Carcinogenicity of aminophenylnorharman, a possible novel endogenous mutagen, formed from norharman and aniline, in F344 rats
Toshihiko Kawamori1,
Yukari Totsuka,
Naoaki Uchiya,
Tomohiro Kitamura,
Hideyuki Shibata,
Takashi Sugimura and
Keiji Wakabayashi2
Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
2 To whom correspondence should be addressed Email: kwakabay{at}gan2.res.ncc.go.jp
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Abstract
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A novel mutagenic compound, 9-(4'-aminophenyl)-9H- pyrido[3,4-b]indole (aminophenylnorharman, APNH), is shown to be formed by the in vitro enzymatic reaction of 9H-pyrido[3,4-b]indole (norharman) and aniline. APNH generates DNA adducts (dG-C8-APNH), and is potently genotoxic to bacteria and mammalian cells. APNH has also been demonstrated to be formed in vivo from norharman and aniline, and suggested to be a new type of endogenous mutagenic compound. To determine its carcinogenic activity, long-term administration of APNH was investigated in 93 male and 90 female F344 rats. Rats were fed diets containing 0, 20 or 40 p.p.m. from 7 weeks of age. All animals were killed after 85 weeks treatment and necropsy was performed. Hepatocellular carcinomas (HCCs) were induced at incidences of 10 and 79% in male rats fed 20 and 40 p.p.m. APNH, and 34% in female rats fed 40 p.p.m. of APNH, respectively. In addition, colon adenocarcinomas were found at incidences of 3 and 9% in male rats, and 4 and 13% in female rats fed 20 and 40 p.p.m. of APNH, respectively. Other tumors, including thyroid carcinomas and mononuclear cell leukemia, were also seen in rats fed APNH. Polymerase chain reactionsingle strand conformation polymorphism analysis revealed ß-catenin gene mutations in 24% of HCCs and K-ras, ß-catenin and Apc gene mutations were found in 22, 44 and 33% of colon cancers induced by APNH, respectively. Most mutations occurred at G:C base pairs. ß-Catenin protein accumulations in the nucleus and cytoplasm were also revealed in both liver and colon tumors. Thus, APNH induced liver and colon cancers with K-ras, ß-catenin and Apc gene mutations in F344 rats.
Abbreviations: APNH, aminophenylnorharman, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole; norharman, 9H-pyrido[3,4-b]indole; HCCs, hepatocellular carcinomas; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; PCRSSCP, polymerase chain reactionsingle strand conformation polymorphism
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Introduction
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On heating tryptophan, a ß-carboline compound, 9H-pyrido [3,4-b]indole (norharman) is produced, together with mutagenic/carcinogenic heterocyclic amines (HCAs),
- and
-carbolines (1,2). Norharman is reported to be present at much higher levels than those of HCAs in cigarette smoke condensate and cooked meat and fish (3). Furthermore, norharman is detected in all urine samples from healthy volunteers eating an ordinary diet, as well as from patients receiving parenteral alimentation (4). It has been found that norharman becomes mutagenic to Salmonella typhimurium TA98 with a metabolic activation system (S9 mix) when incubated with a non-mutagenic aromatic amine, aniline, although norharman itself is not mutagenic to S.typhimurium TA98 and TA100, either with or without an S9 mix (2,5). Aniline is also present in cigarette smoke condensate and some kinds of vegetables (6,7). Moreover, this compound has been reported to be present in human urine and milk samples (810). Thus, it is likely that humans are simultaneously exposed to both compounds in daily life. We have demonstrated that a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH, shown in Figure 1), is formed from norharman and aniline, then converted to the N-hydroxyamino derivative, which produces DNA adducts after esterification to induce mutations in S.typhimurium TA98 and YG1024 (1113). Recently, we clarified that APNH forms DNA adducts primarily at the C-8 position of guanine residues in vitro and in vivo (14). In addition, APNH was found to induce sister chromatid exchanges and chromosome aberrations (15). Moreover, APNH was detected in 24 h urine samples collected from F344 rats administered norharman and aniline (16).
As mentioned above, humans are exposed to both norharman and aniline, so that it is very likely that APNH may be produced in our bodies. In fact, APNH was detected in some 24 h urine samples from smokers (our unpublished data) and therefore could play an important role in human carcinogenesis as a new type of endogenous mutagen. To understand the effects of APNH on human health, it is important to elucidate its carcinogenicity in rodents. We have demonstrated previously that APNH induces glutathione S-transferase placental (GST-P) positive foci, pre-neoplastic hepatic lesions, in the livers of male F344 rats in a short-term experiment (17). In the present study, the carcinogenicity of APNH with long-term administration in both male and female F344 rats was investigated. Tumors were found in the liver and colon, and mutation analysis of cancer-related K-ras, ß-catenin, Apc and p53 genes in the lesions, was also performed using polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) to give points to underlying mechanisms.
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Materials and methods
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Animals and chemicals
A total of 186 weanling F344 rats of both sexes were obtained from Charles River Japan (Atsugi, Japan) and quarantined for 2 weeks. All animals were housed three to a plastic cage. Rats were randomly distributed into three groups and maintained under controlled conditions: 12-h light/dark cycle, 21 ± 2°C room temperature, and 50 ± 10% relative humidity. CE-2, purchased from Japan Clea Laboratory, Tokyo, Japan, was used as the basal control diet. Food and water were available ad libitum throughout the experiment. APNH was purchased from the Nard Institute (Osaka, Japan) and its purity was confirmed to be >99% by HPLC.
Experimental procedure
Starting at 7 weeks of age, the rats were fed either control pellet diet (CE-2) or experimental diets containing 20 or 40 p.p.m. APNH until the termination. Body weights and diet consumption were recorded weekly during the first 14 weeks and then every 4 weeks until the end of the study. When animals were found to be moribund, they were killed and complete autopsy was performed. At 85 weeks after the beginning of the experiment, all surviving animals were killed by ether and similarly processed. Organs/tissues including brain, skinincluding specialized sebaceous glands, oral cavity, esophagus, stomach, intestines, salivary glands, liver, pancreas, kidneys, urinary bladder, thyroid gland, mammary glands, lungs, spleen, thymus, bone marrow, heart, adrenal glands, pituitary gland, male reproductive system including testis, epididymis, prostate, and seminal vesicles or female reproductive system including ovary, uterus and vagina were examined under a dissection microscope for any abnormality. For histological evaluation, all organs were fixed in 10% neutral-buffered formalin, embedded in paraffin blocks, cut into multiple sections, and routinely processed for H&E staining. The histological criteria adapted for diagnoses of tumors were according to Pathology of the Fischer Rat (18). Half of the liver tumors were stocked in liquid nitrogen for subsequent PCRSSCP analyses.
PCRSSCP analysis and direct sequencing
Genomic DNA of liver tumors was obtained from frozen tissue samples using standard procedures involving enzymatic digestion of protein and RNA followed by extraction with phenol and chloroform:isoamyl alcohol (24:1, v/v). In the colon tumors, DNA was extracted from 10 µm thick sections cut from paraffin-embedded materials with TaKaRa DEXPATTM (TaKaRa Shuzo, Kyoto, Japan) according to the manufacturer's instructions. The primers and PCR conditions for the ß-catenin, Apc, p53 and ras family genes examined in this study were the same as those reported previously (1924). One microliter of PCR products was mixed with 9 µl of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol, heated at 90°C for 3 min, then applied to a 10 or 12.5% polyacrylamide gel with or without 5% glycerol. Electrophoresis was carried out at 300 V for 2 h at 20°C and the gel was stained using a silver staining kit (Daiichi Pure Chemicals, Tokyo, Japan). When mutated shifted-bands were observed in the gels, mutation analysis was performed by direct sequencing of DNA fragments extracted from SSCP bands or PCR products using a capillary sequencer (ABI PRISM 310 Genetic Analyzer, Perkin Elmer, Foster City, CA).
Immunohistochemical staining of ß-catenin
Immunohistochemical analysis was performed with the same procedure as described previously, with minor modifications (21). Briefly, after deparaffinization and re-hydration, samples were microwaved (5 min, five times) in 10 mM citrate buffer (pH 6.0), then endogenous peroxidase activity and non-specific reactions were blocked with 0.3% hydrogen peroxide and 5% normal horse serum, respectively. The sections were then incubated overnight with an anti-ß-catenin monoclonal antibody diluted at 1:500 (Transduction Lab., Lexington, KY) at 4°C. Then, the sections were incubated at room temperature with the secondary antibody, biotinated anti-mouse IgG (H+L) raised in a horse, at 1:200 (Vector, Burlingame, CA) for 30 min. Staining was carried out using a Vectastain ABC kit (Vector), 3,3'-diaminobenzidine and hydrogen peroxide. All sections were counterstained with hematoxylin. The bile duct epithelium was considered as a positive control in all liver sections.
Statistical analysis
The Fisher's exact probability test was used for statistical analysis of differences in tumor incidences. Significance was concluded at P < 0.05.
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Results
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General observations
Body weights of control male rats reached 450 g, and those of female rats almost 250 g during the study. Values for male rats fed 40 p.p.m. of APNH were decreased to 95% as compared with the control group at 80 weeks (P < 0.05). Female rats receiving the 40 p.p.m. dose also demonstrated 510% lower body weights than those in the control diet group from 1 month after starting the diet to termination. No change was observed with the 20 p.p.m. dose in either sex. The average consumption of diet per day per rat by males and females was 15 and 10 g, respectively, with no effects from APNH. All male rats fed the control diet survived to the end of the study, although two control females died of metastasis to lungs from fibrosarcoma and uterus bleeding at 1 week before death. One female and one male rat fed APNH at 40 p.p.m. each developed hepatocellular carcinomas (HCCs) at 49 and 53 weeks, respectively, after starting the study. Some animals fed APNH became moribund and were found to have tumors. Effective numbers of rats were defined as those surviving until week 49 of the study when a HCC was first recognized in a female rat given APNH. At termination, survival rates of rats fed APNH at 20 and 40 p.p.m. were 90 and 48% in males and 73 and 48% in females, respectively.
Tumors induced by APNH
At termination, all surviving animals were killed and necropsies were performed. Details for tumors found in both sexes of rats treated with APNH are summarized in Tables I and II, respectively.
Liver tumors
Both male and female rats fed APNH developed liver tumors, including adenomas and HCCs, which were absent in the controls (Table I). The incidences of liver adenomas were 7 and 18% for male rats fed 20 and 40 p.p.m. of APNH, and 9% for female rats fed 40 p.p.m. of APNH, respectively. In the case of HCCs, the incidences were 10 and 79% for male rats fed 20 and 40 p.p.m. of APNH, and 34% for female rats fed 40 p.p.m. of APNH, respectively. In male rats, most of the APNH-induced HCCs were well differentiated (75%), and 32% were moderately differentiated. Only one tumor was diagnosed as poorly differentiated. On the other hand, all APNH-induced HCCs found in female rats were well differentiated. Among 26 HCCs developed in male rats fed 40 p.p.m. APNH, six cases including two well and four moderately differentiated HCCs demonstrated lung metastasis. Male rats fed APNH at 40 p.p.m. also developed cholangiocellular carcinomas at a 12% incidence. Male rats proved more susceptible to induction of liver tumors than females.
Colon tumors
As shown in Table I, rats fed APNH developed colon adenocarcinomas in a dose-dependent manner (3 and 9% in males and 4 and 13% in females at 20 and 40 p.p.m. APNH, respectively). No colon tumors were found in rats fed the control diet.
Other tumors
The incidences of tumors induced by APNH in organs other than the liver or colon are summarized in Table II. Follicular carcinomas and C-cell carcinomas, in the thyroid gland were observed following APNH administration. The incidence of follicular tumors (adenomas and carcinomas) was significantly increased at 40 p.p.m. APNH group, although there were no apparent effects on incidences of C-cell tumors. Mononuclear cell leukemia was also found in both control and APNH-treated rats, the incidence being significantly increased in a dose-dependent manner. In female rats, clitoral gland carcinomas were induced dose-dependently, incidences being 10 and 25% at 20 and 40 p.p.m. of APNH, respectively.
Transitional cell carcinomas in the urinary bladder were found in a male rat fed 20 p.p.m. of APNH and in two female rats fed 40 p.p.m. of APNH with incidences of 3 and 6%, respectively, although the values were not significantly different from the control group. In males, interstitial cell adenomas of the testes were frequently found in all groups at an incidence of 5867% incidence, with no obvious influence of APNH. In addition, preputial gland carcinomas, pituitary gland adenomas, lung carcinomas were observed at incidences of 37%, without significant deviation from the control group. In female rats, endometrial hyperplasia was found to be increased following APNH feeding (20, 36 and 69% at 0, 20 and 40 p.p.m. of APNH, respectively), but no carcinomas were found.
Genetic alterations in liver and colon tumors induced by APNH
Liver. DNA samples obtained from 17 HCCs were examined for mutations of ß-catenin, ras family, p53 and Apc genes using PCRSSCP analysis. While no mutations were identified in the H-ras, K-ras, N-ras, p53 and Apc genes, the ß-catenin gene was found to show mobility-shifted bands in four out of 17 HCC samples (Table III). DNA fragments extracted from those SSCP bands were amplified and subjected to direct sequencing. As shown in Table III, mutations were detected in all four cases: one at the first base of codon 32, two at the second base of codon 37, and one at the second base of codon 41 (a G:C to T:A transversion, C:G to G:C transversions and a C:G to T:A transition, respectively), all leading to amino acid substitutions (Table III).
Immunohistochemical analysis of ß-catenin indicated that some cancer cells showed prominent immunoreactivity in the nucleus, cytoplasm or cell membrane, whereas non-cancerous hepatocytes lacked ß-catenin immunoreactivity in the nucleus and cytoplasm, and showed only weak reactivity, limited to the cell membrane. Accumulation of ß-catenin in the nucleus and cytoplasm, compared with the amount seen in non-cancerous hepatocytes, was detected in six of 17 (35%) HCC samples. Among these, five revealed ß-catenin accumulation in the cytoplasm, and one in the nucleus. Moreover, four out of six HCC samples showing ß-catenin immunoreactivity contained genetic alterations in exon 3 of the ß-catenin gene.
Colon. The findings of nine cases of colon cancers are summarized in Table IV. Four showed mutations at codons 32 and 34 of the ß-catenin gene (one A:T to G:C transition at the second base of codon 32, two G:C to A:T transitions at the second base and one G:C to A:T transition at the first base of codon 34), all leading to amino acid changes. In addition, two mutations of K-ras and three mutations of Apc genes were detected in APNH-induced colon carcinomas. The K-ras mutations were G:C to T:A transversions at the second base of codon 12, with change of Gly to Val. Of the three Apc mutations detected, one was a frameshift mutation with one G deletion in the 5'-GGGGTTT-3' sequence resulting in a truncated product, and the other two were base substitutions including one silent (T:A to G:C at codon 879) and one G:C to C:G transversion, leading to amino acid substitution.
Among the colon cancer samples, three had double mutations with K-ras and ß-catenin (two samples) and Apc and ß-catenin (one sample). The Apc mutation in the colon tumor sample containing the ß-catenin mutation was silent. Moreover, all the samples with Apc and/or ß-catenin gene mutation demonstrated strong nuclear or cytoplasmic immunoreactivity with ß-catenin. Although PCRSSCP analyses of H-ras, N-ras and p53 genes were performed under at least two conditions, no shifted bands were detected in any of the nine colon carcinomas.
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Discussion
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Our results indicated clearly that orally administered APNH is carcinogenic in both male and female F344 rats. As expected from our previous finding of GST-P positive liver foci after 4 weeks of treatment (17), the feeding of APNH at 40 p.p.m. for 85 weeks resulted in the development of HCCs at 79 and 34% in male and female F344 rats, respectively, whereas liver tumors were absent in the controls. Most HCCs found in both sexes were well differentiated.
Genetic alteration of ß-catenin was observed in 24% of the HCCs induced by APNH. However, no mutations were found in ras family, p53 or Apc genes. All of the point mutations detected featured replacement of a serine or threonine residue, encoded by codons 37 and 41 or a contiguous site with serine 33 in exon 3, known to be putative phosphorylation targets of GSK-3ß. In immunohistochemical analysis, all of the HCCs bearing ß-catenin gene mutations demonstrated accumulation of ß-catenin protein in the nucleus and cytoplasm. In addition, two HCC samples without any mutations showed immunoreactivity. The reason for this is not clear, however, we analyzed only limited regions of ß-catenin and Apc genes; therefore, mutations might occur at other regions of these genes or other wnt signaling related genes. On the other hand, there seems to be no relation between ß-catenin accumulation and tumor malignancy. ß-Catenin accumulation and mutation of exon 3 have also been reported in diethylnitrosamine-induced rat liver tumors and also in human liver cancers (2527). With diethylnitrosamine-induced HCCs in rats, mutations were detected in codons 32, 33, 34, 35, 37 and 41 in exon 3 at incidences of 31 and 45% (25,26). In a human series of HCCs, 39% demonstrated accumulation of ß-catenin and 24% gene mutations at codons 32, 34, 35, 37 and 41 (27). Thus, the available data suggest that ß-catenin gene mutations may be related to hepatocarcinogenesis in both rodents and man, although incidences of ß-catenin accumulation and gene mutations observed in human and chemically induced HCCs are much lower than in colon cancers. Therefore, pathways rather than ß-catenin-wnt signaling are presumably involved. Further studies are thus needed to clarify the mechanisms of APNH hepatocarcinogenesis.
As with azoxymethane (AOM)-, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)- and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat and human colon tumors (21,23,28,29), mutations of ß-catenin, K-ras and Apc genes in colon adenocarcinomas were here found to be more frequent than those in HCCs. Missense mutations in K-ras, ß-catenin and Apc genes were found in 22, 44 and 33% of the APNH-induced colon tumors, as compared with 62 (K-ras), 75 (ß-catenin) and 8% (Apc) in AOM-induced colon tumors (21). The respective figures for the IQ-induced ß-catenin and Apc genes are 100 and 15%, and 57 and 50% for PhIP-induced colon tumors (23,28). In the case of human colon cancers, K-ras, ß-catenin and Apc gene mutations have been found in 3336%, 715% and 4348%, respectively (29). The regions for ß-catenin gene mutations detected in APNH-induced colon adenocarcinomas were at codons 32 and 34, which are contiguous with serine 33. These mutations may cause alterations of the ß-catenin protein structure, therefore leading to inhibition of phosphorylation of ß-catenin and blocking of degradation through the ubiquitinproteasome pathway. In addition, Apc gene mutations, except one, led to amino acid change or truncated products, and ß-catenin protein accumulation was evident in the APNH-induced colon cancers.
We have reported previously that the structure of the major APNHDNA adduct was dG-C8-APNH, as is the case with PhIP, IQ and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (14). This adduct has been detected in various organs of rats given 40 p.p.m. APNH for 4 weeks, with levels higher in the liver and colon than in other organs (14). Based on these observations, it is suggested that APNH forms DNA adducts at the C8 position of guanine residues, especially in its target organs. Recently, we have also reported that the gpt mutant frequencies were elevated 10- and 5-fold in the liver and colon of the gpt delta transgenic mouse treated with 20 p.p.m. APNH, respectively (30). APNH induced G:C to T:A trasversions and single G:C deletions in G:C run sequences predominately in the liver of transgenic mice. Similarly, most gene alterations detected in APNH-induced liver and colon tumors in the present study involved G:C base pairs. From these observations, it is suggested that dG-C8-APNH was formed in the target genes, and these adducts might cause the mutations. Therefore, the mutations detected in the APNH-induced tumors were mainly at G or C (opposite position of G) base pairs.
Development of other tumors, such as thyroid adenocarcinomas, was also found to be enhanced by APNH feeding in both males and females in the present study. Moreover, endometrial hyperplasias were increased by feeding of APNH, and tumors of the hematopoietic system, including leukemia, were found to be enhanced in both sexes. The underlying mechanisms of the development of these tumors by APNH are now under investigation in our laboratory.
In conclusion, APNH demonstrated carcinogenicity in various organs, including the liver and colon, in both sexes of F344 rats, at doses almost 10 times lower than those proved to be carcinogenic for HCAs, such as MeIQx (31). As mentioned above, norharman and aniline are abundantly present in our environment and continuous exposure to both compounds during daily life is conceivable. It is reported that APNH can be detected in the urine of rats administered norharman and aniline (16). Moreover, when 24 h urine samples were collected from smokers and non-smokers and analyzed by LC-MS after purification with HPLC, we found that APNH was clearly detectable in some samples from smokers (own unpublished data). Therefore, it is highly conceivable that APNH is a new type of endogenous mutagen/carcinogen, involved in human carcinogenesis.
It has been reported that norharman is mutagenic in the presence of o-toluidine and S9 mix (2,5,32). Moreover, harman (1-methyl-9H-pyrido[3,4-b]indole), another ß-carboline compound, has a similar co-mutagenic activity with aniline or o-toluidine (2). Recently, we reported the mutagenic compounds produced by norharman with o-toluidine, and harman with aniline or o-toluidine, to be 9-(4'-amino-3'-methylphenyl)-9H- pyrido[3,4-b]indole (amino-3'-methylphenylnorharman, 3'-AMPNH), 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole (aminophenylharman, APH) and 9-(4'-amino-3'-methylphenyl)-1-methyl-9H-pyrido[3,4-b]indole (amino-3'-methylphenylharman, AMPH), respectively (3,33). To clarify the effects of APNH and its derivatives on human health, it is important to further elucidate their detailed biological properties. Furthermore, it is very important to determine how much APNH and its derivatives may be produced in our bodies in daily life, and consider preventive studies in accordance.
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Notes
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1 Present address: Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Avenue, Suite 309, Charleston, SC 29425, USA 
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Acknowledgments
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We thank Drs Kunitoshi Mitsumori, Tokyo University of Agriculture, Hidetaka Sato, Japan Food Research Laboratories and Technology, and Hiroyuki Tsuda, Nagoya City University Medical School, for his helpful advice and discussion on tumor histology and Ms Yurika Teramoto for her expert technical support. This work was supported by Grants-in-Aid for Cancer Research, the Second-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare, Japan.
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Received November 30, 2003;
revised May 5, 2004;
accepted May 9, 2004.