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Formation of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in male Fischer-344 rats
Cancer Letters, 67 (1992) 117 - 124 Elsevier Scientific Publishers Ireland Ltd
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Formation of DNA adducts of Z-amino-l-methyl-6phenylimidazo[4,5-blpyridine (PhIP) in male Fischer-344 rats Herman
A.J. Schut and Christopher
R. Herzog
Department of Pathology, Medical College of Ohio, Toledo, Ohio 43699
(USA)
(Received 3 September 1992) (Accepted 7 September 1992)
Summary Z-Amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) is known to induce colon tumors in male Fischer-344 rats. Using 32Ppostlabeling assays, we have examined PhlPDNA adduct formation in various organs and white blood cells (WBCs) of the male Fischer344 rat 24 h after a single oral dose of 0, 0.5, 5 or 50 mg PhlP/kg. Three PhlP-DNA adducts were detected in WBCs and in all organs, except in the liver and stomach which had only two adducts. The extent of adduct formation was dose-related, but at 0.5 mg/kg no adducts could be detected in any of the organs. At 50 mg/kg, adduct levels, expressed
as relative adduct labeling values (RAL x 107, or adducts per 1 O7 nucleotides assuming complete labeling) were highest in the large intestine (5.661, followed by WBCs (5.04), stomach (1.44), small intestine (1.32), kidney (1.16), liver (0.67) and lungs (0.52). It is concluded that orally administered PhlP forms high levels of specific DNA adducts in the large Correspondence to: Herman A.J. Schut, Department of Pathology, Health Education Building, Room 202, Medical Callege of Ohio, 3000 Arlington Ave., Toledo, Ohio 43614, USA. Abbreviations: AIA, amino-imidazoazaarene; IQ, 2-amino-3methylimidazo[4,5-flquinoline; MeIQx, 2-amino-3,&dimethylimidazo[4,5jjquinoxaline; PhlP, 2-amino-1-methyl-6-phenylimidazo[4,5-blpyridine; WBC, white blood cell; IF, intensification factor; RAL, relative adduct labeling.
0304-3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
intestine, the target organ in PhlP carcinogenesis in the male Fischer-344 rat, and that the high level of adducts in WBCs indicates that significant amounts of the ultimate carcinogenic form ojPhlP are present in the circulation.
Keywords: Z-amino- l-methyl-6-phenylimidazo[4,5_b]pyridine; PhIP; 32P-postlabeling analysis; DNA adducts; colon carcinogenesis Introduction A number of mutagens/carcinogens have been isolated from cooked foods [ 131. Among these the heterocyclic amines, in particular the amino-imidazoazaarenes (AIAs) , have been investigated extensively because they comprise a high proportion of the total mutagenic activity present in fried meat [lo]. All of the heterocyclic amines tested to date have been proven to be carcinogenic [2,21]. AIAs can be formed in vitro by heating amino acids with creatine or creatinine [US], or through Maillard reactions involving reducing sugars, amino acids and creatine or creatinine [l&22,26], yielding 2-amino-3-methylimidazo[4,5-flquinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5flquinoline, 2-amino-3&dimethylimidazo[4,5flquinoxaline (MeIQx), 2-amino-3,n,%triIreland Ltd
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methylimidazo[4,5-flquinoxaline
and 2-amino-
[ 111. It is carcinogenic in rodents, causing lymphomas in both male and female CDFl mice [9] and colon adenocarcinomas in male Fischer-344 rats and mammary adenocarcinemas in female Fischer-344 rats [ 171. While both the in vivo and in vitro mammalian cell mutagenicity of AIAs is considered low, that of PhIP is higher than that of the other AIAs [3,301. The results from several in vitro studies have shown that PhIP is activated to its ultimate carcinogenic form by N-hydroxylation [6,16, 19,32,34], a reaction catalyzed by a polycyclic aromatic hydrocarbon-inducible form of microsomal cytochrome P450, principally isozymes IA1 and IA2 [16,19,32,34]. Further esterification/deesterification of N-hydroxyPhIP by sulfotransferase or 0-acetylansferase 27,121 yields the ultimate reactive form, probably the nitronium ion [37], which reacts with DNA to form specific adducts. The formation of PhIP-DNA adducts has been shown in vitro [7] as well as in vivo [27,29,38], using 32Ppostlabeling methods. In vitro, the major adduct formed between N-hydroxy-PhIP and DNA is N*-(2 ‘-deoxyguanosin8yI) -PhIP [ 121. In the present study we have investigated the in vivo formation of PhIP-DNA adducts in the male Fischer-344 rat.
Materials and Methods Animal treatment
and DNA isolation adult male Fischer-344 rats Young (200 - 240 g) were obtained from Harlan Sprague - Dawley (Indianapolis, IN). PhIP was a generous gift from Dr. Richard H. Adamson, National Cancer Institute. Rats (2 per dose) were treated with a single p.o. (gavage) dose of PhIP (0,0.5,5 or 50 mg/kg) using dimethyl sulfoxide-0.05 M sodium phosphate, pH 4.5 (1:3) as the vehicle. The PhIP solution was prepared immediately
before administration and was kept at 37OC to prevent the formation of a gel which occurred at room temperature. Rats were killed 1 day after PhIP administration by first administering a heparin solution (5000 units/kg, i.p.), followed after 15 min by sodium pentobarbital solution (180 mg/kg, i.p.). After complete anesthesia (approximately 5 min) the abdomen was opened and blood was collected from abdominal veins (5 - 7 ml) into lithium heparin tubes. After mixing the blood with 45 ml of Iysis buffer (0.83% ammonium chloride , 0.1% potassium bicarbonate, 0.04 % tetrasodium EDTA, pH 7.3) and leaving the mixture at room temperature for 8 min, the white blood cells (WBCs) were isolated by centrifugation at 3000 x g for 8 min. The supernatant was discarded and the pellet (WBC) was resuspended in 2 ml of nuclei Iysis buffer (10 mM Tris - HCI, 400 mM sodium chloride, 2 mM tetrasodium EDTA, pH 8.2). After removal, the liver, lungs and kidneys were chopped into small pieces (<2 mm*) and a random sample of approximately 0.5 g was taken for DNA isolation. The stomach and the intestines were removed, cut open and rinsed with phosphate-buffered saline containing 1 mM EDTA to remove their contents. The epithelial layer was scraped off from the stomach, the entire small intestine and from the large intestine, including the cecum. Aliquots of 0.5 g were used for isolation of DNA after suspension and homogenization in 5 ml of nuclei lysis buffer. DNA was then isolated from WBCs and the organs by a direct salt precipitation procedure as described by Miller et al. 1201, except that the final DNA pellet was redissolved in 1 ml of 1.5 mM sodium chloride, 0.15 mM sodium citrate (0.01 x SSC) and then incubated with RNase A (100 pg/mI) and RNase Tl (50 units/ml) at 37OC in a shaking water bath for 1 h. After addition of 2 ml of 0.01 x SSC and 3 ml of phenol-chloroform-isoamyl alcohol (25:24: 1) the mixture was vortexed, centrifuged at 4OC for 10 min at 3000 x g, and the aqueous (top) layer was extracted once more with chloroform-isoamyl alcohol (24: 1). DNA was
119
precipitated described measured absorbance absorbance
from the aqueous (top) layer as [ZO] and its concentration was spectrophotometrically using its at 260 nm and a value of 20 A260 units/mg DNA.
32P-postlabe hn * g assay of PhlP-DNA adducts thin-layer Polyethyleneimine-cellulose plates were prepared in the laboratory as described by Gupta et al. [14]. After overnight washing the freshly prepared plates were stored at 4OC. [y-32P]ATP (approximately 4000 Ci/mmol) was freshly prepared for each postlabeling assay [ 141. The intensification version of the 32P-postlabeling assay [23] was used to quantitate PhIP-DNA adducts, as previously described for adducts between DNA and IQ [25,39]. For the determination of the intensification factors (IFS) of the individual PhIP-DNA adducts, the actual (true) adduct leveb were determined by the standard 32Ppostlabeling assay [ 141. In the intensification procedure the amounts of DNA and [32P]ATP were carefully kept constant in each assay, as the ratio of their concentrations determines the IF of the individual PhIP-DNA adducts [23] and, consequently, the accuracy of adduct quantitation. 32P-labe!ed nucleoside bisphosphates were purified on polyethyleneimine-cellulose plates as described before [39], except for modification of the D4 solvent which consisted of 0.8 M lithium chloride, 0.5 M Tris- HCI, 7.4 M urea, pH 8.0. 32P-labeled PhIP-DNA adducts were located on the plates by autoradiography, exposing films at - 70°C for varying periods of time. Radioactive spots (adducts) were cut out and quantitated by Cerenkov counting [ 141. The extent of adduct formation was expressed as relative adduct labeling (RAL) values which were calculated by dividing the counts/min per adduct by the sum of the counts/min in the adduct and the normal nucleotides, making adjustments for dilution and aliquot factors [ 141. < RAL > values obtained under intensification conditions were converted to the actual RAL values (standard conditions), using the IFS for each adduct.
Results and Discussion Three PhIP-DNA adducts could be detected in WBCs and in all organs, except in the liver and stomach which showed only adducts 1 and 2 (Fig. 1). Vehicle-treated animals showed no detectable adducts (Fig. 1 h). The pattern of PhIP-DNA adducts obtained (Fig. 1) was similar to that obtained in the liver of the cynomolgus monkey after repeated gavages (daily for 9 days) with PhIP [27] and in the liver of the Fischer-344 rat after a single oral dose of PhIP [38]. In vitro incubation of Nhydroxy-PhIP with DNA in the presence of mouse liver cytosol and either acetyl coenzyme A or 3’-phosphoadenosine 5’-phosphosulfate also yielded a similar pattern of adducts [7]. Feeding Fischer-344 rats with a diet containing 0.05% PhIP for 2 weeks resulted, however, in a much more complex PhIP-DNA adduct pattern [29], with seven adducts in all organs examined. Therefore, it appears that longterm continuous feeding of the Fischer-344 rat with PhIP results in the formation of additional, i.e. more than three, adducts which are possibly resistant to removal under these experimental conditions. IFS of the three PhIP-DNA adducts varied from 96.1 to 47.1 (Table I). These values are distinct from those determined for other, structurally related, heterocyclic amines such as IQ [25] or MeIQx [8], a finding emphasizing the different affinity of the polynucleotide kinase for structurally different adducts. IFS are also strongly influenced by the relative concentrations of deoxyribonucleoside 3’-monophosphates and [‘y-32P]ATP, which are likely to be different in different laboratories [29]. In most organs adduct 1 was the major adduct, although substantial amounts of adducts 2 and 3 were also present (Table II). Following 9 days of oral administration of PhIP to a cynomolgus monkey, a similar distribution of PhIP-DNA adducts was observed in all tissues examined, including the liver, stomach, small intestine, large intestine, kidney, spleen, pancreas, heart, salivary gland and brain [l]. Using physico-chemical methods, only a
120
_(
0
121
Table 1. Determination of IFS for PhlP-DNA and standard labeling conditions, respectively”.
adducts from < RAL > and RAL values obtained
PhlP-DNA adduct
b (~10~) (mean f S.D., N = 4)
RALC (x 105) (mean S.D., N = 4)
IF
1 2 3
36.51 zt 5.12 18.99 zt 3.10 15.53 zt 3.18
0.38 zt 0.009 0.20 f 0.10 0.33 f 0.10
96.1 95.0 47.1
under intensification
( < RAL > /RAL)
’ IFS determined on DNA from R52-16 cells, transduced with cytochrome P450IA2 DNA [29], exposed to 100 PM PhlP for 24 h. b< RAL> determined under intensification conditions. The DNA digest (deoxyribonucleoside 3’monophosphates equivalent to 3.75 pg DNA) was labeled with 225 FCi [y-32P]ATP (S.A. approx. 4000 Ci/mmol) in a volume of 15.5 pl as described [26] to give a final [Y-~~P]ATP concentration of 3.8 PM. ’ RAL determined under standard conditions. The DNA digest (deoxyrtbonucleoside 3’-monophosphates equivalent to 0.17 pg DNA) was labeled with 250 PCi [-r-32P]ATP (S.A. approx. 4000 Ci/mmol) together with unlabled ATP, in a volume of 10.0 ~1 as described [25,26] to give a final [Y-~~P]ATP concentration of 72.0 PM.
single adduct, N’-(Z’-deoxyguanosin-SyI)PhIP, has been identified after in vitro reaction of IV-hydroxy-PhIP with DNA in the presence of acetic anhydride, or in rat liver in vivo after i.p. administration of [3H]PhIP [El. The reason for this discrepancy with postlabeling methodology is not known, but preliminary evidence (E.G. Snyderwine and H.A.J. Schut, unpublished observations) indicates that adduct 1 (Fig. 1) may be the Cs-guanine adduct identified by Frandsen et al. [ 121. The nature of adducts 2 and 3 (Fig. 1) is not known. It is possible that, in the first step of the postlabeling procedure, incomplete digestion of DNA would result in dimers, i.e. dimers beTable Il. Distribution of individual PhIP-DNA p.o. dose of 50 mg PhIP/kg. Organ
adducts in organs and WBCs of the Fischer-344
rat 24 h after a single
% of total adducts Adduct
Liver Lungs Stomach Small intestine Large intestine Kidneys WBCs
tween an adducted nucleotide and an adjacent unmodified nucleotide, but the chromatographic mobilities of adducts 2 and 3 (Fig. 1) would make this an unlikely possibility. Alternatively, the possibility of degradation of the Cs-guanine adduct during the 32P-postIabeling procedure into two additional adducted products may be considered. Three doses of PhIP (0.5, 5.0 and 50.0 mg/kg) were administered, but at 0.5 mg/kg no adducts could be detected in WBCs or in any of the organs. At 5 and 50 mg/kg PhIPDNA adducts were detectable in a dosedependent fashion in WBCs and in all organs, except in the lungs and small intestine, where
64.9 57.7 61.6 19.3 47.4 48.4 31.4
1
Adduct 2
Adduct 3
35.1 8.1 38.4 48.1 29.4 24.5 41.1
0 34.2 0 32.6 23.2 27.1 27.5
LlJm
SlwMH
SUMLM.
LARGEMl.
KIDNEYS
WBC
Fi2.2. Dose-response and organ distribution of PMP-DNA adducts in male Fischer-344 rats given a single oral dose of 50 mg PhIP/kg by gavage. RAL x lo7 values represent the average of two animals, with a coefficient of variation of < 18%. ‘Non-detectable. Int., intestine.
no adducts were detectable at 5 mg/kg (Fig. 2). A most striking finding in this study was the high level of adduct formation in the colon (Fig. 2), the target organ for PhIP in the male Fischer-344 rat [17]. High levels of colon PhIP-DNA adducts were also found after 2 weeks of feeding male Fischer-344 rats with a 0.05% PhIP diet, but under these conditions adduct levels in the lungs, pancreas and heart exceeded those in the colon [29]. In the cynomolgus monkey given daily oral doses of PhIP for 9 days, the highest PhIP-DNA adduct levels were detected in the heart, followed by the liver, salivary gland, pancreas and kidney, with lower levels in the other organs,, including the large intestine [l]. The carcinogenic@ of PhIP in the cynomolgus monkey is under study; to date, no tumors have been detected (see Ref. 1; R.H. Adamson, pers. commun.).
Under identical experimental conditions, IQDNA adduct formation in the male Fischer344 rat is much higher [39,24] than that of PhIP (Fig. 2) and its distribution in the various organs is quite different. IQ-DNA adducts are lowest in the intestines and in WBCs and are highest in the liver [24], the primary site of IQinduced tumor formation [21]. Thus, it appears that, at least for the heterocyclic amines IQ and PhIP, there is a degree of correlation between the extent of DNA adduct formation and target organ specificity. The biological significance of high PhIPDNA adduct levels in the colon (Fig. 2) is underscored by the induction of colonic aberrant crypts, considered preneoplastic lesions, in Fischer-344 rats fed a diet containing 0.05% of PhIP for up to 16 weeks [28]. It appears that a high proportion of orally administered PhIP
123
is excreted in the feces [35] and that a portion of this may be the N-hydroxy-glucuronide of PhIP, formed in the liver [4]. This conjugate may be hydrolyzed by intestinal bacteria [4] to the hydroxylamine which could react with intestinal DNA after esterification/de-esterification [7,12]. Our values of PhIP-DNA adduct levels in rapidly proliferating tissues such as the small- and large intestine represent minimal estimates as no attempt was made to correct adduct levels for differences in rates of cell proliferation. Also, the relatively high levels of PhIP-DNA adducts in WBCs (Fig. 2) indicates that substantial amounts of the ultimate reactive form of PhIP, probably 2%hydroxy-PhIP, are present in the circulation and that this could contribute to adduct formation in the tissues, including the colon. Orally administered PhIP has also been shown to bind to circulating hemoglobin and serum proteins of Fischer-344 rats [36], but the structures of the bound products are not known. The consumption of cooked meat, in particular heavily browned meat, has been shown to increase the relative risk for colorectal cancer [15] and the presence of PhIP and other heterocyclic amines in the urine of humans on normal diets [33], together with the ability of the human colon to activate Nhydroxy-PhIP to DNA binding species [31], indicates that PhIP may be a potential etiologic agent in human colon cancer. Acknowledgments
We thank Dr. Richard H. Adamson of the National Cancer Institute for a generous gift of PhIP. These studies were supported by grant CA47484 from the USPHS (NIH). References 1
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Formation of DNA adducts of Z-amino-l-methyl-6phenylimidazo[4,5-blpyridine (PhIP) in male Fischer-344 rats Herman
A.J. Schut and Christopher
R. Herzog
Department of Pathology, Medical College of Ohio, Toledo, Ohio 43699
(USA)
(Received 3 September 1992) (Accepted 7 September 1992)
Summary Z-Amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) is known to induce colon tumors in male Fischer-344 rats. Using 32Ppostlabeling assays, we have examined PhlPDNA adduct formation in various organs and white blood cells (WBCs) of the male Fischer344 rat 24 h after a single oral dose of 0, 0.5, 5 or 50 mg PhlP/kg. Three PhlP-DNA adducts were detected in WBCs and in all organs, except in the liver and stomach which had only two adducts. The extent of adduct formation was dose-related, but at 0.5 mg/kg no adducts could be detected in any of the organs. At 50 mg/kg, adduct levels, expressed
as relative adduct labeling values (RAL x 107, or adducts per 1 O7 nucleotides assuming complete labeling) were highest in the large intestine (5.661, followed by WBCs (5.04), stomach (1.44), small intestine (1.32), kidney (1.16), liver (0.67) and lungs (0.52). It is concluded that orally administered PhlP forms high levels of specific DNA adducts in the large Correspondence to: Herman A.J. Schut, Department of Pathology, Health Education Building, Room 202, Medical Callege of Ohio, 3000 Arlington Ave., Toledo, Ohio 43614, USA. Abbreviations: AIA, amino-imidazoazaarene; IQ, 2-amino-3methylimidazo[4,5-flquinoline; MeIQx, 2-amino-3,&dimethylimidazo[4,5jjquinoxaline; PhlP, 2-amino-1-methyl-6-phenylimidazo[4,5-blpyridine; WBC, white blood cell; IF, intensification factor; RAL, relative adduct labeling.
0304-3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
intestine, the target organ in PhlP carcinogenesis in the male Fischer-344 rat, and that the high level of adducts in WBCs indicates that significant amounts of the ultimate carcinogenic form ojPhlP are present in the circulation.
Keywords: Z-amino- l-methyl-6-phenylimidazo[4,5_b]pyridine; PhIP; 32P-postlabeling analysis; DNA adducts; colon carcinogenesis Introduction A number of mutagens/carcinogens have been isolated from cooked foods [ 131. Among these the heterocyclic amines, in particular the amino-imidazoazaarenes (AIAs) , have been investigated extensively because they comprise a high proportion of the total mutagenic activity present in fried meat [lo]. All of the heterocyclic amines tested to date have been proven to be carcinogenic [2,21]. AIAs can be formed in vitro by heating amino acids with creatine or creatinine [US], or through Maillard reactions involving reducing sugars, amino acids and creatine or creatinine [l&22,26], yielding 2-amino-3-methylimidazo[4,5-flquinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5flquinoline, 2-amino-3&dimethylimidazo[4,5flquinoxaline (MeIQx), 2-amino-3,n,%triIreland Ltd
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methylimidazo[4,5-flquinoxaline
and 2-amino-
[ 111. It is carcinogenic in rodents, causing lymphomas in both male and female CDFl mice [9] and colon adenocarcinomas in male Fischer-344 rats and mammary adenocarcinemas in female Fischer-344 rats [ 171. While both the in vivo and in vitro mammalian cell mutagenicity of AIAs is considered low, that of PhIP is higher than that of the other AIAs [3,301. The results from several in vitro studies have shown that PhIP is activated to its ultimate carcinogenic form by N-hydroxylation [6,16, 19,32,34], a reaction catalyzed by a polycyclic aromatic hydrocarbon-inducible form of microsomal cytochrome P450, principally isozymes IA1 and IA2 [16,19,32,34]. Further esterification/deesterification of N-hydroxyPhIP by sulfotransferase or 0-acetylansferase 27,121 yields the ultimate reactive form, probably the nitronium ion [37], which reacts with DNA to form specific adducts. The formation of PhIP-DNA adducts has been shown in vitro [7] as well as in vivo [27,29,38], using 32Ppostlabeling methods. In vitro, the major adduct formed between N-hydroxy-PhIP and DNA is N*-(2 ‘-deoxyguanosin8yI) -PhIP [ 121. In the present study we have investigated the in vivo formation of PhIP-DNA adducts in the male Fischer-344 rat.
Materials and Methods Animal treatment
and DNA isolation adult male Fischer-344 rats Young (200 - 240 g) were obtained from Harlan Sprague - Dawley (Indianapolis, IN). PhIP was a generous gift from Dr. Richard H. Adamson, National Cancer Institute. Rats (2 per dose) were treated with a single p.o. (gavage) dose of PhIP (0,0.5,5 or 50 mg/kg) using dimethyl sulfoxide-0.05 M sodium phosphate, pH 4.5 (1:3) as the vehicle. The PhIP solution was prepared immediately
before administration and was kept at 37OC to prevent the formation of a gel which occurred at room temperature. Rats were killed 1 day after PhIP administration by first administering a heparin solution (5000 units/kg, i.p.), followed after 15 min by sodium pentobarbital solution (180 mg/kg, i.p.). After complete anesthesia (approximately 5 min) the abdomen was opened and blood was collected from abdominal veins (5 - 7 ml) into lithium heparin tubes. After mixing the blood with 45 ml of Iysis buffer (0.83% ammonium chloride , 0.1% potassium bicarbonate, 0.04 % tetrasodium EDTA, pH 7.3) and leaving the mixture at room temperature for 8 min, the white blood cells (WBCs) were isolated by centrifugation at 3000 x g for 8 min. The supernatant was discarded and the pellet (WBC) was resuspended in 2 ml of nuclei Iysis buffer (10 mM Tris - HCI, 400 mM sodium chloride, 2 mM tetrasodium EDTA, pH 8.2). After removal, the liver, lungs and kidneys were chopped into small pieces (<2 mm*) and a random sample of approximately 0.5 g was taken for DNA isolation. The stomach and the intestines were removed, cut open and rinsed with phosphate-buffered saline containing 1 mM EDTA to remove their contents. The epithelial layer was scraped off from the stomach, the entire small intestine and from the large intestine, including the cecum. Aliquots of 0.5 g were used for isolation of DNA after suspension and homogenization in 5 ml of nuclei lysis buffer. DNA was then isolated from WBCs and the organs by a direct salt precipitation procedure as described by Miller et al. 1201, except that the final DNA pellet was redissolved in 1 ml of 1.5 mM sodium chloride, 0.15 mM sodium citrate (0.01 x SSC) and then incubated with RNase A (100 pg/mI) and RNase Tl (50 units/ml) at 37OC in a shaking water bath for 1 h. After addition of 2 ml of 0.01 x SSC and 3 ml of phenol-chloroform-isoamyl alcohol (25:24: 1) the mixture was vortexed, centrifuged at 4OC for 10 min at 3000 x g, and the aqueous (top) layer was extracted once more with chloroform-isoamyl alcohol (24: 1). DNA was
119
precipitated described measured absorbance absorbance
from the aqueous (top) layer as [ZO] and its concentration was spectrophotometrically using its at 260 nm and a value of 20 A260 units/mg DNA.
32P-postlabe hn * g assay of PhlP-DNA adducts thin-layer Polyethyleneimine-cellulose plates were prepared in the laboratory as described by Gupta et al. [14]. After overnight washing the freshly prepared plates were stored at 4OC. [y-32P]ATP (approximately 4000 Ci/mmol) was freshly prepared for each postlabeling assay [ 141. The intensification version of the 32P-postlabeling assay [23] was used to quantitate PhIP-DNA adducts, as previously described for adducts between DNA and IQ [25,39]. For the determination of the intensification factors (IFS) of the individual PhIP-DNA adducts, the actual (true) adduct leveb were determined by the standard 32Ppostlabeling assay [ 141. In the intensification procedure the amounts of DNA and [32P]ATP were carefully kept constant in each assay, as the ratio of their concentrations determines the IF of the individual PhIP-DNA adducts [23] and, consequently, the accuracy of adduct quantitation. 32P-labe!ed nucleoside bisphosphates were purified on polyethyleneimine-cellulose plates as described before [39], except for modification of the D4 solvent which consisted of 0.8 M lithium chloride, 0.5 M Tris- HCI, 7.4 M urea, pH 8.0. 32P-labeled PhIP-DNA adducts were located on the plates by autoradiography, exposing films at - 70°C for varying periods of time. Radioactive spots (adducts) were cut out and quantitated by Cerenkov counting [ 141. The extent of adduct formation was expressed as relative adduct labeling (RAL) values which were calculated by dividing the counts/min per adduct by the sum of the counts/min in the adduct and the normal nucleotides, making adjustments for dilution and aliquot factors [ 141. < RAL > values obtained under intensification conditions were converted to the actual RAL values (standard conditions), using the IFS for each adduct.
Results and Discussion Three PhIP-DNA adducts could be detected in WBCs and in all organs, except in the liver and stomach which showed only adducts 1 and 2 (Fig. 1). Vehicle-treated animals showed no detectable adducts (Fig. 1 h). The pattern of PhIP-DNA adducts obtained (Fig. 1) was similar to that obtained in the liver of the cynomolgus monkey after repeated gavages (daily for 9 days) with PhIP [27] and in the liver of the Fischer-344 rat after a single oral dose of PhIP [38]. In vitro incubation of Nhydroxy-PhIP with DNA in the presence of mouse liver cytosol and either acetyl coenzyme A or 3’-phosphoadenosine 5’-phosphosulfate also yielded a similar pattern of adducts [7]. Feeding Fischer-344 rats with a diet containing 0.05% PhIP for 2 weeks resulted, however, in a much more complex PhIP-DNA adduct pattern [29], with seven adducts in all organs examined. Therefore, it appears that longterm continuous feeding of the Fischer-344 rat with PhIP results in the formation of additional, i.e. more than three, adducts which are possibly resistant to removal under these experimental conditions. IFS of the three PhIP-DNA adducts varied from 96.1 to 47.1 (Table I). These values are distinct from those determined for other, structurally related, heterocyclic amines such as IQ [25] or MeIQx [8], a finding emphasizing the different affinity of the polynucleotide kinase for structurally different adducts. IFS are also strongly influenced by the relative concentrations of deoxyribonucleoside 3’-monophosphates and [‘y-32P]ATP, which are likely to be different in different laboratories [29]. In most organs adduct 1 was the major adduct, although substantial amounts of adducts 2 and 3 were also present (Table II). Following 9 days of oral administration of PhIP to a cynomolgus monkey, a similar distribution of PhIP-DNA adducts was observed in all tissues examined, including the liver, stomach, small intestine, large intestine, kidney, spleen, pancreas, heart, salivary gland and brain [l]. Using physico-chemical methods, only a
120
_(
0
121
Table 1. Determination of IFS for PhlP-DNA and standard labeling conditions, respectively”.
adducts from < RAL > and RAL values obtained
PhlP-DNA adduct
RALC (x 105) (mean S.D., N = 4)
IF
1 2 3
36.51 zt 5.12 18.99 zt 3.10 15.53 zt 3.18
0.38 zt 0.009 0.20 f 0.10 0.33 f 0.10
96.1 95.0 47.1
under intensification
( < RAL > /RAL)
’ IFS determined on DNA from R52-16 cells, transduced with cytochrome P450IA2 DNA [29], exposed to 100 PM PhlP for 24 h. b< RAL> determined under intensification conditions. The DNA digest (deoxyribonucleoside 3’monophosphates equivalent to 3.75 pg DNA) was labeled with 225 FCi [y-32P]ATP (S.A. approx. 4000 Ci/mmol) in a volume of 15.5 pl as described [26] to give a final [Y-~~P]ATP concentration of 3.8 PM. ’ RAL determined under standard conditions. The DNA digest (deoxyrtbonucleoside 3’-monophosphates equivalent to 0.17 pg DNA) was labeled with 250 PCi [-r-32P]ATP (S.A. approx. 4000 Ci/mmol) together with unlabled ATP, in a volume of 10.0 ~1 as described [25,26] to give a final [Y-~~P]ATP concentration of 72.0 PM.
single adduct, N’-(Z’-deoxyguanosin-SyI)PhIP, has been identified after in vitro reaction of IV-hydroxy-PhIP with DNA in the presence of acetic anhydride, or in rat liver in vivo after i.p. administration of [3H]PhIP [El. The reason for this discrepancy with postlabeling methodology is not known, but preliminary evidence (E.G. Snyderwine and H.A.J. Schut, unpublished observations) indicates that adduct 1 (Fig. 1) may be the Cs-guanine adduct identified by Frandsen et al. [ 121. The nature of adducts 2 and 3 (Fig. 1) is not known. It is possible that, in the first step of the postlabeling procedure, incomplete digestion of DNA would result in dimers, i.e. dimers beTable Il. Distribution of individual PhIP-DNA p.o. dose of 50 mg PhIP/kg. Organ
adducts in organs and WBCs of the Fischer-344
rat 24 h after a single
% of total adducts Adduct
Liver Lungs Stomach Small intestine Large intestine Kidneys WBCs
tween an adducted nucleotide and an adjacent unmodified nucleotide, but the chromatographic mobilities of adducts 2 and 3 (Fig. 1) would make this an unlikely possibility. Alternatively, the possibility of degradation of the Cs-guanine adduct during the 32P-postIabeling procedure into two additional adducted products may be considered. Three doses of PhIP (0.5, 5.0 and 50.0 mg/kg) were administered, but at 0.5 mg/kg no adducts could be detected in WBCs or in any of the organs. At 5 and 50 mg/kg PhIPDNA adducts were detectable in a dosedependent fashion in WBCs and in all organs, except in the lungs and small intestine, where
64.9 57.7 61.6 19.3 47.4 48.4 31.4
1
Adduct 2
Adduct 3
35.1 8.1 38.4 48.1 29.4 24.5 41.1
0 34.2 0 32.6 23.2 27.1 27.5
LlJm
SlwMH
SUMLM.
LARGEMl.
KIDNEYS
WBC
Fi2.2. Dose-response and organ distribution of PMP-DNA adducts in male Fischer-344 rats given a single oral dose of 50 mg PhIP/kg by gavage. RAL x lo7 values represent the average of two animals, with a coefficient of variation of < 18%. ‘Non-detectable. Int., intestine.
no adducts were detectable at 5 mg/kg (Fig. 2). A most striking finding in this study was the high level of adduct formation in the colon (Fig. 2), the target organ for PhIP in the male Fischer-344 rat [17]. High levels of colon PhIP-DNA adducts were also found after 2 weeks of feeding male Fischer-344 rats with a 0.05% PhIP diet, but under these conditions adduct levels in the lungs, pancreas and heart exceeded those in the colon [29]. In the cynomolgus monkey given daily oral doses of PhIP for 9 days, the highest PhIP-DNA adduct levels were detected in the heart, followed by the liver, salivary gland, pancreas and kidney, with lower levels in the other organs,, including the large intestine [l]. The carcinogenic@ of PhIP in the cynomolgus monkey is under study; to date, no tumors have been detected (see Ref. 1; R.H. Adamson, pers. commun.).
Under identical experimental conditions, IQDNA adduct formation in the male Fischer344 rat is much higher [39,24] than that of PhIP (Fig. 2) and its distribution in the various organs is quite different. IQ-DNA adducts are lowest in the intestines and in WBCs and are highest in the liver [24], the primary site of IQinduced tumor formation [21]. Thus, it appears that, at least for the heterocyclic amines IQ and PhIP, there is a degree of correlation between the extent of DNA adduct formation and target organ specificity. The biological significance of high PhIPDNA adduct levels in the colon (Fig. 2) is underscored by the induction of colonic aberrant crypts, considered preneoplastic lesions, in Fischer-344 rats fed a diet containing 0.05% of PhIP for up to 16 weeks [28]. It appears that a high proportion of orally administered PhIP
123
is excreted in the feces [35] and that a portion of this may be the N-hydroxy-glucuronide of PhIP, formed in the liver [4]. This conjugate may be hydrolyzed by intestinal bacteria [4] to the hydroxylamine which could react with intestinal DNA after esterification/de-esterification [7,12]. Our values of PhIP-DNA adduct levels in rapidly proliferating tissues such as the small- and large intestine represent minimal estimates as no attempt was made to correct adduct levels for differences in rates of cell proliferation. Also, the relatively high levels of PhIP-DNA adducts in WBCs (Fig. 2) indicates that substantial amounts of the ultimate reactive form of PhIP, probably 2%hydroxy-PhIP, are present in the circulation and that this could contribute to adduct formation in the tissues, including the colon. Orally administered PhIP has also been shown to bind to circulating hemoglobin and serum proteins of Fischer-344 rats [36], but the structures of the bound products are not known. The consumption of cooked meat, in particular heavily browned meat, has been shown to increase the relative risk for colorectal cancer [15] and the presence of PhIP and other heterocyclic amines in the urine of humans on normal diets [33], together with the ability of the human colon to activate Nhydroxy-PhIP to DNA binding species [31], indicates that PhIP may be a potential etiologic agent in human colon cancer. Acknowledgments
We thank Dr. Richard H. Adamson of the National Cancer Institute for a generous gift of PhIP. These studies were supported by grant CA47484 from the USPHS (NIH). References 1
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