P < 0.01). The lacI mutation spectra of the liver, colon and kidney from IQ-treated rats were similar. These were characterized by an increase in GC transversions in the liver and colon and an increase in the proportion of 1 bp G:C deletions in the liver and kidney. A single G deletion in the sequence 5'-CGGGA-3', characteristic of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine exposure, was detected in the liver and colon. A 2 bp GC deletion was identified at an identical position in the liver, colon and kidney. The colon was the only organ to contain two larger deletions of 13 and 33 bp. A preference was observed for base substitution mutations at guanine in the sequence 5'-CGC/T-3' and for 1 bp deletions at the guanine doublet in the sequence 5'-CGGA-3', especially in the liver and colon. Using the lacI gene as marker in the Big Blue rat model, the mutations identified in the IQ spectra have similarities to those identified for other HAAs studied in the same experimental system, but not to mutations identified in IQ-induced tumours.
Abbreviations: dG-C8-IQ, N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline; dG-N2-IQ, 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline; HAAs, heterocyclic aromatic amines; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MF, mutant frequency; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PBS, phosphate-buffered saline.
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Introduction
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2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a member of the group of heterocyclic aromatic amine compounds (HAAs) which may be formed in cooked meats. HAAs are potent genotoxic rodent carcinogens (13). Chronic feeding with IQ in rats induces tumours in the Zymbal gland, liver, small and large intestines and skin (46). Furthermore, IQ is a strong hepatic carcinogen in non-human primates (7). Because daily exposure to IQ and other HAAs in food may be a potential carcinogenic risk to humans, it is important to understand the mechanism of its carcinogenicity.
The interaction of IQ with DNA has been studied in a variety of in vitro systems. IQ is metabolically activated via cytochrome P450 mediated N-oxidation to 2-(hydroxyamino)-3-methylimidazo[4,5-f]quinoline (811), which reacts with DNA forming mainly N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ), and to a minor extent 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f] quinoline (dG-N2-IQ) adducts (Figure 1
; 12,13). Reversion assays have identified frameshift mutations of one or two G:C base pairs induced by IQ in bacterial genes (14,15). IQ induces predominantly GC
TA transversions in the Escherichia coli supF gene in human fibroblasts (16) and the yeast URA3 gene in E.coli (17). Using the hprt locus as target, GC
CG and AT
CG transversions were detected in CHO-K1 cells (18) and transversions at GC nucleotides in human lymphoblastoid TK6 cells (19). This latter study also identified single guanine deletions in a run of six guanines, the same hotspot which was identified for 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) in the same system (20).
Transgenic animal mutagenesis systems have recently been developed (21,22) enabling the measurement of in vivo induced lacI mutations in any organ of interest. The advantages of this system are that the fate of the test chemical depends not on artificial in vitro criteria but on endogenous molecular and cellular pathways. In this study, we used the Big Blue® rat system to determine mutational events due to feeding of IQ and to attempt to explain the organ-specific induction of tumours by IQ.
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Materials and methods
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Chemicals
IQ (99% pure) was obtained from Toronto Research Chemical (Downsview, Ontario, Canada). Agar and NZYM medium were purchased from Difco through Chemie Brunschwig (Basel, Switzerland). All other Big Blue assay materials were obtained from Stratagene (Basel, Switzerland).
Animals and treatment
Male Big Blue F344 rats were purchased from Stratagene (La Jolla, CA) at the age of 4 weeks. They were fed on a diet of Nalfag 890 and water ad libitum and treated with IQ after ~1 week acclimatization. IQ was administered by gavage as its hydrochloride salt in 1 ml of phosphate-buffered saline (PBS). Each treatment group consisted of four animals. One group was given a single dose of 20 mg/kg IQ and the second group 20 mg/kg IQ for 5 consecutive days. The control group was given one dose of PBS by gavage. Two weeks after the last treatment the animals were killed and the liver, kidneys and colon mucosa (23) were removed, frozen immediately in liquid nitrogen and stored at 80°C.
Determination of lacI mutant frequencies
Genomic DNA from liver, colon and kidney was isolated using the Recoverease DNA isolation method (Stratagene). The
shuttle vector harbouring the lacI marker gene was packaged using Transpack packaging extract, infected into E.coli SCS-8 and plated onto NZYM agar plates containing X-gal according to the Stratagene Big Blue Instruction Manual. At least 300 000 plaques were plated for each organ from each rat of each treatment group. Full blue plaques were taken for mutation analysis.
Analysis of lacI mutations
Blue plaques were isolated and replated at low density on X-gal containing NZYM agar plates to confirm the mutant phenotype. The mutant phages were eluted in SM buffer (0.1 M NaCl, 8 mM MgSO4, 50 mM TrisHCl, pH 7.5) containing 10% chloroform. PCR amplification of the lacI target gene was performed using high fidelity Pfu polymerase (Stratagene) and the two primers 5'-CCCGACACCATCGAATG-3' (positions 53 to 37) and 5'-ACAATTCCACACAACATAC-3' (positions 1201 to 1185). The PCR product was purified using the Wizard PCR Preps DNA purification kit (Promega). Sequencing was performed either by the fluoro-cycle sequencing method as described (24) or with the Cyclist Exo Pfu DNA sequencing kit and Big Blue sequencing primers (Stratagene). The latter was performed according to the instruction manual incorporating [
-35S]dATP (Amersham) to visualize the sequencing products on denaturing acrylamide gels. The full listing of all mutations identified in this study can be found in the Big Blue lacI database maintained by J.de Boer, University of Victoria, Canada (http//www.darwin.ceh.uvic.ca/bigblue).
Statistical analysis
Mutant frequencies were compared using Student's t-test assuming unequal variances. Mutational spectra were compared with
2 tests.
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Results
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Mutant frequencies (MFs)
The mutant frequencies of each organ from the three different treatments are summarized in Table I
. The mean spontaneous lacI mutant frequencies from the control animals were 2.8 ± 1.1x105 in the liver, 2.7 ± 1.2x105 in the colon and 3.3 ± 1.3x105 in the kidney. IQ induced a dose-dependent increase in the lacI mutant frequencies in all three organs. At the highest dosing regime (5x20 mg/kg) the mean MFs were significantly increased above background (P < 0.05), with the highest frequencies observed in the liver (12.9 ± 6.2x105), followed by the colon (7.4 ± 1.4x105) and kidney (5.9 ± 0.8x105).
Mutational spectra
Over two-thirds of all spontaneous lacI mutants recovered from the liver, colon and kidney controls were sequenced (87, 91 and 70%, respectively). Mutants from the highest dosing regime of 5x20 mg/kg IQ were taken for mutation analysis. Half of the lacI colon (59%) and kidney (56%) mutants and 28% of the liver mutants were sequenced. The data were corrected for clonality, i.e. identical mutations occurring at a single locus in the same organ of a single rat were scored only once.
The mutational spectra are shown in Table II
. The spontaneous spectra in liver, colon and kidney were significantly different from each other (P < 0.01). The most frequent types of mutations detected both in the liver and colon were GC
AT transitions (38 and 44%, respectively), followed by GC
TA transversions (25%) in the liver and 1 bp G:C deletions (28%) in the colon. Spontaneous mutations in the kidney consisted mainly of GC
TA transversions (52%), followed by GC
AT transitions (39%).
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Table II. Mutational spectra at the lacI locus in the liver, colon and kidney of control and IQ-treated Big Blue rats
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The mutational spectra in the liver from animals treated with IQ showed significant differences to the organs from control animals (P < 0.01). GC
TA transversions predominated, increasing from 25 to 52%, with a large increase in 1 bp G:C deletions (from 3 to 19%). The mutational spectrum in the colon of IQ-treated rats was not significantly different to that of the colon from control rats (P = 0.07). The proportion of 1 bp deletions was similar (28 and 24%), but GC
TA transversions increased from 16 to 33%. The colon was the only organ to show two deletions of 13 and 33 bp after IQ treatment. The IQ-induced mutational spectrum in the kidney was not significantly different to the spontaneous kidney spectrum (P = 0.15). The proportion of GC
TA transversions actually decreased (52 to 36%), but an increase in 1 bp G:C deletions from 4 to 18% was observed. One identical 2 bp GC deletion was observed in all three IQ-treated organs. The IQ-induced mutation spectra in the three organs were not significantly different (P = 0.24) from each other.
Sequence specificity
The target in 94% of spontaneous base substitutions was a G:C pair; 51% of these were transitions. The target in 87% of the base substitutions from organs of IQ-treated rats was a G:C pair; 75% of these were transversions. The nearest neighbour bases of all G:C substitution mutations from the organs of IQ-treated rats were examined (Table III
). A preference for a 5'-C was observed in the liver (64%) and colon (58%) and either 5'-C or 5'-G in the kidney (41 and 44%). The preferred 3' base was a C or T in the liver (34 and 39%) and colon (both 35%) and C or A in the kidney (both 33%).
A total of nine spontaneous 1 bp deletions (1 frameshift) were identified; three were A:T deletions and six were G:C deletions. All of the 33 1 bp deletions (1 frameshift) identified in the liver, colon and kidney from IQ-treated rats were G:C deletions. Over 50% of these were at a 5'-GG-3' doublet (Table IV
). Nearly 70% of 1 bp deletions in both the liver and colon were preceded by a 5'-C (69 and 67%), whereas 5'-T was preferred in the kidney (72%). A 3'-A was preferred in the liver, colon and kidney (56, 44 and 43%, respectively). The two larger deletions of 13 and 33 bp identified in the colon were both flanked by GC-rich regions. Only one type of 5'-GC-3' 2 bp deletion was identified, within an identical sequence in all three organs.
A total of six identical substitutions (GC
AT transitions) and one GC
TA transversion were identified at position 93 of the lacI gene in the liver and kidney from control animals (Figure 2a
). Transitions at this nucleotide were also identified in the colon and kidney from IQ-treated animals. No hotspots unique to either the liver, colon or kidney from IQ-treated rats were identified (Figure 2b
). Base substitutions were identified at nucleotide 92 in the livers of IQ-treated animals, and also in the liver from one control animal. A total of six GC
TA transversions were observed at position 86 and seven substitutions at position 95 were observed in all three organs for IQ-treated rats. These sites are also targets for spontaneous mutation in the liver, colon and kidney. Only two sites of mutation appear to be IQ specific. These are a G:C deletion in the 5'-GGGA-3' sequence at positions 877880, identified twice in the liver and once in the colon, and the 2 bp GC deletion at the unique run 5'-GCGCGC-3' at positions 790795, identified once in all three organs. This latter region also shows a clustering of different mutations for IQ-treated organs: a G insertion after position 790 and a substitution at 791 was identified in the kidney, two substitutions at 792 in the liver and two deletions at 793 in the colon. In addition, deletions at the doublet 795/796 were identified in the liver and kidney. A second clustering of base substitutions was observed in the 5'-GCGC-3' sequence at positions 380383 in the IQ-treated liver and colon. Mutations at positions 86 and 95 are within the preferred 5'-CGT-3' sequence. Position 92 and the deletions at 877/879 are within a 5'-CGGGA-3'. The clustering of mutations at 380383 and 790795 are at runs of G:C and C:G.
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Discussion
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IQ forms two types of DNA adducts: dG-C8-IQ and dG-N2-IQ. In rats treated with a single dose of IQ, both adducts were detected in the liver and colon, as well as the kidney (25). Both adducts were removed at similar rates in the colon, however, the dG-C8-IQ adduct is removed more rapidly in the liver and kidney (26). This differential adduct removal does not therefore help to explain the tissue specificity of IQ-induced carcinogenesis. As reported here, we have observed increased MFs at the lacI locus in the liver and colon as well as in the kidney from Big Blue rats treated with IQ. The time allowed for fixation of adducts into mutations was 2 weeks after treatment; this may not be optimal for all the three tissues. A high proportion of spontaneous 1 bp deletions was detected in the colon and of GC transversions in the kidney from untreated rats. The IQ mutational spectra were similar for all three organs and were characterized by GC transversions (4659% total transversions) and 1 bp G:C deletions (1824%). A difference in the mutational spectra from IQ-treated and control rats was statistically significant in the liver only. Although the number of mutants analysed was small, it is however worth noting differences in the sequence contexts. The preference for a cytosine 5' of the mutated base is not as great in the kidney as in both the liver and colon, neither is the preference for a 3' cytosine or thymine. The only other in vivo mutagenesis study using IQ was with the lacZ MutaMouse® model where IQ increased the mutation frequency in the liver (27). Unfortunately, no other organ was examined and no sequence data are available. Few previous Big Blue studies have specifically examined the kidney. Dimethylnitrosamine induces tumours in mouse liver, kidney and lung. Increased lacI mutation frequencies were observed in all three target tissues, whereas no induction was observed in non-target organs (28). The flame retardant tris(2,3-dibromopropyl)phosphate is a kidney-specific carcinogen and induces lacI mutants in the kidney, but also in the liver. Sequencing, however, showed that the mutational spectra from treated and untreated livers were similar, while treated kidneys were different from the control with an increase in GC deletions (29).
Using the lacI gene as marker in the Big Blue rat model, the sites of specific types of mutation identified in the IQ spectra of the liver, colon and kidney have similarities to hotspots identified for other HAAs studied in the same experimental system. Transversions at positions 86, 92 and 95 have previously been noted as 2-amino-9H-pyrido[2,3-b]indole mutational hotspots in the Big Blue mouse colon (31). Position 92 was a hotspot for 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in the Big Blue mouse liver, bone marrow (32) and colon and the 2 bp GC deletion at positions 790795 was also noted in the colon (31). These two latter hotspots, together with the 1 bp deletion in the 5'-GGGA-3' sequence found at positions 877880 of the IQ-treated liver and colon, were identified in the colon of male and female Big Blue rats treated with PhIP (30).
No direct correlation between DNA adduct level, MeIQ MFs at the lacI locus and MeIQ-induced cancer has been determined in different mouse organs (33,34). In the mouse, MeIQ induces tumours in the liver and not in the bone marrow. Increased MFs were observed in both tissues (35). However, the mutational spectra differed in that a high frequency of transversions was observed in the liver, while the bone marrow showed comparable frequencies of all types of mutation (31). High frequencies of total GC transversions (50 and 59%) and lower frequencies of 1 bp G:C deletions (7 and 9%) appear to be the characteristic mutational spectra of MeIQ in the colon and liver (30,31). GC transversions are also indicated in MeIQ-induced squamous cell tumours in the rat Zymbal gland and the mouse forestomach, where identical GC
TA transversions in codon 13 of the Ha-ras gene (36,37) were identified.
PhIP induces colon tumours in male rats but not in females. High MFs were found in the colon from both male and female Big Blue rats treated with PhIP, with no differences in mutational spectra. Transversions (37 and 42% total transversions), a high proportion of 1 bp G:C deletions (39 and 33%) and deletions in the 5'-CGGGA-3' sequence were identified in both sexes (32). This latter deletion is considered as a characteristic hotspot for PhIP exposure in the Big Blue mouse and rat colon (30,32). Guanine deletions in the sequence 5'-GGGA-3' in the Apc gene of PhIP-induced rat colon tumours have also been reported (38) and, therefore, this mutation is regarded as important in colon carcinogenesis.
Our present study indicates that both tranversions and deletions are characteristics of the IQ-induced mutational spectra at the lacI locus of Big Blue rats. However, mutations in the Apc gene were detected in only two out of 13 colon tumours induced by IQ, which were not deletions in the 5'-GGGA-3' sequence, but T
C and C
T transitions (39). Out of 15 Zymbal gland tumours from IQ-treated rats, the GC
TA transversion in codon 13 of the Ha-ras gene (characteristic of MeIQ-induced tumours) was detected only twice; other mutations were GC
CG transversions and GC
AT transitions. Similarly, both transistions and transversions were identified in the Ka-ras gene (40). Analysis of the p53 gene from the same IQ-induced tumours identified two GC
TA transversions, one GC
CG transversion, one GC
AT transition and one G:C deletion in four of the 15 tumours (41). These observations suggest that the spectrum of IQ-induced mutations in tumour genes are broader and not as specific as for the mutational spectra from either PhIP- or MeIQ-induced tumours. In addition, the PhIP and MeIQ lacI mutational spectra seem to reflect the spectra in tumour genes (deletions for PhIP, transversions for MeIQ). This seems not to be the case for IQ.
Whilst accepting the limitations of these types of transgenic in vivo mutation assays (42), they are a useful tool to determine exposure to chemical carcinogens. However, a greater database of MFs, mutational spectra and mutation sequence context, from target and non-target organs, in addition to the analysis of actual tumour genes, is invaluable for the investigation of tissue-specific carcinogenic mechanisms. The available data from target and non-target organs of HAA carcinogenicity suggests that while DNA adduct formation and mutation are important for the initiation of events leading to carcinogenesis, other cellular events are critical for specific tumour initiation and proliferation.
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Acknowledgments
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We thank Dr B.W. Glickman (University of Victoria, Canada) for helpful discussions and André Rytz (Nestec Ltd) for statistical advice.
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Notes
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2 To whom correspondence should be addressed Email: anne.constable{at}rdls.nestle.com 
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Received February 11, 1999;
revised September 20, 1999;
accepted September 23, 1999.