Metabolism of the food mutagen 2-amino-3-methylimidazo [4, 5-f

Bethesda, Maryland 20892, and Nestis Research Centre, Nestec Ltd., P.O. Box 44, ... carcinogen which also produces hepatocellular carcinoma in monkeys...
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Chem. Res. Toxicol. 1992,5, 843-851

843

Metabolism of the Food Mutagen 2-Amino-3-methylimidazo[4,5-flquinoline in Nonhuman Primates Undergoing Carcinogen Bioassay Elizabeth G. Snyderwine,*JDieter H. Welti,t Lament B. Fay,$ Hans Peter Wiirzner,f and Robert J. Tureskyt Laboratory of Experimental Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892,and Nest16 Research Centre, Nestec Ltd., P.O. Box 44, Vers-chez-les-Blanc, CH-1000Lausanne 26,Switzerland Received June 17,1992

2-Amino-3-methylimidazo[4.5-flquinoline (IQ)is a potent bacterial mutagen and rodent carcinogen which also produces hepatocellular carcinoma in monkeys. The metabolism and disposition of this procarcinogen were investigated in monkeys undergoing carcinogen bioassay and in monkeys given an acute dose of I&. Analysis of urine, feces, and bile revealed that I& was extensively metabolized. A number of metabolites in urine were purified by high-performance liquid chromatography and characterized by 'H NMR and mass spectroscopy. Metabolites resulted from cytochrome P450-mediated ring oxidation a t the C-5 position or N-demethylation. These metabolites could be further transformed by conjugation to sulfate or 8-glucuronic acid. Glucuronidation and sulfamate formation a t the exocyclic amine group were other major routes of metabolism. Enteric bacteria also contributed to I&biotransformation by forming the 7-oxo derivatives of I& and N-demethyl-IQ. The metastable W-glucuronide conjugate of the carcinogenic metabolite, 2-(hydroxyamino)-3-methylimidazo[4,5-flquinoline, was found in urine. This indicates that metabolic activation through cytochrome P450-mediated N-oxidation occurs in vivo and that glucuronidation is a means of transport of the carcinogenic metabolite to extrahepatic tissues. Introduction Diet is an important factor in the occurrence of human cancers (11. The identification of mutagenic/carcinogenic heterocyclic aromatic amines (HAA)l in meat and fish cooked by ordinary methods (2-4)has led to the hypothesis that nutritionally linked cancers common in the western world, such as colorectal and breast cancers, could be associatedwith exposure to these compounds (5,6). More than a dozen mutagenic HAAs have been identified in cooked beef and fish at the low parts per billion level (24). 2-Amin~3-methylimid[4,5flquinoline(I&)is among one of the first HAAs identified in these staples (7). When administered as part of the diet to rodents, I&produced tumors at multiple sites including the liver, large and small intestine, lung, breast, and clitoral and Zymbal's glands (8). Other HAAs also cause multisite tumors in rodents (8).In a recent study in nonhuman primates, I&was found to be a potent liver carcinogen (9). Thus, despite the occurrence of HAAs in only trace amounts, their presence in many daily staples suggests that human exposure can be significant and HAAs may be involved in the etiology of human cancers. * Correspondenceshould be addressedto this author at the Laboratory of Experimental Carcinogenesis, Division of Cancer Etiology, Building 37, Room 3C28, National Cancer Institute, Bethesda, MD 20892. + National Cancer Institute. 8 Nestec Ltd. Abbreviations: HAA,heterocyclic aromatic amines; IQ, 2-amino-3methylimidazo[4,5-flquinoline; N-hydroxy-IQ, 2-(hydroxyamino)-3methylimidazo[4,5-flquinoline; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-flquinoxaline; PhIP, 2-amino-l-methyl-6-phenylimidazo[4,5-blpyridine; NOE, nuclear Overhauser effect; COSY, homonuclear correlation NMR spectroscopy; FAB, fast atom bombardment; DMSO, dimethyl sulfoxide; TMS, tetramethylsilane;HPC, (hydroxypropy1)cellulose.

Metabolic processing is an important factor in determining the carcinogenicity of many genotoxins. I& is a procarcinogen that requires metabolic activation to exert its genotoxicity and form DNA adducts (10-12). Experimental studies using liver preparations from a number of species including rodents and humans have shown that metabolic activation of HAAs occurs primarily via cytochrome P450 1A22-mediated N-oxidation to form the mutagenic arylhydroxylamines (12-17). The major DNA adduct formed from the reaction of N-hydroxy-I&with DNA has been identified as N-(deoxyguanosin-8-yl)-IQ (18). This adduct has also been identified by the 32Ppostlabeling method in both rodent and monkey tissues, suggesting that metabolic activation through N-oxidation also occurs in vivo (19,20). The major routes of I& biotransformation have been elucidated in the rat. Cytochrome P450-mediated ring oxidation at the 5 position of I& followed by conjugation to sulfate or @-glucuronicacid is representative of two major routes of detoxification (21). Direct conjugation to the exocyclicamine group through W-glucuronidationand sulfamate formation is representative of other important routes of inactivation in rats (21,221.Although metabolic activation of I& through N-oxidation has been demonstrated with rat, human, and monkey liver microsomes (11,12,17,23), neither N-hydroxy-IQ nor its metastable metabolites have been detected in biological fluids of animals (21,221.In the light of possible species differences in metabolism between rats and monkeys, and the phylogenetic association of the monkeys to man, we have examined the in vivo metabolic processing of I& in cynomolgus monkeys which are undergoing carcinogen For cytochrome P450 nomenclature, see ref 45.

This article not subject to U.S.Copyright. Published 1992 by the American Chemical Society

844 Chem. Res. Toxicol., Vol. 5, No. 6,1992

bioassay (9). I& metabolites were identified in urine, bile, and feces of monkeys given a carcinogenic dose regimen of I&,and also in urine and feces of monkeys given a single acute dose of I&. The results of this study reveal both similarities and important differences between rodents and monkeys in the metabolism of I&. In addition, the metastable N-glucuronide conjugate of the carcinogenic metabolite N-hydroxy-IQ, a metabolite not reported in rats, was detected in urine of monkeys and demonstrates that glucuronidation participates in the transport of the carcinogenic metabolite to extrahepatic tissues. Our results confirm that metabolic activation through N-oxidation occurs in vivo and support the role of N-hydroxyI&in the hepatocellular carcinogenicityof I& in nonhuman primates.

Materials and Methods Materials. Caution: IQ is carcinogenic to rodents and nonhuman primates and should be handled carefully. [2-14C]IQ (56 mCi/mmol, >98% radiochemical purity) was purchased from Chemsyn Science Laboratories (Lenexa, KS). Unlabeled IQ was purchased from the Nard Institute, Ltd. (Osaka, Japan). @-Glucuronidasetype VI1 and arylsulfatase type VI11 (from abalone entrails) were obtained from Sigma Chemical Co. (St. Louis, MO). Solvable tissue solubilizer and Formula 989 and Aquasol scintillation cocktailswere purchased from New England Nuclear-Dupont (Boston, MA). Spectroscopy. 1H NMR spectra were obtained with a 360MHz Bruker spectrometer with a 5-mm lH probehead. Samples were prepared under an Nz atmosphere in a glove box in DMSOde using TMS as an internal standard. One-dimensional spectra, nuclear Overhauser effect difference spectra, and COSY measurements were performed as previously described (24). I t is important to note that the spectra of severalmetabolites exhibited significant sensitivity to the final solvent conditions or pH. In some instances spectra were acquired twice, with ammonium acetate buffer or water and methanol as final HPLC solvents. Mass spectra were acquired with a Finnigan/MAT 8430/SS300 mass spectrometer. E1 spectra were obtained by direct sample introduction at 70 eV with an EI/CI source at 180 "C. Positive and negative ion FAB-MS were obtained with the same source at 60 OC using diethanolamine or glycerol as a matrix. Accurate mass measurements were performed with a resolution of about 3000. Perfluorokerosenewas the referencecompound in E1 mode, and the matrix was the reference in the FAB mode. Chemical Standards and Syntheses. 7-Oxo-IQ was kindly provided by Dr. R. L. Van Tassel1 (Department of Anaerobic Microbiology,Virginia PolytechnicInstitute and State University, Blacksburg, VA). IQ-sulfamate was prepared by reaction of the parent amine with chlorosulfonic acid as previously described (22). W-Acetyl-IQ was prepared by reaction of IQ in pyridine containing 20% acetic anhydride (17). N-Demethyl-IQ was a generous gift from Dr. S. R. Tannenbaum (Department of Chemistry, MIT, Cambridge, MA). Its synthesis was previously described (25). The lH NMR spectrum showed there was tautomerism which led to occurrence of double signals with an integral ratio of 0.3:0.7 on the H-5 and H-7 and on the D2Oexchangeable NH and NHz resonances. This tautomerism was not further investigated ['H NMR, 6.23 ppm (ca. 0.6 H, s br, NHz), 6.33 (ca, 1.4 H, s br, NH2), 7.42 (1H, dd, J 7 , 8 4.1 Hz, J 8 , g 8.3Hz, H-8), 7.51 (ca. 0.7 H, d, H-5), ca. 7.59 (ca. 0.3 H, br, H-51, 7.64 (1 H, d, 5 4 5 8.7 Hz, H-4) 8.53 (1H, ddd, J 7 , g = 1.8 Hz, J 5 , g 0.7 Hz, H-9), 8.68 (ca. 0.3 H, br, H-7), 8.70 (ca. 0.7 H, br, H-7), 11.16 (0.7 H, s br, NH), 11.86 (0.3 H, s br, NH); MS m/z obsd 184.0749,calcd 184.07481. Nitro-IQ was synthesized as described by Grivas for the synthesis of the nitro derivative of MeIQx (26) ['H NMR, 4.27 ppm (3 H, s, 3-CH3), 7.75 (1H, dd, 57.8 4.3 Hz, JB,g8.3Hz,H-8),8.14(1H,dd,J4,59.3H~,J5,90.7H~,H-5),8.24 (1H, d, H-4), 8.88 (1H, ddd, 57.9 1.8 Hz, H-9),9.00 (1H, dd, H-7); MS mlz obsd 228.0647, calcd 228.06531. N-Hydroxy-IQ was prepared by reduction of the nitro derivative with ascorbic acid

Snyderwine et al. (17) and was characterized by mass spectrometry following reaction with nitrosobenzene (24). Animals and Treatments. Cynomolgus monkeys (Macaca fascicularis) and rhesus monkeys (Macaca mulatta) born in a closed colony and mother-reared were used for this study. Animals were housed in an AAALAC-accredited facility at Hazleton Laboratories,VA, under contract to the National Cancer Institute and in compliance with "The Guide for the Care and Use of Laboratory Animals". The protocol was reviewed and approved by the NIH Animal Care and Use Committee. Details on the housing and diet have been described previously (9). Male Sprague-Dawley rats (250-300 g) were obtained from Iffa Credo, L'Arbresle (France). Biliary metabolites were obtained from rodents given an ip injection of IQ (20 mg/kg) as previously described (22). The sulfamate and the 5-0-glucuronide and 5-sulfate conjugates were purified by the HPLC conditions describedbelow. These biliary metabolites were spectroscopically compared to metabolites obtained from monkeys. Blood Levels and Excretion of [WIIQ. Blood levels and excretion of radioactivity following [I4C]IQadministration were examined at two dosages. Under the first dosage protocol, two naive female cynomolgus monkeys received a single dose of 2.2 pmol/kg [14C]IQ (120 pCi) weight dissolved in 5 mL of distilled water by gavage. These monkeys were 2 l/2 years old and weighed 2.5 and 2.3 kg. Under the second dosage protocol one female cynomolgus monkey (5.2 kg, 6 l/z years old) was given IQ chronically for carcinogenicitytesting [5 days per week at 10 mg (50 pmol)/kg in HPC] for 5 years as previously described (9)and then received [14C]IQ(50 pmol(10 pCi)/kg in 1mL of HPC by gavage). The monkey did not have tumors at the time of study and was in good health (as assessed by the parameters described in ref 9). The cumulative dose of IQ given up to the time of [14C]IQ administration was 47.4 g. Twenty-four hours after administration of radiolabeled I&,the monkey resumed its dosing with unlabeled IQ (10mg/kg). Monkeys dosed with [WIIQ were housed in metabolism cages; urine and feces were collected as voided at 1, 3, 8, 24, 48, and 72 h. Urine was collected on wet ice and both urine and feces were stored at -70 "C prior to metabolite analysis. Blood samples (2 mL) were obtained from the femoral vein at these same time intervals. Monkeys received IQ in the morning prior to breakfast and resumed normal diets after dosing. All radioactivity measurements were done by liquid scintillation counting using a Beckman LS5000TD counter programmed for quench corrections. Radioactivity present in urine (50-200 pL) was measured in Aquasol following addition of 1mL of distilled water. Feces were homogenizedand assayed in triplicate (20-50 mg). Samples of blood (25-150 pL) and feces were dissolved in Solvable and decolorized with 30% hydrogen peroxide according to the technical information provided by Dupont. Formula 989 was added to dissolved blood and fecal samples 24 h prior to scintillation counting to allow sufficient time for chemiluminescence to decay. Purification of Urinary Metabolites for Spectroscopic Characterization. Overnight urine was collected from four cynomolgus monkeys (2 males and 2 females) that were being treated chronically with IQ at 10 mg/kg, 5 days per week (for 5 years), for carcinogen bioassay. The monkeysdid not have tumors at the time this metabolism study was carried out. Urine was concentrated from approximately 1000 mL to 30 mL using a rotary evaporator at 37 "C. The concentrate was then spiked with 5 mL of urine from a [14C]IQ-treatedmonkey. Particulates were removed by centrifugation (5000g, 10 min) and filtration (0.45 pM). Initial fractionation of metabolites was performed with a Waters HPLC system (18)(MilliporeCorp., Milford, MA). Step 1: Urinary metabolites were fractionated with a Waters Bondapak C18 preparative column (19 mm X 150 mm, 10-pm particle size). The solvent conditions were 99% 100 mM ammonium acetate (pH 5.8) and 1%methanol. The concentration of methanol was increased to 100% at 60 min using a convex curve (number 07), and the flow rate was 4 mL/min. Metabolite fractions were then pooled and concentrated by rotary evaporation. Step 2: Metabolites were further purified on a Supelco C18 reverse-phase column (4.6 mm i.d. X 25 cm, 5-pm

Metabolism of IQ in Monkeys particle size) (Supelco, Inc., Bellefonte, PA). The solvent conditions were isocratic from 0 to 15 min at 91% 50 mM ammonium acetate (pH 7.5) and 9 % methanol. The percentage of methanol was increased linearly to 18% at 45 min, to 90% at 55 min, and to 100% at 60 min. The flow rate was 1 mL/min throughout the run. Metabolite fractions were collected and evaporated to dryness. SubsequentHPLC purification steps 3-6 were performed with a Hewlett Packard 1090M system employing a diode array detector at 264 nm. Step 3: Metabolites were resuspended in water or a minimal volume of DMSO and then further purified using the Supelco C18 reverse-phase column described above. The solvent conditions were isocratic at 90% 50 mM ammonium acetate (pH 5.8) and 10% methanol for 10 min. The percentage of methanol was increased linearly to 16% at 45 min, to 90% at 60 min, and to 100% at 70 min. The flow rate was 1 mL/min throughout the run. Step 4: The metabolites were subjected to HPLC using an LC-NH2column (Supelco, 4.6 mm i.d. X 25 cm, 5-pm particle size) in a normal-phase mode. All samples were resuspended in DMSO. The solvent conditions were isocratic at 90% acetonitrile and 10% 50 mM ammonium acetate (pH 6.8) for 10 min, which then increased linearly to 100% ammonium acetate at 40 min at a flow rate of 1mL/min. Step 5: Metabolites were run on the Supelco C18 reverse-phase column using a linear gradient from 10% to 100% methanol in 50 mM ammonium acetate (pH 6.8) over 40 min at 1 mL/min. Step 6: A further purification using a linear gradient from 10%to 100% methanol in water over 20 min was done for IQ-sulfamate, W-glucuronides, and ring-hydroxylatedglucuronic or sulfuric acid conjugates.The bacterial derived metabolites were purified with this final gradient system employing 50 mM ammonium acetate (pH 6.8) instead of water. Metabolites were placed under vacuum (0.02 mbar) and then characterized by proton NMR and mass spectroscopy. Purification of Biliary Metabolites. Bile was obtained upon necropsy from three monkeys (one female cynomolgus, one male rhesus, one female rhesus) that developed hepatocellular carcinoma following chronic IQ treatment (20 mg/kg, 5 days per week), with the final dose of IQ being administered 2 h prior to death (9). The bile was added to 3 volumes of ethanol and placed on ice for 30 min. The precipitated proteins were removed by centrifugation at 15000g for 10 min, and the supernatant was concentrated by rotary evaporation. The metabolites were then purified by HPLC steps 4-6 described above. Analysis of Fecal Metabolites. Three grams of feces were homogenized in 25 mL of distilled HzO. The mixture was centrifuged at 5000g for 10 min and the pellet extracted once more with water. This was followed by extraction of the pellet with 50:50methanol/water (twice)and then with 100% methanol (twice). Approximately 30-40 % of the radioactivity in feces could be recovered by this procedure. Further extraction of the fecal pellet with methanol and/or water recovered very little additional radioactivity. The pooled supernatants were concentrated by rotary evaporation and analyzed by two HPLC conditions outlined in steps 2 and 3 above. Complete spectral analyses were performed on urinary metabolites and the biliary 5-0-glucuronide and 5-sulfate conjugates. Other biliary and fecal metabolites were characterized by UV spectroscopy, by HPLC comigration with purified urinary metabolites or synthetic standards, and by chemical and enzymatic hydrolysis experiments. Enzyme and Chemical Hydrolysis Assays. Sensitivity of metabolites toward @-glucuronidaseand arylsulfatase was tested as previously described, and the produds were analyzed by HPLC (24). Metabolites were also subjected to acid hydrolysis by incubation in 1 N HCl at 60 "C for 2 h. Mutagenicity Testing. 7-Oxo-IQ and N-demethyl-7-0~0IQ were tested for mutagenicity by the Ames assay, modified to include a preincubation step, using Salmonella typhimurium TA98 strain with or without liver S9 fraction from Aroclor-1254treated rats (10). Up to 10 pg of N-demethyl-7-oxo-IQ and 2.5 pg of 7-oxo-IQ (50 and 12.5 nmol equiv of IQ) were assayed for mutagenicity.

Chem. Res. Toxicol., VoZ. 5, No. 6, 1992 846

A. Monkey 1 Monkey 2

0

0

-----

f! 1.0 m

E

0,5-\

-

13 8

24

48

72

TIME (Hours)

0 0

d

'I

20

13 8

24

48

72

TIME (Hours)

Figure 1. [14C]IQ absorption and blood levels of radioactivity following administration of the acute dose of 2.2 pmol/kg (A) or chronic dose of 50 pmollkg (B). Each monkey is represented individually.

Results Blood Levels,Excretion,and MetabolicProcessing. I&is rapidly adsorbed from the gastrointestinal tract of monkeys. As shown in Figure 1, peak blood levels of radioactivity were observed within 1-3 h after administration of 14C-labeled I&. Blood clearance was rapid, and there appeared to be little binding of reactive I& metabolites to blood proteins 72 h postexposure. Less than 0.15% of the dose remained in the blood at this time. A similar observation was made in the rodent, where blood protein binding of I& was also quite low (27). Similar excretion patterns were observed for both dosages. Urine was the primary route of excretion, accounting for 5 0 4 0 % of the dose within 72 h, while elimination through feces accounted for approximately 10% of the dose (Figure 2). A rapid excretion of I& and ita metabolites in urine occurred within the first 24 h and accounted for 90% of the total radioactivity excreted. In contrast to the results with monkeys which showed that approximately 60-70 % of the dose was eliminated in urine and feces by 72 h, studies in rata showed that up to 90% of a dose of I&can be recovered in urine and feces within 24 h (27). The metabolism of I& in monkeys is extensive. Less than 3% of the dose given at 2.2 pmollkg was excreted in urine as the unmetabolized compound. The identity of unmetabolized I& was confirmed by HPLC comigration with synthetic I& and by ita UV spectrum which was indistinguishable from that of IQ. The E1 mass spectra for both the isolated product and IQ were identical (data not shown). The HPLC analysis of urinary metabolites is presented in Figure 3. Nine metabolites were spectroscopically characterized in urine or bile of monkeys, and their structures are shown in Figure 4.

846 Chem. Res. Toxicol., Vol. 5, No. 6,1992

Snyderwine et al.

PERCENT "%IO DOSE EXCRETED IN MONKEYS AFTER 2.2pmol/kg ( 0 )AND 50pmolikg ( 0 )

H

W

H

N-deme~yl-lQ-h"-glucuronide 12)

IQ-h'-glucuronide

13)

dbC

]Feces I

OS&-

I

24

IQ-5-0-glucuronide (41

72 151

N-hydroxy-lQ-h'.glucuronide (61

IQ-5sulfate I71

N-demethyl-7-oxo-lQ (81

7-oxo-IQ 191

N-demethyl-lQ 1101

IQ-sulfamate

TIME (Hours) AFTER DOSING

Figure 2. Excretion of [14C]IQ in urine and feces of monkeys given the acute dose of 2.2 pmol/kg (mean of two monkeys) or chronic dose of 50 pmollkg (one monkey). 0 H

D

tt I II; 2

,A 10

4111

9

B.

8

L 10

20

9

A

30 40 50

60

TIME (min)

Figure 4. Structuresof fully identified I&metabolites found in urine, bile, and feces of monkeys. Table I. Percent of IQ and IQ Metabolites Excreted in Urine of Three Cynomolgus Monkeys over a 24-h Period dose of IQ (umol/kg)a metabolite no.6 50 2.2 2.2 metabolite 1 1 2.5 4.4 2.5 N-dem-IQ-N-gluc 2 5.0 7.2 20.3 IQ-N-gluc 3 6.8 4.8 3.9 IQ-5-O-gluc 4 9.5 13.8 7.3 IQ-sulfamate 5 5.6 18.6 25.9 N-OH-IQ-N-gluc 6 3.2 1.2 1.5 IQ-5-sulfate 7 23.6 14.4 13.4 N-dem-7-oxo-IQ 8 4.4 2.0 4.1 7-0xo-IQ 9 6.5 3.4 6.0 N-dem-IQ 10 14.8 9.1 11.0 5.3 2.7 2.8 IQ

Figure 3. HPLC profile of IQ metabolites in urine of monkeys given the acute dose of 2.2 pmollkg (A) or chronic dose of 50 pmol/kg (B).The urine examined was voided over a 24-h period. The HPLC conditions are those described under step 3 in Materials and Methods. Approximately the same amount of radioactivity was injected in panels A and B. The numbers correspond to IQ metabolites whose structures are shown in Figure 4.

a One monkey received 50 umc x P4CIIQafter having receive a total cumulative dose of 4714 g of @for c&cinogenicit~bioassay. This monkey was not tumor-bearing at the time of this study. Two other monkeys received a single dose of 2.2 pmol/kg [14C]IQ. All three were female cynomolgus monkeys. The percentage of each metabolite was determined by HPLC. b The metabolite number corresponds to the structures shown in Figure 4.

Six of the metabolites found in urine were also detected in bile including the following: IQ-sulfamate, IQ-Wglucuronide and N-demethyl-IQ-W-glucuronide, and the 5-sulfate and the 5-0-glucuronideconjugates of 5-hydroxyIQ and N-demethyl-7-oxo-IQ. Notably, neither 7-oxo-@ nor N-hydroxy-IQ-W-glucuronide was found in bile. However, the effect of hepatocellular carcinoma on the metabolism in monkeys assayed for biliary metabolites is not known. IQ-sulfamate, 'I-oxo-IQ, and N-demethyl-7oxo-IQ were the three metabolites which survived passage through the intestinal tract and were identified in feces of healthy monkeys. Approximately 5 5% of the radioactivity recovered in feces was attributed to I&,which arose from either the bacterial cleavage of phase I1 conjugates or from incomplete absorption of the administered compound. Other biliary metabolites appeared to be either reabsorbed during passage through the intestinal tract or further transformed by the bacterial flora in the gut to fecal bound material.

Mutagenicity testing showed that N-demethyl-7-0~0IQ was not mutagenic in the presence or absence of S9 activation (data not shown). In our hands, 7-oxo-IQ was mutagenic only in the presence of the S9 fraction (mutation assays were done independently in two different laboratories), which is in contrast to results previously reported where this metabolite was found to be a direct-acting mutagen (28). Table I shows the distribution of metabolites found in urine from two previouslyuntreated female monkeys given IQ a t 2.2 pmol/kg and one monkey undergoing carcinogen bioassay given multiple doses of IQ at 50 pmol/kg. Predominating metabolites included N-demethyl-IQ and its N-glucuronide conjugate, IQ-sulfamate, the 5-sulfate, and the 5-0-glucuronide. After a single dose of IQ, approximately 16-22 % of the metabolites recovered in urine were N-demethylated. In the monkey given multiple doses of I&,N-demethyl-IQ and its W-glucuronide were the major metabolites found in urine, and together they accounted for approximately 35 % of the radioactivity

Metabolism of IQ in Monkeys

Chem. Res. Toricol., Vol. 5, No.6, 1992 847

Table 11. 360-MHz 1H NMR SDectral Data for IQ and Metabolites. ~

I&

proton H-9 Js,s 57,s J5,s

H-8 Je,9 J7,e Js,~~.H

H-7 57,s 51,s

H-5 J4,5 J5,9

H-4 J4,5

2-NH2 2-NH

8.54 ddd 8.3 1.8 0.7 1.43 dd 8.3 4.2 8.72 dd 4.2 1.8

7.51 dd 8.8 0.7 7.71 d 8.8 6.56 s sl br

8.71 dd 4.2 1.8 7.57 d(d) 8.8

nm 7.70d 8.8

3 8.64 ddd 8.3 1.7

4 8.60 dd 8.4 1.7

0.7 7.42dd 8.3 4.2

7.53dd 8.4 4.2

8.73 dd 4.2 1.7 7.60 dd 8.8

8.72 dd 4.2 1.7

7.16 d 8.8

8.79 dd 4.1 1.6 1.72 d(d) 9.0 -0.5

0.7

7.54 s

7.85 d 9.0

6.55 s a1 br 7.64 d sl br 7.37 d -9.0 9.3

JH-Y,N-H

NH (quinoline) or HO HS-CH,q H-1' (glucuronide)

2 8.60 d(d) 8.3 nm nm 7.44dd 8.3 4.2

metabolites 5 8.68 d sl br -8.0 nm nm 7.53dd 8.3 4.2

6 8.77 d(d) -8.3 nm nm 1.53dd 8.3 4.2 8.81 dd 4.2 1.8

7.76 dd 9.0 0.6 7.92 d 9.0

7 8 8.49 dd 8.13 d 8.3 9.5

9 8.13 dd 9.5

1.8

nm 7.41 dd 6.39d 8.3 9.5 4.1 nm 8.70 dd 4.1 1.8 6.80d 8.4 nm 7.81 s 7.26d 8.4 6.41 s 6.35 s sl br

0.4 6.40dd 9.5 1.6

6.84dd 8.5 0.4 7.32d 8.5 6.62 s

8.19 s sl br 11.48 s br

11.50 s sl br

3.64 s

3.70 s 3.62 s 3.81 s 3.94s 3.57 8 3.52 s 4.88 Y" 5.13 "t" 5.02 d 4.97 d JH-V,H.Z~ JaTb -8.6 Javgb -8.9 7.8 8.4 a Shifts in ppm relative to internal TMS;couplingconstants: absolute values in Hz (*0.2 Hz). Abbreviations: nm = not measurable because of line broadening, br = broad, sl br = slightly broad, s = singlet, d = doublet, t = triplet, "..." = approximate description, (...) =just detectable. Javg = (JH-I,,H-Z~ + JH-l',N-H)/2.

excreted. As seen in Table I, there is large animal variation in IQ metabolism. In view of the small number of animals available for this study, the effect of dose and chronic administration cannot be adequately evaluated. Structural Identificationof Metabolites in Urine. (A) 2-Aminoimidazo[4,5-fquinoline (N-DemethylIQ). Metabolite 10 comigrated with N-demethyl-IQ by HPLC, and the online UV spectrum was identical to that of the synthetic chemical. The E1 mass spectrum of the isolated product revealed a molecular ion at mlz 184.0747, in excellent agreement with the mass of the calculated molecular ion mlz 184.0748 of N-demethyl-IQ ( C ~ O H ~ N ~ ) , indicating a loss of a methyl group from IQ. The E1 mass Figure 5. UV spectra of 7-oxo-IQ and N-demethyl-7-oxo-IQ. spectrum for synthetic compound was in agreement with N-demethylated analogue of metabolite 9,7-oxo-IQ. The that obtained from the isolated product. (B) 2-Amino-3,6-dihydro-3-methyl-7H-imidazo[4,6- E1 mass spectra support these structures. The mass f quinolin-7-one and 2-Amino-3,6-dihydro-7H-imida- spectrum of metabolite 9,7-oxo-IQ, displayed a molecular ion peakat mlz 214.0851 (C11Hlfl40, calculated 214.0854)) zo[4,5-fquinolin-7-one (7-Oxo-IQ and N-Demethyl16mass units greater than that observed for IQ, indicating 7-oxo-IQ).Metabolite 9 was identified as 7-oxo-IQbased oxidation. The mass spectrum of synthetic '7-oxo-IQ was upon HPLC comigrationwith the authentic standard (29) in agreement with a molecular ion peak at mlz 214.0810. and by comparison of the respective UV and lH NMR The mass spectrum of metabolite 8 displayed a molecular spectra which were identical. The completely assigned ion at mlz 200.0702, which agreed well with the calculated lH NMR data are given in Table 11. The data compare mass of the molecular ion for N-demethyl-7-oxo-IQat mlz well with those reported on the synthetic compound in 200.0698 (CloHaN40). the same solvent (30).3 The spectrum is characterized by ( C ) 2-Amino-3-methylimidazo[4,5-fquinolin-6-y1 the absence of the H-7 signal and significant shift changes Sulfate (IQ-5-sulfate).Metabolite 7 could be hydrolyzed of all aromaticprotons with respect to I&. The assignments by arylsulfatase, but not by 8-glucuronidase, suggesting are based on the characteristic small H-5, H-9 coupling the metabolite contained a sulfuric acid conjugate. This and on the fact that the H-8 coupled with a single DzOmetabolite was chromatographically and spectroscopically exchangeable resonance at 11.50 ppm. The UV chromocompared to the same derivative isolated from rat bile. phore of metabolite 8 was very similar to that of 7-oxo-IQ The 1H NMR spectrum on metabolite 7 acquired from (Figure 5). The proton NMR spectra also revealed a monkey bile showed that all the aromatic resonances were striking resemblance to that of 7-oxo-IQ except that the very similar to those reported by Luke et al. (21). Slight N-CH3 signal was absent. The H-5, H-9 coupling which shift differences for the 2-NHz and the N3-CHz may be could not be resolved in metabolite 8 could be demonattributed to the different chromatographic conditions strated in a COSY experiment after D20 exchange. The employed for purification. The negative ion FAB mass spectra of metabolite 8 are therefore consistent with an spectrum supported the structure as IQ-5-sulfate. An ion [M - HI- was detected at mlz 293 with a fragment ion at After correction of a misprint on the H-5 signal: 6.81 instead of 6.51 ppm; personal communication, Dr. R. Van Tassell. mlz 80, due to [SOa-I.

Snyderwine et al.

848 Chem. Res. Toxicol., Vol. 5, No. 6, 1992

spectrum showed that the signals at 4.94 and 4.89 ppm belong to two of the glucuronide hydroxyl groups. After D20 exchange, they disappeared, and the glucuronide H-4 protons were better separated and further removed from H-7 H -7 H-5 the dominating HDO signal, which was shifted to 3.77 OH-3' ppm. The COSY spectrum of the glucuronide moiety obtained after addition of D20, shown in Figure 6B, confirmed the identity of the @-glucuronide(vicinal PFM couplingconstants between 8.9 and 9.9 Hz) and permitted the unequivocal assignment of all sugar protons in spite B N-CIHj HDO H;5' * of the significant level of contaminants in that spectral H-1' region (5.02 ppm, d, J1',2f 8.9 Hz, H-1'; 3.60 ppm, t, Javg 9.0 Hz, H-2'; 3.55 ppm, d, J4t.5-t 9.9 Hz, H-5'; 3.37 ppm, t, Jaw 8.9 Hz, H-3'; 3.22 ppm, dd, Jaw 9.4 Hz, H-4'). The similarity I I to the spectrum of the analogous MeIQx metabolite is 3 . 2 HH striking (24). The glucuronic acid must be conjugated to the exocyclic amine group of I&. A remaining NH group, - 3.4 normally seen between 6.50 and 8.20 ppm, was not - 3.6 detected. The assignmentas anN-hydroxy-N-glucuronide 4-5' rather than an N-0-glucuronide is based upon the me- 3.8 tabolite's relativestability,which contraststhe high lability reported for 0-glucuronides of arylhydroxylamines (31). - 4.0 This assignment is further supported by the positive test with pentacyanoamine ferroate, indicating a free NHOH group (24,31). An ion [M - HI- at m/z 389 in the negative ion FAB mass spectrum (Figure6C) indicatesa glucuronide conjugate with incorporation of an oxygen atom in the molecule. A small fragment at ion [M - H - 03- at m/z 373 was also detected; such a fragmentation could not occur for an 0-glucuronide and supports the proposed structure as an HO-N-glucuronide. Hydrolysis of the conjugate in 0.5 N HC1released N-hydroxy-IQ (confirmed a by HPLC, data not shown). Identification of N-hydroxyI PPM I I&was also confirmed by reaction with nitrosobenzene to 5.0 418 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 form the azoxy derivative 2- [phenyl(N,OJV)azoxy]-3methylimidazo[4,5# quinolineand by high-resolution MS (C17H13N50, found 303.1121, calculated 303.1120). (E) N-( 3-Methylimidazo[4,5-fJquinolin-2-yl)sulfamic Acid (IQ-sulfamate). Metabolite 5 was shown to be the sulfamate on the basis of HPLC comigration with NEG-FAB 3c : matrix the synthetic standard and by comparison of UV spectra H Na 1under neutral and alkaline pH (22). I&was recovered in quantitative fashion upon incubation of this metabolite + > under acid. Allowing for influencing factors such as 4 temperature and pH sensitivity, the lH NMR spectrum U is in good agreement with that observed for IQ-sulfamate (note that the H-4 and H-5 protons are reassigned on the basis of a COSY experiment (22). The negative ion FAB mass spectrum revealed an ion [M - HI- at m/z 277.0397 LUU L3U 3uu m u m,z 4uu (calculated 277.0395), in agreement with the calculated molecular weight of the sulfamate at 278 (CllH10N404S). Figure 6. 360-MHz lH NMR spectrum of N-hydroxy-IQ-WFragment ions were also detected, ([M - HI - SO& at m/z glucuronide(metabolite6). Impurities are marked with asterisks (A). Detail from the two-dimensional homonuclear correlation 197 and [SO& at m/z 80,and indicated the presence of spectrum (COSY)showing the region of the glucuronide proton a sulfate group. signals after DzO exchange. The proton correlations are circled (F) 2-Amino-5-(~-1-glucosiduronyloxy)-3-methylimand labeled (B). Negative ion FAB mass spectrum (C). idazo[ 4,5-fJquinoline(IQ-5-0-glucuronide). Metab(D) N1-(~-1-Glucosiduronyl)-N-hydroxy-2-amino- olite 4 was labile to @-glucuronidase. The lH NMR 3-methylimidazo[ 4,5-f)quinoline(N-Hydroxy-IQ-N2spectrum revealed the presence of the H-7, -8,and -9 protons of I&in agreement with earlier data (21),as well glucuronide). The proton NMR spectrum of approxias the slightly shifted 2-NH2 and the N3-CH3 groups. A mately 100 nmol of metabolite 6 is shown in Figure 6A. single resonance accounting for 1proton was detected at Due to the low level of metabolite, several contaminants 7.54 ppm, indicating oxidation at either the H-4 or -5 which are attributable to HPLC buffers and solvents are position of I&. As was previously observed for the present in the spectrum. All the aromatic resonances and corresponding MeIQx metabolite (24), an NOE of the the N3-CH3 group of I& are intact. The presence of a aromatic singlet could not be detected upon irradiation of glucuronic acid is suggested by the resonance at 4.97 ppm, the N-CH3 group and the site of oxidation is not una doublet with a coupling constant of 8.4 Hz. A COSY

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Metabolism of ZQ in Monkeys

Chem. Res. Toxicol., Vol. 5, No. 6, 1992 849

/ 1. N-Hydroxylation equivocal. However, in a recent metabolism study conNHz N-Sul fation ducted in the rat using I& labeled with tritium in the 5 N-Glucuronidation position, the tritum label was completely eliminated from the 0-glucuronide conjugate, proving the site of oxidation a t the 5 position (21). The anomeric proton of the glucuronic acid was readily detected as a doublet a t 5.02 / 2 . N-Demethvlation ppm (J 7.8 Hz). The glucuronide moiety was confirmed 4. C-Hydroxylation by a COSY experiment after D2O exchange. The negative 3. C-Hydroxylation ion FAB mass spectrum of metabolite 3 confirmed this 0-Sul fation 0-Glucuronidation proposed structure. An ion [M - HI- was detected at mlz Figure 7. Major routes of IQ metabolism in the monkey. 389, with a minor fragment [M - H - 1761-detected a t mlz 213, attributed to loss of the glucuronide moiety. Discussion (G) N2-(~-1-Glucosiduronyl)-2-amino-3-methylimidazo[4,5-flquinoline (IQ-Wglucuronide). MetaboI& undergoes extensive metabolic processing in the lite 3 was resistant toward hydrolytic enzymes, but it was monkey. Ten urinary metabolites of I&have been isolated labile to acid with quantitative recovery of I&. The 'H from animals given a carcinogenic dose regimen or single NMR spectrum revealed that the metabolite contained a acute dose, and nine have been fully characterized by @-glucuronicacid conjugated to the exocyclic amine group. spectroscopy. As shown in Figure 7, the pathways of I& Most signals were in excellent agreement with literature metabolism in monkeys involve cytochrome P-450 reacvalues (21). However, the remaining 2-NH proton of I& tions, phase I1conjugations,and enteric bacterial oxidation as well as combinations of these pathways. was found at 7.37 ppm. It coupled with the anomeric proton of the glucuronic acid a t 5.13 ppm, which was a There are both species similarities and differences "triplet" (a doublet of doublets with nearly equal coupling between rodents and monkeys in the metabolism and constants). Upon addition of D20, this triplet collapsed excretion of IQ. In monkeys, urine is the major route of to a doublet. This proves that the 2-N position of I& is excretion of I& and its metabolites, accounting for substituted with @-glucuronicacid. We recently observed approximately50-60 % of the dose. Excretion via the feces a similar coupling of the remaining 2-NH proton with the accounts for only about 10% of the dose over a 72-h period. anomeric glucuronic acid proton in the W-glucuronide of In contrast, the percentages of the dose eliminated via MeIQx (24). The glucuronic acid moiety was confirmed urine and feces of the rat are nearly equal (28). In both species, I& was effectivelydetoxified by direct conjugation by a COSY experiment. The negative ion FAB mass of the exocyclic amine group, forming the W-glucuronide spectrum supported this structure. An ion [M - HI- was and the sulfamate derivatives (21,22). Cytochrome P450detected at mlz 373, with a minor fragment ion [M - H mediated ring oxidation of IQ at the 5 position followed - 1761- at mlz 197, due to loss of glucuronic acid. (H) NL-(~-l-Glucosiduronyl)-%-aminoimidazo[4,5- by conjugation to sulfate or glucuronic acid were two other major routes of detoxification of this procarcinogen in both flquinoline (N-Demethyl-IQ-NL-glucuronide).Mespecies (21). Trace amounts of W-acetyl-IQ were detected tabolite 2 was hydrolyzed by both 0-glucuronidase and in urine of IQ-fed rats (32,33);however, this metabolite acid to yield N-demethyl-IQ, which suggested that the was not found in monkeys. Neither glutathione nor metabolite was an W-glucuronide conjugate of N-demercapturic acid conjugates of I& have been found in methyl-IQ. The 'H NMR spectrum was consistent with rodents (21,22,32,33). These conjugates were not found this. All the aromatic resonances of I&were present, but in either urine or bile of monkeys, indicating that the N-CH3 was missing. The remaining 2-NH signal at conjugation with glutathione does not play a significant 7.64 ppm coupled to the anomeric proton of the glucuronic role in the metabolism of I&. Interestingly, N-demethacid, seen as a poorly resolved "triplet" at 4.88 ppm, which ylation, which is a very minor route of I& metabolism in collapsed to a doublet upon addition of D2O. The negative rats (32), is a major route of IQ metabolism in monkeys. ion FAB mass spectrum supported this structure. An ion Three N-demethylated metabolites of I&found in monkeys [M - HI- was detected at mlz 359 and a major fragment were as follows: N-demethyl-IQ, N-demethyl-IQ-Wion [M - H - 1761- at mlz 183,which is due to the cleavage glucuronide, and N-demethyl-7-oxo-IQ. Approximately of the glucuronide moiety. 16-22% percent of the dose of I& recovered in urine (I) Metabolite 1. Metabolite 1 has been partially underwent N-demethylation in the two monkeys given IQ characterized. The lH NMR spectrum revealed that all at 2.2 pmollkg. This metabolic pathway appeared to be of the aromatic protons were intact; however, the N3-CH3 even more prominent in the monkey chronically fed I&a t normally seen for I&and the other identified metabolites 50 pmollkg. Mutagenicity studies have suggested that a t 4.0-3.5 ppm was missing. Instead, another CH3 signal N-demethylation is a route of detoxification. N-Demethylwas found at 2.78 ppm which coupled to a DzO-exchangeI& is only weakly mutagenic: its mutagenic potency is able signal (J 3.1 Hz). A glucuronide conjugate was about 70-fold less than that of I& (33). However, the formaldehyde produced during N-demethylation has been suggested by a broadened resonance at 4.74 ppm which shown to cause DNA strand breaks, and cross-links shifted to 4.86 ppm and became a doublet upon DzO between DNA and protein (34). Formaldehyde also exchange. This signal is attributed to the anomeric proton. inhibits the repair of Oe-methylguanine adducts and A COSY experiment proved the presence of the glucuronic potentiates the mutagenicity of the alkylating agent acid moiety. The negative ion FAB mass spectrum N-methyl-N-nitrosourea (34, 35). The possible role of displayed an ion at [M - HI- mlz 391, which is two mass formaldehyde in I& hepatocarcinogenicity in nonhuman units greater than the ring-hydroxylated 5-0-glucuronide primates merits study. or N-hydroxy-N-glucuronide conjugates. Additional studThe enteric bacteria of monkeys appear to have a greater ies and comparisons to synthetic derivatives are required role than the enteric bacteria of rodents in the metabolism for unequivocal structure identification of this metabolite.

Y 4

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850 Chem. Res. Toxicol., Vol. 5, No. 6, 1992

of IQ. Both the mutagen 7-oxo-IQ (29) and its N-demethylated homologue N-demethyl-7-oxo-IQ, which has not been previously reported, were found in monkey urine and feces. Carmen et al. (29),recently demonstrated that anaerobic cultures of human intestinal bacteria hydroxylate IQ at the 7 position to produce 7-oxo-@. This metabolite was subsequently identified in human feces following consumption of fried meat (36). N-Demethyl7-oxo-IQ may arise analogously from the enteric bacterial metabolism of N-demethyl-IQ. 7-Oxo-IQ was reported to be a direct-acting bacterial mutagen in the Ames reversion assay (29); however, we have found that 7-oxo-@ is mutagenic only in the presence of S-9 (experiments conducted independently in two different laboratories). The analogue N-demethyl-7-oxo-IQ was not even mutagenic in the presence of S-9,and thus, it appears to be a detoxified metabolite. The presence of both metabolites in urine demonstrates that they are reabsorbed into the systemic circulation. N-Demethyl-7-oxo-IQ was also detected in bile, indicating that it entered the enterohepatic circulation. Neither N-demethyl-7-oxo-IQ nor 7-oxo-IQ appears to contribute to the DNA adducts found in tissues of monkeys fed IQ since 32P-postlabelinganalysis shows that DNA adducts in liver, and extrahepatic tissues including colon, are identical to those obtained by the reaction of N-hydroxy-IQ with DNA in vitro (19, 20). Therefore these compounds are probably not involved in the initiation of hepatocellular carcinoma observed in monkeys. However, further toxicological evaluation of these metabolites is warranted. Metabolic activation of I& and other aromatic and heterocyclic aromatic amines by mammalian enzymes occurs through N-oxidation of the exocyclic amine group that is principally catalyzed by cytochrome P450 1A2 (121 7 ) . The arylhydroxylamines may react directly with DNA, or following conjugation with acetate or sulfate (12), via highly reactive nitrenium ion intermediates (37). 32PPostlabeling analysis of DNA from monkeys exposed to I& reveals that DNA adducts are formed in many extrahepatic tissues, including kidney, colon, stomach, and urinary bladder (19,20). The adducts found in extrahepatic tissues are the same as those found in the liver, and they appear to be derived from N-hydroxy-IQ. The presence of DNA adducts in extrahepatic tissues indicates that the N-hydroxy metabolite is transported to these organs either directly or as the metastable N-glucuronide conjugate; alternatively, formation of this carcinogenic metabolite may occur within the extrahepatic tissues. We have shown that transport of N-hydroxy-IQ may occur through the metastable N-hydroxy-IQ-W-glucuronide conjugate which was found in urine of monkeys. This metabolite has not been identified in urine or bile of rodents, although N-hydroxy-N-glucuronide conjugates of the structurally related food-borne mutagens MeIQx and PhIP have been reported ( 3 4 3 9 ) . Target tissues of carcinogenesis induced by arylamines in animal models are often the colon and urinary bladder (40). Tumor formation in these two tissues is in part determined by the extent of the conversion of the arylamine to the N-glucuronide of the arylhydroxylamine and by the relative excretion of this metabolite in urine versus bile (41,421. The mildly acidic pH of urine may cleave the conjugate and liberate the reactive arylhydroxylamine, which then may initiate the neoplastic process in the urinary bladder (41). The acid stability of the N-glucuronide conjugatesof N-hydroxy-IQ and N-hydroxy-MeIQx

Snyderwine et al. (24)in urine is considerably greater than that reported for N-glucuronide conjugates of several arylhydroxylamines (41, 42), and the reactive N-hydroxy metabolites of IQ and MeIQx are not liberated. IQ-DNA adducts are formed only at very low levels in monkey bladder, and this low level of DNA modification may be due in part to the stability of N-hydroxy-IQ-W-glucuronidein urine. We could not detect the N-hydroxy-N-glucuronide conjugate of IQ in bile of monkeys. Thus, if this metastable metabolite is excreted in bile, it must account for a very low percentage of the dose (level of detection estimated at approximately 1% of total metabolites found in bile). The N-hydroxy-N-glucuronide conjugates of IQ and MeIQx are sensitive to 8-glucuronidase and the carcinogenic N-hydroxy metabolite is liberated which is able to react with DNA (24).4 The presence of DNA adducts in colon of monkeys (19) suggests that very low amounts of this metastable metabolite is excreted in bile or that N-hydroxy-IQformation occurs to a minor extent directly in the intestinal tract. IQ is a potent liver carcinogen in monkeys with the period from initial dose of IQ to diagnosis of tumors being similar to that found for induction of tumors in monkeys receiving oral doses of the carcinogen N-nitrosodiethylamine (9,43, 44). Despite considerable detoxification of IQ in monkeys undergoing carcinogen bioassay, a significant amount of activation occurs in vivo to induce liver tumor development. Our results show that IQ is metabolized to the reactive metabolite N-hydroxy-IQ in vivo which may be the initiator of carcinogenesis. Further studies on the effect of dose on IQ tumorigenesis, as well as studies concerning the metabolic processing and DNA adduction of N-hydroxy-IQ in monkeys, may provide additional insight into other possible target organs and into the role of N-hydroxy-IQ in tumor initiation. Such investigations may permit better extrapolation for the risk of dietary consumption of trace levels of carcinogenic heterocyclic amines by humans.

Acknowledgment. We thank Dr. Dan Dalgard and Mrs. Jeannette Reeves at Hazleton Laboratories America and Drs. Richard Adamson and Unnur Thorgeirsson, National Cancer Institute, Bethesda, for their expertise and assistance with the monkey colony and Mrs. Francia Arce Vera, Nestec Ltd., Lausanne, for technical assistance in NMR spectroscopy. The authors also thank Drs. Richard Adamson and Snorri Thorgeirsson, National Cancer Institute, Dr. Paul Skipper, Division of Toxicology, MIT, and Drs. Shozo Takayama and Takashi Sugimura, National Cancer Center Research Institute, Tokyo, for stimulating discussions and input into heterocyclic amine research. This work was supported in part by the National Cancer Institute Contract N01-CP-51013 to Hazleton Laboratories.

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Unpublished observation.

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