DNA adduct formation by 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) in rat colon Chun-xing Lin, Yasumasa Monden and Atsushi Umemoto Second Department of Surgery, The University of Tokushima School of Medicine, Tokushima, Japan Abstract: A food-born carcinogen, 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) induces cancer in the rat colon. The mechanism for colonic DNA adduct formation leading to cancer by IQ was studied using a colostomized F344 rat model. In this model, the transverse colon of the rat was colostomized, which produced a fecal stream-positive proximal colon and a negative distal colon were produced. When IQ (50 mg/kg) was administered into the distal colon of the colostomized rats (n=5), the ratio of the DNA adduct level of the distal colonic mucosa to the paired muscular layer 24 hr after dosage was 2.02, whereas that was 1.51 and 1.37 when IQ was administered into the stomach (n=6) and the vein (n=5), respectively. This suggested that luminal exposure of IQ induced DNA adduct formation. Since IQ (an amine form) has no reactivity toward DNA, these findings suggested that IQ was immediately activated in the absorbed mucosal cells and reacted with DNA. However, most of the IQ absorbed was metabolically activated in the liver, distributed by blood circulation, and formed DNA adducts in the colonic mucosa and muscular layer. J. Med. Invest. 48:102-108, 2001 Keywords:IQ, DNA adduct, colon, 32P-postlabeling, metabolism INTRODUCTION Accumulating evidence demonstrates that a series of genetic alterations are involved in multi-stage colon carcinogenesis (1). In addition, it has been recognized that various dietary carcinogens play an important role in the development of colon cancers (2-4). Our previous study showed that unknown DNA adducts were formed in the human colonic mucosa (5) at a level apparently higher than that of the small intestinal mucosa which shows a much lower incidence of cancer (6). Such DNA adducts may be the cause of critical mutations in cancer-related genes. A heterocyclic amine 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) (Fig. 1) is shown to develop a high incidence of tumors in the small and large intestine, liver, Zymbal gland, clitoral gland and skin in F344 rats (7). IQ is suggested to be generated from creatine, free amino acids and hexoses, present in raw meat, via the Maillard reaction during the cooking process (8), and so is recognized as an unavoidable dietary component for humans. IQ forms N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo-[4,5-f]quinoline (dG-C8-IQ) and 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ) in vitro and in vivo (9-11) (Fig. 1). Although IQ-DNA adduct levels in F344 rats were highest in the liver, followed, in order, by the lungs, kidneys, stomach, colon, white blood cells and small intestine (12), the carcinogenic targets were only the liver, small intestine and colon. The threshold for the initiation of carcinogenesis by the adducts, repair of the adducts and cell turnover rate in each tissue may be responsible for this discrepancy. Thus, the relationship between the DNA adduct level and tumorigenesis is not a simple matter of cause and effect. However, it has been demonstrated that IQ induced point mutations in H-ras and K-ras genes in Zymbal gland tumors (13) and in Apc and β-catenin genes in colon tumors (14, 15). Thus, the DNA adduct formation and subsequent gene mutations are presumably the major mechanism for the rat colon carcinogenesis by IQ. There are three theoretical routes by which IQ, administered by gavage, can come into contact with colonic mucosal DNA (16):(i) Luminal exposure: IQ in the stomach travels through the intestinal lumen and reaches the colonic mucosal cells from the luminal direction, (ii) Circulatory exposure : IQ is absorbed from the gastrointestinal tract, metabolized in the liver, and transferred to the colonic cells through systemic blood circulation, (iii) Biliary exposure : after absorption from the GI-tract, IQ is metabolized in the liver, and excreted into bile, reaching the luminal surface of the colonic mucosa. However, it is not yet thoroughly clarified how these routes work for DNA adduct formation in the colon. In the present study, the colostomized rats having normal proximal colon and fecal stream-excluded distal colon were used to clarify the role of each route. MATERIALS AND METHODS Chemicals IQ-HCl was from Dr. Keiji Wakabayashi, National Cancer Center Research Institute, Japan. RNase A, RNase T1, micrococcal nuclease, spleen phosphodiesterase and phosphodiesterase I were purchased from Worthington Biochemical Co. Ltd. (Freehold, NJ). T4 polynucleotide kinase was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Nuclease P1 was purchased from Yamasa Shoyu Co., Ltd. (Choshi, Japan). Proteinase K and apyrase were purchased from Sigma (St. Louis, MO). [α-32P]adenosine-5'-triphosphate (>7,000 Ci/mmol) was obtained from ICN Radiochemical (Irvine, CA). Polyethyleneimine-cellulose sheets (POLYGRAM CELL 300 PEI) were purchased from Machery-Nagel (Duren, Germany). Animals Male Fischer 344 rats (4-6 weeks old) were purchased from Nippon SLC Co. (Hamamatsu, Japan) and housed in polycarbonate cages, two to three animals per cage, for one week prior to use in the pathogen-free room of our animal facilities. They were kept under constant conditions of temperature (22±2°C) and humidity (55±5%) with a 13 hr light/11 hr dark cycle. They were fed a commercial diet MF (Oriental Yeast, Co., Ltd., Tokyo, Japan) and provided tap water ad libitum. Study design and surgery Male F344 rats underwent surgery to differentiate the transportation routes in rat body for IQ (Fig. 2). In all the experimental studies, laparotomy was performed under general anesthesia with intraperitoneal administration of pentobarbital sodium (50 mg/kg b.w.). In Study 1, rats (6 weeks old) were colostomized at the middle of the transverse colon, and the divided colonic stumps were separately anchored through the abdominal wall to the exterior (Fig. 3A-C). Using this procedure, the distal side of the colon was completely separated from the fecal stream and naturally became empty within a few days. The distal colon was further cleaned up by enema with 0.9% NaCl solution from the stoma for three days before dosing. A single dose of IQ-HCl (50 mg/kg, 20 mg/ml of water solution) was administered into the stomach (n=6) in Study 1A, into the excluded distal colon (n=5) in Study 1B and intravenously (n=5) in Study 1C two weeks after colostomy. In Study 1B, immediately after administration of IQ into the distal colon via the stoma, the stoma and the anus of the distal colon were stitched to avoid leakage. In Study 2, the bile duct of the rats (8 weeks old) was catheterized under laparotomy (n=5) and bile was drained for 24 hr after intravenous administration of IQ-HCl (50 mg/kg) (Fig. 3D). In all experiments, the rats were sacrificed 24 hr after administration of IQ by carbon dioxide asphyxiation, and the large intestines were collected. The intestines were incised in a longitudinal direction and immediately rinsed free of their contents with 0.9% NaCl solution. The mucosal layer was scraped off, and the separated mucosal layers and muscle layers were stored at -80°C until use. 32P-postlabeling of IQ-DNA adducts DNA was isolated by the method reported previously (5, 17). The concentration of DNA was determined by measuring the absorbance at 260 nm (using a value of 20 absorbance units/mg DNA) and adjusted to a final concentration of 2 mg/ml. IQ-DNA adducts were detected by 32P-postlabeling analysis with the intensification method (18). Briefly, 10 μg of DNA was digested to deoxynucleoside 3'-monophosphates with micrococcal nuclease (3 units) and spleen phosphodiesterase (0.03 units) in a total volume of 10 μl of 20 mM sodium succinate, 10 mM CaCl2, pH 6.0 at 37°C for 3.5 hr (19). The DNA digest was diluted 2-fold with water. Then, 10 μl of the digest was taken and incubated with 5 μl of labeling cocktail containing 1.5 μl of kination buffer (300 mM Tris-HCl, pH 9.5, 100 mM MgCl2, 100 mM dithiothreitol and 10 mM spermidine), 1.0 μl of [α-32P]ATP (150 mCi/ml), 0.5 μl (5 units) of T4 polynucleotide kinase and2.0μl of water at37°C for 1 hr. The labeled digest was further treated with 2 μl potato apyrase (5 units/ml) and 1 μl water for 45 min at 37°C. To purify the IQ-bound nucleotides from normal nucleotides, the 32P-labeled nucleoside bisphosphates were spotted onto PEI-cellulose sheets and developed with 1.7 M sodium phosphate (pH 6.0) at 22°C for 16 hr. The origin was cut out and attached to a new PEI-cellulose sheet using a magnet, and developed with 2.7 M lithium formate/5.1 M urea (pH 3.5) from bottom to top. This was further developed in 0.96 M LiCl/6.4 M urea/0.4 M Tris-HCl (pH 8.0), followed by 1.0 M sodium phosphate (pH 6.0) from left to right, with a 3.5 cm paper wick (Whatman 3MM chromatography paper). The radioactive spots on a thin-layer chromatography (TLC) sheet were visualized and their radioactivity was quantified by BAS-1500 Bio Imaging Analyzer and BAS-IIIs Imaging Plate (Fuji Photo Film Co., Ltd, Tokyo, Japan). For the total nucleotide count, an aliquot of the above DNA digest was labeled, treated with apyrase, spotted on the PEI-cellulose sheet and developed with 0.5 M lithium chloride (17). The relative adduct labeling (RAL) values were calculated according to the formula : RAL = adducts level / (total nucleotides levelxdilution factor). All experiments for quantification of the IQ-DNA adduct level by 32P-postlabeling were performed in triplicate. To estimate DNA adduct levels as accurately as possible, all samples in a study group were analyzed at once, and the analysis of samples was performed in a random order. Statistical analysis was performed by ANOVA. RESULTS In study-1, the preoperative body weight of the rats (140±2 g) at 6 weeks old slightly decreased to 137±9 g one week after colostomy, but increased again to 162±6 g when IQ was administered at 8 weeks of age. When IQ was administered into the stomach (Study 1A), the IQ-DNA adduct level of the proximal mucosa was significantly higher than that of the distal mucosa (p<0.05), and that of the proximal muscular layer was significantly higher than that of the distal muscular layer (p<0.05) (Table 1). In the same study, the IQ-DNA adduct level of the proximal mucosa was significantly higher that of the proximal muscular layer (p<0.05), and that of the distal mucosa was significantly higher than that of the distal muscular layer (p<0.05). When IQ was administered into the distal colon (Study 1B), the IQ-DNA adduct level of the proximal mucosa was similar to that of the distal mucosa, but that of the proximal muscular layer was significantly higher than that of the distal muscular layer (p<0.05) (Table 1). In the same study, the IQ-DNA adduct levels of the proximal and distal mucosa were significantly higher than those of the proximal and distal muscular layer, respectively (p<0.05). As a whole, the DNA adduct level of Study 1B dosed in the distal colon was similar to that of Study 1A dosed in the stomach. In contrast, DNA adduct level of Study 1C dosed intravenously was about three-fold higher than that of Study 1A and B. When IQ was administered intravenously (Study 1C), the IQ-DNA adduct level of the proximal mucosa was significantly higher than that of the distal mucosa (p<0.05), but that of the proximal muscular layer was no difference compared with that of the distal muscular layer (Table 1). In the same study, the IQ-DNA adduct level of the proximal mucosa was significantly higher that of the proximal muscular layer (p<0.05), and that of the distal mucosa was not significantly higher than that of the distal muscular layer. In Study 2, when IQ was administered intravenously to the bile-drained rats, the IQ-DNA adduct level of the mucosa was about 2-fold higher that of the muscular layer (p<0.05) (Table 1). In all studies, approximately 5-6 DNA adduct spots were detected on a TLC sheet by 32P-postlabeling analysis as reported previously (16). DISCUSSION Among three theoretical exposure routes of IQ to the colon (luminal, circulatory and biliary exposures), all routes should be potentially active in the colonic mucosa if IQ is administered into the stomach of the non-operated rats. However, the luminal and biliary exposures to the distal colonic mucosa were blocked by the colostomy (Study 1A). When IQ was administered into the distal colon of the colostomized rats, luminal exposure to the proximal mucosa alone was blocked, in contrast, heavy luminal exposure to the distal mucosa occurred (Study 1B). When IQ was administered intravenously to the colostomized rats, luminal exposure to the mucosa was blocked (Study 1C). When IQ was administered intravenously to the bile-drained rats, only circulatory exposure was active (Study 2). Thus, a combination of these experimental models provided important information to clarify the active exposure routes to the colon. The colostomy procedure was an acceptable stress for the rats, and the rats were healthy when IQ was administered. Since the absolute values of 32P-postlabeling have a wide error range in all experiments, the samples to be compared should be analyzed together. However, the number of samples for one time analysis is limited. In the present study, to compare the mucosal DNA adduct levels in the different studies, the paired mucosa-adjacent muscular layer which allows only circulatory exposure was analyzed as a good standard. The general patterns of the DNA adduct levels in four types of colonic tissues (mucosa/muscular layer of the proximal/distal colon) were similar in Studies 1A, 1B and 1C. However, one difference was that the DNA adduct level of the distal mucosa in Study 1B alone was higher than that in Study 1A or 1C:i.e. the ratio of the DNA adduct level of the distal mucosa to the paired muscular layer was 2.02 in Study 1B, whereas it was 1.51 in Study 1A and 1.37 in Study 1C. This suggests that luminal exposure of IQ induced DNA adduct formation. Since IQ (an amine form) has no reactivity toward DNA, these findings imply that IQ was immediately activated in the absorbed mucosal cell and reacted with DNA. This idea is not recognized in other heterocyclic amines (20). The principal metabolic pathways of heterocyclic amines leading to DNA adducts involve cytochrome P450 (CYP) mediated N-oxidation of exocyclic amine nitrogen (phase I) and subsequent esterification by phase II enzymes (21). In hepatic tissue, heterocyclic amines are known to be activated to the N-hydroxy form mainly by CYP1A2, but this enzyme activity is absent in the colon (22). However, in the case of IQ, we previously demonstrated, using different rat models, that IQ was partly activated to form DNA adduct in the colonic mucosal cells (16). The present findings support our previous observations. Thus, except CYP1A2, there should be another metabolic activation mechanism such as CYP1B1 (22) and prostaglandin H synthase (23) in the colonic mucosa. However, the major activation was presumably catalyzed by hepatic CYP1A2 as previously recognized, because most DNA adduct was formed via circulatory exposure in the present study. A comparison of DNA adduct levels of the proximal mucosa between Study 2 and Study 1A-C showed the effect of biliary exposure by IQ metabolites on colonic DNA adduct formation. Although the biliary exposure to the proximal mucosa was excluded by bile drainage in Study 2, the DNA adduct level of the proximal mucosa did not decrease compared with that in Study 1.This suggests that the contribution of biliary IQ metabolites to the colonic adduct formation was negligible. Therefore, the finding that the IQ-DNA adduct level of the proximal mucosa was significantly higher than that of the distal mucosa in Study 1C was caused mostly by circulatory exposure. The IQ-DNA adduct level of the proximal muscular layer was also higher than that of the distal colon in all Study 1 experiments. In addition, a preferential DNA adduct formation was shown in the mucosa compared with the paired muscular layer in all studies. The differences in the activities of metabolic activation/inactivation enzymes, the transport of the cell membrane and DNA repair may be the causes of these preferences of DNA adduct formation. In conclusion, the findings of the present study suggest that most of the part of IQ (an amine form) passes through the colonic mucosal cells without being metabolized when absorbed, is activated to an N-hydroxy form in hepatic tissue mainly by CYP1A2, is transported via the systemic circulation to the colon, and forms DNA adducts (Fig. 4). Some of the IQ, however, receives another metabolic activation allowing DNA adduct formation in colonic cells. The role of biliary IQ metabolites in colonic DNA adduct formation is negligible. ACKNOWLEDGMENTS This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan. A doctoral fellowship to Lin Chun-Xing from the Ministry of Education, Science, Sport and Culture of Japan is gratefully acknowledged. We are grateful to Dr. K. Wakabayashi for providing IQ-HCl. REFERENCES 1. Fearon ER, Vogelstein B : A genetic model for colorectal tumorigenesis. Cell 61 : 759-760, 1990 2. Nagao M, Sugimura T : Carcinogenic factors in food with relevance to colon cancer development. Mutat Res 290:43-51, 1993 3. Sugimura T : Nutrition and dietary carcinogens. Carcinogenesis 21:387-395, 2000 4. Sugimura T, Nagao M, Wakabayashi K : How we should deal with unavoidable exposure of man to environmental mutagens : cooked food mutagen discovery, facts and lessons for cancer prevention. Mutat Res 447:15-25, 2000 5. Umemoto A, Kajikawa A, Tanaka M, Hamada K, Seraj MJ, Kubota A, Nakayama M, Kinouchi T, Ohnishi Y, Yamashita K, Monden Y : Presence of mucosa-specific DNA adduct in human colon :possible implication for colorectal cancer. Carcinogenesis 15:901-905, 1994 6. Hamada K, Umemoto A, Kajikawa A, Tanaka M, Seraj MJ, Nakayama M, Kubota A, Monden Y:Mucosa-specific DNA adducts in human small intestine : a comparison with the colon. Carcinogenesis 15:2677-2680, 1994 7. Takayama S, Nakatsuru Y, Masuda M, Ohgaki H, Sato S, Sugimura T:Demonstration of carcinogenicity in F344 rats of 2-amino-3-methyl-imidazo[4,5-f]quinoline from broiled sardine, fried beef and beef extract. Gann (Jpn J Cancer Res) 75:467-470, 1984 8. Jagerstad M, Laser Reutersward A, Olsson R, Grivas S, Nyhammer T, Olsson K, Dahlqvist A :Creatin(in)e and Maillard reaction products as precursors of mutagenic compounds : effects of various amino acids. Food Chemistry 12 : 239-244, 1983 9. Snyderwine EG, Roller PP, Adamson RH, Sato S, Thorgeirsson SS : Reaction of N-hydroxylamine and N-acetoxy derivatives of 2-amino-3-methylimidazolo[4,5-f]quinoline with DNA. Synthesis and identification of N-(deoxyguanosin-8-yl)-IQ. Carcinogenesis 9:1061-1065, 1988 10. Turesky RJ, Rossi SC, Welti DH, Lay JO Jr, Kadlubar FF : Characterization of DNA adducts formed in vitro by reaction of N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and N-hydroxy-2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline at the C8 and N2 atoms of guanine. Chem Res Toxicol 5:479-490, 1992 11. Ochiai M, Nakagama H, Turesky RJ, Sugimura T, Nagao M : A new modification of the 32P-post-labeling method to recover IQ-DNA adducts as mononucleotides. Mutagenesis 14:239-242, 1999 12. Schut HAJ, Herzog CR, Cummings DA : Accumulation of DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in tissues and white blood cells of the Fischer-344 rat after multiple oral dosing. Carcinogenesis 15:1467-1470, 1994 13. Nagao M, Ushijima T, Toyota M, Inoue R, Sugimura T : Genetic changes induced by heterocyclic amines. Mutat Res 376:161-167, 1997 14. Kakiuchi H, Watanabe M, Ushijima T, Toyota M, Imai K, Weisburger JH, Sugimura T, Nagao M :Specific 5'-GGGA-3'→5'-GGA-3' mutation of the Apc gene in rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Proc Natl Acad Sci USA 92:910-914, 1995 15. Dashwood RH, Suzui M, Nakagama H, Sugimura T, Nagao M:High frequency of beta-catenin (ctnnb 1) mutations in the colon tumors induced by two heterocyclic amines in the F344 rat. Cancer Res 58:1127-1129, 1998 16. Kajikawa A, Umemoto A, Hamada K, Tanaka M, Kinouchi T, Ohnishi Y, Monden Y : Mucosa-preferential DNA adduct formation by 2-amino-3-methylimidazo-[4,5-f]quinoline in the rat colonic wall. Cancer Res 55:2769-2773, 1995 17. Beach AC, Gupta RC : Human biomonitoring and the 32P-postlabeling assay. Carcinogenesis, 13 :1053-1074, 1992 18. Randerath E, Agrawal HP, Weaver JA, Bordelon CB, Randerath K : 32P-postlabeling analysis of DNA adducts persisting for up to 42 weeks in the skin, epidermis and dermis of mice treated topically with 7,12-dimethylbenz[a]anthracene. Carcinogenesis 6:1117-1126, 1985 19. Gupta RC, Reddy MV, Randerath K : 32P-postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis 3:1081-1092, 1982 20. Kaderlik KR, Minchin RF, Mulder GJ, Ilett KF, Daugaard-Jenson M, Teitel CH, Kadlubar FF: Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues. Carcinogenesis 15 :1703-1709, 1994 21. King RS, Kadlubar FF, Turesky RJ : In vivo metabolism. In : Nagao M, Sugimura T, eds. Food Borne Carcinogens. John Wiley & Sons, Ltd, Chichester, 2000, pp. 90-111 22. Shimada T, Hayes CL, Yamazaki H, Amin S, Hecht SS, Guengerich FP, Sutter TR : Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res 56:2979-2984, 1996 23. Wolz E, Wild D, Degen GH:Prostaglandin-H synthase mediated metabolism and mutagenic activation of 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). Arch Toxicol 69 : 171-179, 1995 Received for publication December 26, 2000;accepted January 19, 2001. Address correspondence and reprint requests to Atsushi Umemoto, M.D., Ph.D., Second Department of Surgery, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan and Fax:+81-88-633-7144.