Potent genotoxicity of aminophenylnorharman, formed from non-mutagenic norharman and aniline, in the liver of gpt delta transgenic mouse

Ken-ichi Masumura1, Yukari Totsuka2, Keiji Wakabayashi2 and Takehiko Nohmi1,3

1 Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga Setagaya-ku, Tokyo 158-8501, Japan and 2 Cancer Prevention Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan


    Abstract
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 Abstract
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 Material and methods
 Results
 Discussion
 References
 
Aminophenylnorharman (APNH) is formed from non-mutagenic norharman and aniline, and is mutagenic to Salmonella typhimurium TA98 with S9 mix. Norharman and aniline are present in cigarette smoke and cooked foods and both compounds are detected in human urine samples, suggesting that APNH could be a mutagenic and carcinogenic human risk factor. The purpose of the present study was to determine the in vivo mutagenicity of APNH. Male gpt delta transgenic mice were fed a diet containing 10 or 20 p.p.m. APNH for 12 weeks. The gpt mutant frequency (MF) in the liver increased 10-fold in 20 p.p.m. APNH-treated mice, which was almost equivalent to the MF observed in the liver of the same transgenic mice treated with 300 p.p.m. 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline for 12 weeks. In the colon mucosa, the gpt MF increased ~5-fold in 20 p.p.m. APNH-treated mice. Our results suggest that APNH is a strong hepatic mutagen in mice. The APNH-induced gpt mutations in the liver were dominated by G:C to T:A transversions, followed by G:C to A:T transitions. They also included single G:C deletions in G:C run sequences and 2 bp deletions: GCGC to GC and CGCG to CG. The Spi- deletion MF in the liver was 13-fold higher in 20 p.p.m. APNH-treated mice, relative to the control, and were dominated by single base pair deletions, in particular, in G:C run sequences. Large deletions were rare. The mutational characteristics induced by APNH are compared with those induced by other heterocyclic amines, and the human risk of APNH is discussed.

Abbreviations: APNH, aminophenylnorharman; MeIQx, 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline; MF, mutant frequency; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 6-TG, 6-thioguanine


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Humans are exposed to a variety of environmental chemicals. Some of these chemicals are mutagens and/or carcinogens associated with human risk (1). A non-mutagenic ß-carboline compound, 9H-pyrido[3,4-b]indole (norharman), is widely detected in cigarette smoke and cooked foods at much higher concentrations than those of known mutagenic/carcinogenic heterocyclic amines (2,3). Norharman itself is not mutagenic to Salmonella typhimurium TA98 and TA100 with or without S9 mix. However, norharman becomes mutagenic to TA98 and YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline (48). Aminophenylnorharman [APNH: 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole], which is formed from norharman and aniline by metabolic activation systems and in vivo, was recently identified as a mutagenic heterocyclic amine (Figure 1) (7,8). Both norharman and aniline are present in cigarette smoke and cooked foods (3,9) and are detected in human urine and milk samples (1012). APNH is detected in the rat urine when norharman and aniline are administrated by oral gavage (13). In addition, APNH is detected in the reaction mixture of norharman and aniline in the presence of human microsome fractions (13). Therefore, APNH may be a human risk factor if it is mutagenic in vivo. To examine this possibility, the genotoxicity of APNH was examined using gpt delta transgenic mice. In this mouse model, point mutations, such as base substitutions and frameshift mutations, are detected by 6-thioguanine (6-TG) selection using the Escherichia coli gpt gene and deletions including frameshifts are detected by Spi- selection using the red/gam genes of lambda phage (14,15). These two genetic selections are complementary and identify different types of mutations. In previous studies, we characterized the in vivo mutations induced by mutagenic and carcinogenic heterocyclic amines such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in the same mouse model (1618). The highest mutant frequencies (MFs) were observed in the colon and liver, respectively, in the PhIP- and MeIQx-treated mice. Here, we report for the first time the in vivo mutagenicity of APNH in the liver and the colon. APNH is more mutagenic in the liver than in the colon, and its mutagenicity in the liver is much stronger than that of MeIQx in the same organ. The possible carcinogenic risk of APNH is discussed.



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Fig. 1. Formation of aminophenylnorharman from norharman and aniline in the presence of S9 mix.

 

    Material and methods
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 Material and methods
 Results
 Discussion
 References
 
Treatment of mice and rescue of transgene
APNH was purchased from the Nard Institute (Osaka, Japan). Transgenic mice gpt delta (C57BL/6 J background) were fed a diet containing 20 or 10 p.p.m. of APNH for 12 weeks. Control animals were fed a basal diet of CE-2 (Japan Clea Laboratory, Tokyo, Japan). After the treatments, the mice were fed a basal diet for another 2 weeks and then killed. Each group consisted of five 7-week-old male mice. Genomic DNA was extracted from liver and colon mucosa using RecoverEaseTM DNA Isolation Kit (Stratagene, La Jolla, CA). Lambda EG10 phages were rescued from genomic DNA by in vitro packaging method using TranspackTM packaging extract (Stratagene). The experimental protocol was approved by the Committee for Ethics of Animal Experimentation of National Cancer Center Research Institute.

Mutation assay and sequencing analysis
The gpt mutation assay was performed as described previously (15). The rescued phages were infected to E.coli YG6020 expressing Cre recombinase to convert the transgene to plasmid. Infected cells were mixed with molten soft agar and poured onto agar plates containing chloramphenicol (Cm) and 6-TG. The plates were incubated at 37°C for the selection of colonies harboring plasmid carrying the mutated gpt gene. Infected cells were also poured on the plates containing Cm without 6-TG to determine the number of rescued plasmids. The gpt MF was calculated as described previously (15). The selected 6-TG-resistant mutants were cultured and collected. A 739 bp DNA fragment containing the mutated gpt gene was amplified by polymerase chain reaction (PCR) as described previously (15). DNA sequencing of the gpt gene was performed with BigDyeTM Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, CA) on ABI PRISMTM 310 Genetic Analyzer (Applied Biosystems). The sequencing primer was gptA2 primer (5'-TCTCGCGCAACCTATTTTCCC-3').

The Spi- mutation assay was performed as described previously (15) with some modification. We added 10 mM MgSO4 to both agar plates and soft agar to improve the detection efficiency of Spi- plaques as described previously (19). The rescued phages were infected to E.coli XL1-Blue MRA (P2). Infected cells were mixed with molten soft agar, poured onto lambda-trypticase agar plates and incubated at 37°C. The plaques detected on the plates (Spi- candidates) were suspended in 50 µl of SM buffer. The suspension was spotted on the two types of plates where XL1-Blue MRA (P2) or WL95 (P2) strain was spread. The plates were incubated for 24 h at 37°C. The numbers of mutants that made clear spots on both strains were counted as confirmed Spi- mutants. Phage lysates of the recovered Spi- mutants were used as templates for PCR analysis (20,21). The PCR primers were: primer 001 (5'-CTCTCCTTTGATGCGAATGCCAGC-3'); primer 002 (5'-GGAGTAATTATGCGGAACAGAATCATGC-3'); primer 005 (5'-CGTGGTCTGAGTGTGTTACAGAGG-3'); primer 006 (5'-GTTATGCGTTGTTCCATACAACCTCC-3'), and primer 012 (5'-CGGTCGAGGGACCTAATAACTTCG-3'). The appropriate primers for DNA sequencing were selected based on the results of PCR analysis. The sequencing primers have been described previously (2022). The entire sequence of lambda EG10 is available at http://dgm2alpha.nihs.go.jp.

Statistical analysis
All data are expressed as mean ± SD. Differences between two groups were tested for statistical significance using Student's t-test. A P value <0.05 denoted the presence of a statistically significant difference.


    Results
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Mutagenicity of APNH was assayed in the liver and colon of APNH-treated and untreated male mice (Figure 2). For the gpt assay, we analyzed 346 000 to 1 098 000 colonies derived from the rescued phages per organ per mouse. For the Spi- assay, we analyzed 611 000 to 4 110 000 rescued plaques per organ per mouse. The gpt MF in the liver of the 20 p.p.m. APNH-treated mice was 68 x 10-6, which was about 10 times higher than that of untreated mice (6.6 x 10-6). Furthermore, ~6-fold increase was observed in the 10 p.p.m. APNH-treated mice (38 x 10-6). In the colon, the gpt MFs of the 10 or 20 p.p.m. APNH-treated mice were 17 x 10-6 or 29 x 10-6, respectively. Those were 3 or 5 times higher than that of untreated mice (6.2 x 10-6).



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Fig. 2. The gpt and Spi- mutant frequencies in the liver and colon mucosa of the mice fed a diet containing APNH for 12 weeks. Data are mean ± SD. *P < 0.01, relative to the control.

 
To characterize the APNH-induced gpt mutations, we analyzed 73 and 51 mutants recovered from the liver of the 20 p.p.m. APNH-treated mice and untreated mice, respectively (Tables I and II). The APNH-induced gpt mutations recovered from the liver were dominated by single base substitutions (56/73 = 77%): G:C to T:A transversions (37/56 = 66%) predominated, followed by G:C to A:T transitions (17/56 = 30%). Deletions were also observed (12/73 = 16%). Most of them were 1 or 2 bp deletions at G:C base pairs (9/12 = 75%). They included four single G:C deletions in G:C run sequences, two single G:C deletions without run sequence, two GCGC to GC and one CGCG to CG deletions. Other types of mutations included tandem base substitutions and one base deletion with base substitution. In untreated mice, 78% (40/51) of total mutations were base substitutions. G:C to A:T transitions predominated (21/40 = 53%) and 57% (12/21) of them occurred at 5'-CpG-3' sites. G:C to T:A transversions (7/40 = 18%) and A:T to G:C transitions (5/40 = 13%) were also observed. Deletions amounted to 18% (9/51). The predominant types of deletion were single-base deletions at A:T run sequences (6/9 = 67%). One insertion and one tandem base substitution were observed.


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Table I. Mutation spectra of gpt mutations recovered from the liver of APNH-treated gpt delta mice

 

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Table II. Summary of gpt mutations recovered from the liver of APNH-treated gpt delta mice

 
The Spi- MF in the liver of the 10 or 20 p.p.m. APNH-treated mice were 12 x 10-6 or 16 x 10-6, respectively, which were 10 or 13 times higher than that of the untreated mice (1.2 x 10-6). To characterize Spi- deletion mutations induced by APNH, 40 and 11 Spi- mutants recovered from the livers of the 20 p.p.m. APNH-treated mice and untreated mice, respectively, were analyzed (Tables III and IV). In the APNH-treated group, 73% (29/40) were single G:C base pair deletions. The majority of these events occurred in G:C run sequences (20/29 = 69%). The mutational hot spots, defined as nucleotide positions where more than four mutations were observed in three or more mice, were at nucleotides 188–190, 238–241 and 286–289 in the gam gene; the sequence changes were GGG to GG and GGGG to GGG. In addition to single base pair deletions, five deletions and one insertion were observed. They include -2 (x2), -13, -2690 and -3452 bp deletions and +29 bp insertion. In untreated mice, 55% (6/11) of the sequenced Spi- mutants were single base pair deletions. Other five mutations were -14, -22, -777 and -4061 bp deletions and +149 bp insertion.


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Table III. Mutation spectra of Spi- mutations recovered from the liver of APNH-treated gpt delta mice

 

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Table IV. Summary of Spi- mutations recovered from the liver of APNH-treated gpt delta mice

 

    Discussion
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 Material and methods
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APNH is a newly identified mutagenic heterocyclic amine formed by coupling of norharman with aniline in the presence of S9 mix (7,8). APNH displays acute testicular toxicity in F344 rats, although neither norharman nor aniline alone induces such testicular toxicity (23). APNH also induces sister chromatid exchanges and chromosome aberrations in cultured mammalian cells (24). In the present study, we investigated the genotoxicity of APNH and its molecular characteristics in vivo, by applying the mutagenicity assay using gpt delta transgenic mice.

The gpt and Spi- mutations in the liver and colon were induced in a dose-dependent manner by subchronic exposure of mice to APNH (Figure 2). The gpt MF increased 10-fold in the liver of mice treated with 20 p.p.m. APNH for 12 weeks. This is almost the same level of MF observed in the liver of mice treated with 300 p.p.m. MeIQx for 12 weeks (Figure 3). These results demonstrate that APNH is a strong hepatic mutagen in mice. In the colon mucosa, the gpt MF increased ~5-fold in 20 p.p.m. APNH-treated mice. Considered together, our results suggest that liver is a more sensitive organ relative to the colon with respect to the mutagenicity of APNH in mice. We also compared the gpt MFs in the liver and colon of mice treated with APNH, MeIQx or PhIP-treated mice (Figure 3). MeIQx induces hepatocellular carcinoma, adenoma and neoplastic liver nodules in F344 rats and CDF1 mice (25,26). Our results showed that MeIQx increased gpt MF in the liver, which is a major target organ for carcinogenesis (18). These results suggest that APNH may be a potential hepatocarcinogen. In line with this, Kawamori et al. (27) reported that APNH induced glutathione S-transferase positive placental form-positive foci in the liver of male F344 rats. Induction of MFs in the colon could also be indicative of the carcinogenicity of APNH in the colon. However, the organ specificity and sensitivity of carcinogenesis could also be due to a different susceptibility or cell division kinetics in the later stages of carcinogenesis. For example, the colon is a major target of MF induction for PhIP in both mice and rats (16,28), but PhIP does not induce colon cancer in mice (29). It induces colon and prostate cancers in male F344 rats and mammary carcinoma in female rats (30,31). To investigate the carcinogenicity of APNH, studies involving long-term exposure of rodents to APNH are currently underway.



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Fig. 3. Comparison of gpt mutant frequencies in the liver and colon of mice treated with the heterocyclic amines. Five male mice were used for each group of the experiments of APNH and MeIQx and three male mice for each group of PHIP. APNH was mixed with the diet at 20 p.p.m. for 12 weeks and the liver and colon mucosa were recovered after the following 2 weeks on basal diet without APNH (this study). MeIQx was mixed with the diet at 300 p.p.m. for 12 weeks and the liver and colon were recovered (18). PhIP was mixed with the diet at 400 p.p.m. for 13 weeks and the liver and colon mucosa were recovered after the following 2 weeks on basal diet without PhIP (16). Data are mean ± SD.

 
The APNH-induced gpt mutations were dominated by G:C to T:A transversions, followed by G:C to A:T transitions (Table I), although single G:C base pair deletions at G:C run sequences were also observed. The G:C base pair substitutions dominated by G:C to T:A and the frameshifts at G:C base pairs are known as typical characteristics of the mutation spectra in rodents induced by various heterocyclic amines, such as PhIP, MeIQx, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-9H-pyrido[2,3-b]indole (A{alpha}C) (17,18,3234). There is sufficient evidence indicating that metabolic activation leading to formation of DNA adducts is critical for the mutagenicity and carcinogenicity of these amines. The heterocyclic amines are enzymatically activated by N-oxidations catalyzed by P450 in the liver of rodents and humans (3537), and their N-hydroxy products are further activated by acetyltransferases or sulfotransferases (38,39). They form DNA adducts at C8 and N2 positions of guanine bases (40,41). Recently, deoxyguanosine-C8-APNH adducts was identified as a major product formed in various tissues of APNH-treated rats by 32P-postlabeling method (42). This finding suggests that APNH shows mutagenicity in vivo by forming DNA adducts at guanine residues, like other carcinogenic heterocyclic amines. In the APNH-induced mutation spectrum, G:C to C:G transversion formed a small percentage (1%) in contrast with that of PhIP (13%) (17). These results suggest that the different structures of adducts could result in different preferences of misincorporation opposite adducts on the template strand during DNA replication. In addition to DNA adduct formation, oxidative DNA damage may also play a role in the genotoxicity of APNH (43). APNH-induced deletions in the gpt gene also contained 2 bp deletions; GCGC to GC and CGCG to CG. These preferences of APNH-induced frameshift mutations could explain the high mutagenic activity of APNH in TA98 and YG1024, indicators of the frameshifts: 2 bp deletions of a GC or CG within the sequence CGCGCGCG (7,8). In untreated mice, 78% (40/51) of total mutations were base substitutions. G:C to A:T transitions were predominant (21/40 = 53%) and 57% (12/21) of such substitutions occurred at 5'-CpG-3' sites. These results suggest that deamination of the methylated cytosine at CpG sites contributes to spontaneous mutations in vivo (44). G:C to T:A transversions (7/40 = 18%) may reflect oxidative damage such as 8-oxoguanine or abacic sites in DNA (45,46). Deletions were 18% (9/51); with the predominant types being single base deletions at A:T run sequences (6/9 = 67%), in contrast to that of APNH-treated group.

Spi- mutations in the liver of APNH-treated mice were induced in a dose-dependent manner (Figure 2). Spi- selection positively detects deletion mutations, in which both gam and red gene functions are simultaneously inactivated or frameshifts in the upstream gam gene (15,17,47). The reason for the appearance of the latter class of single base pair deletions in the gam gene is that these frameshifts not only inactivate the gam gene function but also interfere with the start of translation of the downstream redBA genes, thereby functionally inactivating both the gam and redBA genes. In this study, the WL95 (P2) strain was used for confirmation of the Spi- candidate plaques (see Materials and methods). This is because we observed in the previous study that some Spi- candidate plaques that were recovered from the selection agar plates had no deletions in the gam gene, but base substitutions (17). They occupy ~12% of total Spi- mutants recovered from the colon of PhIP-treated mice (11/96 = 11.5%). Most of these mutants without deletions do not display Spi- phenotype in another P2 lysogen WL95 (P2) (48) and do not exhibit Fec- phenotype; they could make plaques on a recA strain, whereas other typical types of Spi- deletion mutants could not. We suspect the appearance of such substitution-type mutants is due to inadequate selectivity of the XL1-Blue MRA (P2) strain used in the Spi- assay. By confirmation test of the candidate plaques using WL95 (P2), we could eliminate almost all ‘irregular’ substitution-type mutants before the calculation of Spi- MF and further sequencing analyses.

The Spi- MF in the liver increased 13-fold in 20 p.p.m. APNH-treated mice, and the majority of APNH-induced Spi- mutations were single G:C base pair deletions that occurred at G:C run sequences rather than large deletions (Tables III and IV). In addition, nucleotides 166–170 in the gam gene, 5'-GCGCG-3' sequence, were a target of 1 bp deletion with base substitutions and 2 bp deletions. Similar characteristics were observed in the colon of PhIP-treated mice (17). On the other hand, the second major type of Spi- deletions observed in PhIP-treated mice, G:C base pair beside T, C and A run sequences (24%), was not a prominently induced class in the liver of APNH-treated mice (8%). This result suggests that the frameshift mutation beside run sequences was not a major target in APNH-induced mutagenesis. The large deletions were not frequently observed in the Spi- mutants recovered from APNH-treated mice, similar to the finding in PhIP-treated mice (17). PhIP induces sister chromatid exchange, but not chromosomal aberrations in bone marrow and only weakly induces micronucleus in bone marrow and peripheral blood in C57BL/6 mice (49). Because of the similarity of the Spi- mutation spectra between APNH and PhIP, we suspect that APNH may not efficiently induce chromosome aberrations or micronucleus in vivo. However, APNH is reported to potently induce sister chromatid exchanges and chromosome aberrations in vitro (24). Thus, further studies are necessary to conduct micronucleus or chromosome aberration assays in bone marrow or in liver to clarify the apparent discrepancy regarding the clastogenic activity of APNH in vivo and in vitro.

Humans are exposed to a variety of chemicals, and hence it is not difficult to imagine that more than one compound displays combined effects. For example, simultaneous exposure to two non-mutagenic/carcinogenic compounds may induce genotoxicity and/or carcinogenicity in vivo. Such combined effects have not yet been thoroughly investigated, despite the mechanistic importance. In this study, we demonstrated that APNH, formed from non-mutagenic norharman and aniline, showed a strong mutagenicity in the liver and colon of mice. We suggest that APNH may be a human risk when it is produced even in a small amount in our body. There are possible reasons for the strong mutagenicity of APNH. One is the high efficiency of DNA replication error on the template strand containing the APNH–DNA adducts [50]. However, the characteristics of the mutational spectra of APNH observed were largely similar to those of other heterocyclic amines, although they somewhat differed from those of PhIP. Another possible reason is the strong reactivity of APNH and its derivatives, which could contribute to the formation of the higher amounts of APNH–DNA adducts. Previous studies showed severe testicular toxicity in APNH-treated F344 rats (23), and cultured mammalian cells treated with APNH show induction of sister chromatid exchange and chromosome aberrations (24). These results suggest the strong reactivity of APNH to various cellular components including DNA and proteins. Further studies are important to determine the mechanism of the strong mutagenicity of APNH by estimating the amount of DNA-adducts induced by APNH and comparing these levels with those of other heterocyclic amines such as PhIP.


    Notes
 
3 To whom correspondence should be addressed Email: nohmi{at}nihs.go.jp Back


    Acknowledgments
 
This study was supported by Grants-in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare of Japan.


    References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received July 10, 2003; revised August 29, 2003; accepted September 2, 2003.