Formation and Differential Removal of C-8 and N2-Guanine Adducts

Chronic feeding of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the diet results in ... Humans metabolically activate HAAs to genotoxins at levels ...
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Chem. Res. Toxicol. 1996, 9, 397-402

397

Formation and Differential Removal of C-8 and N2-Guanine Adducts of the Food Carcinogen 2-Amino-3-methylimidazo[4,5-f]quinoline in the Liver, Kidney, and Colorectum of the Rat Robert J. Turesky,* Jovanka Markovic, and Jean-Marc Aeschlimann Nestec Ltd., Research Centre, P.O. Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland Received July 20, 1995X

Chronic feeding of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the diet results in tumor formation of the liver and colorectum, but does not induce tumorigenesis in the kidney of male Fischer-344 rats. The formation and rate of removal of DNA adducts were investigated in rats given an oral dose of IQ (20 mg/kg) to determine if adduct persistence affects the tissue susceptibility to IQ-induced tumorigenesis. Analysis of DNA adducts by 32P-postlabeling showed the formation of two 2′-deoxyguanosine (dG) adducts, N-(deoxyguanosin-8-yl)-2-amino3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) and 5-(deoxyguanosin-N2-yl)-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ). The pattern and distribution of these dG adducts were similar in all tissues; dG-C8-IQ and dG-N2-IQ accounted for approximately 70% and 15-20%, respectively, of the observed radioactivity. Maximal DNA binding was observed in liver (7.64 ( 1.08 adducts per 107 bases) and in colorectum (1.08 ( 0.22 adducts per 107 bases) 24 h following IQ treatment, while optimal binding appeared in kidney (2.41 ( 0.47 adducts per 107 bases) 72 h after treatment. Greater than 50% of the dG-C8-IQ adduct was removed from DNA of liver and kidney within 1 week of treatment. In contrast, the dG-N2-IQ adduct persisted and was the principal lesion remaining in liver and kidney 4 weeks after treatment with IQ. There was no evidence for selective removal of either adduct in the colorectum over a 3 week period, and adduct removal appeared to be attributed to cell turnover and not due to excision repair processes. Therefore, the relative persistence of dG-C8-IQ and dG-N2-IQ adducts does not appear to explain tissue susceptibility to IQ-induced neoplasia. The slow disappearance of IQ-DNA adducts suggests that adducts may accumulate during chronic exposure to IQ. Further investigations on DNA adduct formation and removal in animals chronically exposed to this carcinogen may help to explain the susceptibility of various organs to IQ-induced tumorigenesis.

Introduction Heterocyclic aromatic amines (HAAs)1 are potent genotoxic rodent carcinogens (1-3) formed at the low part per billion level in cooked meats and fish (4, 5) and in cigarette smoke condensate (6). 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is structurally representative of this class of genotoxins, and recently IQ has been reported to be a potent hepatocarcinogen in nonhuman primates (7). Metabolic activation of HAAs to genotoxic species occurs through mammalian cytochrome P450 mediated N-oxidation to form the arylhydroxylamine, which can bind to DNA (8-14), or may undergo further * Correspondence should be addressed to this author at Nestec Ltd., Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland. 021-785-8833 (phone), 021-785-8553 (fax). X Abstract published in Advance ACS Abstracts, February 1, 1996. 1 Abbreviations: dG, 2′-deoxyguanosine; 3′-dGMP, 2′-deoxyguanosine 3′-monophosphate; HAAs, heterocyclic aromatic amines; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; N-acetoxy-IQ, N2-acetoxyamino-3-methylimidazo[4,5-f]quinoline; dG-N2-IQ, 5-(deoxyguanosin-N2yl)-2-amino-3-methylimidazo[4,5-f]quinoline; dG-C8-IQ, N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline; MeIQx, 2-amino3,8-dimethylimidazo[4,5-f]quinoxaline; AAF, 2-(acetylamino)fluorene; dG-C8-AAF, N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene; dG-N2AAF, 3-(deoxyguanosin-N2-yl)-AAF; 2-AF, 2-aminofluorene; dG-C8-AF, N-(deoxyguanosin-8-yl)-2-aminofluorene; MAB, N-methyl-4-aminoazobenzene; dG-N2-MAB, 3-(deoxyguanosin-N2-yl)-MAB; dG-C8-MAB, N-(deoxyguanosin-8-yl)-MAB; PNK, polynucleotide kinase; MNSPD, micrococcal nuclease spleen phosphodiesterase; SPD, spleen phosphodiesterase; RAL, relative adduct labeling.

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activation by esterification to produce highly reactive species which react with DNA (13, 15-17). Humans metabolically activate HAAs to genotoxins at levels comparable to animal species which develop tumors following long-term feeding studies with these mutagens (8-17). Therefore, there is concern that HAAs may be involved in the etiology of human cancers (2, 18). The covalent binding of genotoxic carcinogens to DNA is regarded as a critical event in cancer initiation (19, 20). In the case of IQ, reaction of a putative carcinogenic metabolite, N2-acetoxyamino-3-methylimidazo[4,5-f]quinoline (N-acetoxy-IQ), with 2’-deoxyguanosine (dG) results in the formation of two dG adducts. The major lesion is a C-8 guanine adduct, N-(deoxyguanosin-8-yl)-2-amino3-methylimidazo[4,5-f]quinoline (dG-C8-IQ), with adduction occurring at the exocyclic amino group of IQ (21, 22). The second adduct is 5-(deoxyguanosin-N2-yl)-IQ (dG-N2IQ), where adduction occurs at the C-5 atom of the IQ heteronucleus (Figure 1). Analysis by the 32P-postlabeling technique (23) has also shown that both IQ-guanyl adducts are present in tissues of rodents following a single dose of IQ (24-31). The liver and colon are two tissues in the rat which develop tumors during chronic exposure to IQ as part of the diet; however, tumorigenesis has not been reported in the kidney (1, 2). In these three tissues, the dG-C8-IQ and dG-N2-IQ lesions account for approximately 50-70% and 15-20%, respectively, of the adducts measured by 32P-postlabeling (32). In this study, © 1996 American Chemical Society

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Turesky et al.

Figure 1. Chemical structures of IQ, and the DNA adducts dG-C8-IQ and dG-N2-IQ. The C8 adduct is drawn in the syn conformation and the N2 adduct is in the anti conformation.

we have examined the formation and removal of dG-C8IQ and dG-N2-IQ adducts in liver, kidney, and colorectum to determine if differences in organ susceptibility to IQinduced tumorigenesis may be attributed to adduct persistence or differences in rates of adduct removal.

Materials and Methods Chemicals. Caution: IQ and several of its derivatives are carcinogenic to rodents and should be handled carefully. The following chemicals were obtained from Sigma Chemical Co. (St. Louis, MO): calf thymus DNA, 2′-deoxyguanosine (dG), 2′deoxyguanosine 3′-phosphate (3′-dGMP), ATP, bicine, calcium chloride, urea, sodium succinate, spermidine, proteinase K, micrococcal nuclease, nuclease P1, and spleen phosphodiesterase (type I). IQ and [2-14C]IQ, specific activity 10 mCi/mmol, radiochemical purity >98%, were obtained from Toronto Research Chemical (Downsview, Ontario, Canada). Cloned T4 polynucleotide kinase was obtained from New England Biolabs (Beverly, MA). [32P]ATP (7000 Ci/mmol) was obtained from ICN Chemicals (Irvine, CA). PEI-cellulose thin-layer plates were from Machery-Nagel (Du¨ren, Germany). Qiagen tip-2500 columns were purchased through Kontron Instruments (Lausanne, Switzerland). Isolute C18 end-capped cartridges (100 mg) were purchased from ICT AG (Basel, Switzerland). All other chemicals were reagent grade unless specified. Chemical Syntheses of DNA Adduct Standards. IQmodified calf thymus DNA, 3′-dGMP and dG-3′,5′-bisphosphate adduct standards of IQ were prepared and characterized as previously described (22, 31, 32). Animal Experiments. Male Sprague Dawley rats (200250 g) were obtained from Iffa Credo (L’Arbresle, France) and given tap water and chow ad libitum. IQ was administered by gavage as its hydrochloride salt in 1 mL of water at a dose of 20 mg/kg. Animals (4 per group) were sacrificed by anesthesia with sodium pentobarbital (60 mg/kg ip) at 6 h or 1, 2, 3, 7, 10, 14, 21, or 28 days following exposure. Nuclear DNA from colorectum, liver, and kidney was isolated by Qiagen chromatography (31, 32). DNA Digestion and Adduct Enrichment. DNA (30 µg) in 150 µL of 2 mM sodium succinate, 1 mM CaCl2, and 1 mM Tris-HCl (pH 8.1) was hydrolyzed with 3.0 U/0.38 U of micrococcal nuclease/spleen phosphodiesterase (MNSPD) at 37 °C for 8 h. DNA adducts were then enriched by solid phase extraction (31, 32) prior to postlabeling. 32P-Postlabeling. The 32P-postlabeling and relative adduct labeling (RAL) calculations were performed as described by Randerath et al. (23) with modifications (31, 32). Adducts were resolved with the following TLC solvents: D1, 1.0 M NaH2PO4, (pH 5.8); D2, omitted; D3, 3.6 M lithium formate, 8.5 M urea (pH 3.5); D4, 1.0 M LiCl, 0.5 M Tris-HCl, 8.5 M urea (pH 8.0); D5, 1.7 M NaH2PO4, (pH 6.0). Adduct visualization and quantification were determined with a Packard Instant Image Analyzer (32). Adduct analyses were performed in duplicate for each animal (4 animals per group, N ) 8), except in the case of kidney (day 7) and colon (day 14, 21) which were done in quadruplicate (N ) 16).

Figure 2. 32P-Postlabeling profiles of the following: (A) liver DNA of an untreated rat; (B) 500 fmol of 3′,5′-bisphospho-dGN2-IQ and 3’,5’-bisphospho-dG-C8-IQ; (C1) liver DNA of a rat treated with IQ (20 mg/kg) 24 h and (C2) 4 weeks after IQ exposure; (D1) kidney DNA at 24 h and (D2) 4 weeks after IQ treatment; (E1) colorectal DNA at 24 h and (E2) 3 weeks after IQ treatment. Table 1. Maximal Levels of IQ-DNA Adducts Formed in Rat Tissuesa tissue colorectum kidney liver a

time after total adducts dG-C8-IQ dG-N2-IQ treatment (h) (RAL × 10-7) (RAL × 10-7) (RAL × 10-7) 24 72 24

1.08 ( 0.22 2.41 ( 0.47 7.64 ( 1.08

0.87 ( 0.21 1.38 ( 0.25 5.39 ( 1.14

0.21 ( 0.03 0.48 ( 0.09 1.91 ( 0.33

Male Fischer-344 rats given IQ (20 mg/kg) (N ) 4 ( SD).

Results The TLC profiles of DNA adduct standards, liver DNA from an untreated rat, and DNA from liver, kidney, and colorectal tissues 24 h and 3 or 4 weeks after a single exposure to IQ (20 mg/kg) are shown in Figure 2. A similar adduct pattern is observed in all tissues. Maximal DNA binding was observed in liver and in colorectum 24 h following treatment with IQ, while maximal adduct levels were found in kidney 72 h after treatment (Table 1). At maximal DNA binding, the dG-C8-IQ adduct is the predominant lesion in all tissues and it accounts for approximately 50-70% of the observed radioactivity, followed by dG-N2-IQ which accounts for 15-20% of the radioactivity. Several other lesions are observed, particularly in kidney, which appear to be incompletely digested oligomers of dG-C8-IQ (31). The identity of dGC8-IQ and dG-N2-IQ was corroborated by HPLC analysis

IQ-DNA Adduct Formation and Persistence

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of the excized TLC lesions with unlabeled synthetic 3′,5′bisphosphate-dG adduct standards as visual markers (data not shown) (31, 32). The stability of dG-C8-IQ and dG-N2-IQ in liver, kidney, and colorectum was examined over a 4 week period (Figure 3). The persistence of the adducts in the respective tissues is different, and the rates of adduct removal, as measured by the ratio of dG-N2-IQ/dG-C8IQ, are not identical. Greater than 50% of the dG-C8IQ adduct observed in kidney and liver tissue at maximal binding is removed within 7 days, and 28 days after IQ treatment, only 8 -11% of the dG-C8-IQ adduct is remaining. The dG-N2-IQ is removed less rapidly, and by 4 weeks after IQ treatment, it is the principal lesion remaining in the liver and at comparable levels to dGC8-IQ in the kidney. The median values of dG-N2-IQ/ dG-C8-IQ at maximal DNA binding levels, in liver and kidney, are 0.34 and 0.36, respectively, and by 4 weeks after IQ treatment, the median has increased 3- to 4-fold (1.00 for kidney and 1.29 for liver). In kidney tissue, days 2 and 7 in particular, a wide range in the dG-N2-IQ/dGC8-IQ ratio is observed. This variation appears to be attributed to an incomplete digestion and recovery of the dG-8-IQ lesion, which results in an over estimated median.2 The enzymatic hydrolysis and recovery of the dG-C8-IQ adduct from the kidney was complete at later time points and comparable to that observed in liver and colorectum where the dG-C8-IQ and dG-N2-IQ adducts consistently accounted for 90% of the observed postlabeled material (Figure 2). A significant increase in the ratio of dG-N2-IQ/dG-C8-IQ over day 1 was observed in both liver and kidney from 7 days after treatment with IQ, which demonstrates that the dG-C8-IQ lesion is preferentially removed. In contrast to liver and kidney, DNA adduct removal in colorectum is more rapid, presumably through the high mitotic index of this tissue. Approximately 50% of both C-8 and N2-guanyl adducts are removed within 3 days, and 95% of the lesions are removed within 21 days of IQ treatment. There is no evidence for selective removal of dG-C8-IQ in colorectal tissue. The median value of dGN2-IQ/dG-C8-IQ 24 h following treatment with IQ is 0.25, and after 21 days the median is 0.22; these values are not different with statistical analysis (T test or robust statistics). Therefore, adduct removal appears to be attributed to cell turnover and not to excision repair processes.

Discussion In this study, DNA adduct formation and persistence have been examined in male Fischer-344 rats following administration of a single oral dose of IQ (20 mg/kg) which, when given chronically to this species, results in tumorigenesis in liver and colorectum, but not in kidney (1, 2). A similar pattern and distribution of DNA adducts are observed in all tissues with highest adduct levels formed in liver, followed by kidney and then colorectum. Therefore, total DNA adducts formed, and the selective formation of either dG-C8-IQ or dG-N2-IQ in liver and colorectum, but not in the kidney, cannot explain the susceptibility of these tissues to IQ-induced tumorigenesis. The relatively high levels of DNA adducts formed in rat liver correlate well with tumor induction in this tissue by IQ; however, the absence of tumorigenesis in kidney appears inconsistent with the high levels of DNA 2

R. Turesky, unpublished observations.

Figure 3. Removal of dG-C8-IQ and dG-N2-IQ from liver, kidney, and colorectal tissue of rats given a single oral dose of IQ (20 mg/kg). Data are the average ( SD of 4 animals. Lower panels depict the individual measurements of the ratio of dG-N2IQ/dG-C8-IQ over time. The ratios in kidney and liver tissue 7 days after IQ treatment and beyond were statistically different from the observed values 1 day after treatment with IQ using either the T test or robust statistics (p < 0.02). There was no difference in the dG-N2-IQ/dG-C8-IQ ratio as a function of time in the colon.

adducts present. Therefore, factors in addition to DNA adduct formation, such as adduct persistence, error prone

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repair, cell division, and tumor promotion, must contribute to tumorigenesis (33-35). Adduct persistence is believed to be an important feature of chemical carcinogenesis (33-35). dG-C8-IQ and dG-N2-IQ persist in liver, kidney, and colorectum, and both adducts are readily detected 21-28 days following treatment with IQ. However, the persistence of these adducts and their rate of removal in the respective tissues are different. In the case of liver and kidney, approximately 50% of the dG-C8-IQ adduct is removed within 3-7 days of maximal DNA binding, and by day 14, 70-80% of the adduct is removed. Adduct removal occurs more slowly during days 15-28 post treatment with IQ, and approximately 10% of the adducts initially observed at maximal DNA binding remain present after 4 weeks. The dG-N2-IQ lesion is removed at a slower rate in both tissues, and approximately 25-40% of the adducts initially observed at maximal DNA binding are present 28 days after treatment. The data suggest that adduct removal may be biphasic or more complex, but there are an insufficient number of data points to develop a rigorous mathematical model. A similar trend for total IQ-DNA adduct removal was observed previously in rat liver following a single dose of IQ, although differential removal of IQ-guanine adducts was not noted (25). Following chronic administration of the stuctural related food mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to rats, total DNA adduct removal from liver also appeared to be biphasic, with an initial rapid removal of adducts followed by a slower rate (36). Further investigations on dG-C8-IQ and dG-N2-IQ DNA adduct formation and persistence in liver and kidney following chronic exposure to IQ may help to explain the differences in the susceptibility of these tissues to IQinduced tumorigenesis. DNA adducts of IQ also persist in colorectal tissue. Data presented here, and elsewhere (25), show that IQDNA adducts in colorectum epithelial cells are present up to 21 days after exposure to IQ. Although IQ-DNA adducts formed in colorectal tissue are relatively low compared to those formed in kidney and liver, the high incidence of colorectal cancer may be attributed, in part, to the high rate of cell division and DNA synthesis which results in fixation of the initiated DNA lesions (37, 38). There is no evidence for selective removal of either adduct in the colorectum; 50% of the adducts are removed within 3 days of IQ treatment, and 95% of the dG-C8-IQ and dG-N2-IQ adducts are removed within 3 weeks of dosing. If dG-C8-IQ was preferentially removed from colorectal tissue at rates comparable to liver or kidney tissues, an increase in the dG-N2-IQ/dG-C8-IQ ratio should have been detected by 3 weeks following IQ treatment. dGC8-IQ is removed more rapidly than dG-N2-IQ from DNA of human lymphoblasts (3 adducts per 107 bases) over 24 h, during which time the cells have undergone division.3 Therefore, the postlabeling analysis is sufficiently sensitive to discern differences in IQ-DNA adduct removal in rapidly dividing cells. The cell turnover in colon and rectal epithelial cells of male Fischer344 rats has been estimated at approximately 98% over 20 days based upon the loss of incorporated [methyl-3H]thymidine (39). Thus, the removal of IQ-DNA adducts in colorectal tissue appears to closely parallel the high mitotic index and rapid cell replication ocurring in this tissue. 3 R. Turesky and P.-M. Leong Morgenthaler, unpublished observations.

Turesky et al.

Numerous investigations have shown that the conformation of the glycosidic linkage of adducted DNA is an important factor in adduct persistence (40-45) and the toxicological properties of DNA adducts (46-50). For example, the aromatic amine and hepatocarcinogen 2-(acetylamino)fluorene (AAF) forms three principal lesions in rat liver DNA, including N-acetyl-N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AAF), 3-(deoxyguanosin-N2-yl)-2-(acetylamino)fluorene (dG-N2-AAF), and the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) (33, 34). The dG-C8-AF adduct exists preferentially in the normally occuring anti structure of dG, while the acetylated dG-C8-AAF adduct adopts the syn form (40, 41). These conformations also appear to exist in DNA and affect the relative persistence of these adducts (42, 43). Because dG-C8-AAF exists in the syn form, it induces a greater distortion of the DNA helix at the site of carcinogen modification than either dG-C8-AF or dG-N2-AAF (40, 41). Consequently, dG-C8AAF is rapidily removed from DNA, while dG-C8-AF and dG-N2-AAF are persistent lesions and accumulate with multiple dosing in rodents (42, 43). N-Methyl-4-aminoazobenzene (MAB) also forms two guanine adducts: 3-(deoxyguanosin-N2-yl)MAB (dG-N2-MAB) and N-(deoxyguanosin-8-yl)MAB (dG-C8-MAB), which represent respectively 25% and 70% of the total MAB bound to rat liver DNA (44, 45). The dG-C8-MAB lesion is rapidly removed from the DNA within 72 h; however, dG-N2MAB is a persistent lesion (45). The more rapid removal of dG-C8-MAB also may be explained, in part, by differences in the DNA adduct conformation. Based upon 1H-NMR spectroscopy, dG-C8-IQ and dGN2-IQ have been shown to reside preferentially in the syn and anti forms, respectively (22). Although, the conformation of these adducts in DNA is currently not known, the biological data presented here clearly show that the stability and persistence of these guanyl adducts in liver and kidney tissue are different. There are also differences in formation and persistence of these IQ-guanyl adducts in nonhuman primates which develop liver tumors following chronic treatment with IQ (51). dGC8-IQ is the principal lesion in the liver of nonhuman primates following a single dose of IQ (20 mg/kg), and it accounts for approximately 60-80% of the total adducts, followed by dG-N2-IQ, which accounts for approximately 30% of the lesions (51). Following long-term exposure to IQ, dG-N2-IQ accumulates in the liver and in other slowly dividing tissues to become the principal (51). The types and frequencies of mutations induced by C-8 and N2-guanine adducts of aromatic amines are different, and their biological effects are influenced by the local DNA sequence context, the conformation of the adduct, and induced helix stucture at the site of carcinogen modification (33-35, 46-50). Further studies evaluating the genetic and structural effects of dG-C8-IQ and dGN2-IQ on DNA are warranted to assess their contribution to the potent carcinogenicity of this food mutagen.

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