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A chromatographic technique for the analysis of oxidized metabolites: Application to carcinogenic N-hydroxyarylamines in urine
120-125 (1981)
ANALYTI~ALBI~HEMISTRY
118,
A Chromatographic Application
Technique for the Analysis of Oxidized Metabolites: to Carcinogenic ~~ydroxyarylamines in Urine
CLAY B. FREDERICK,
JOANN B. MAYS, AND FRED F.
KADLUBAR
Division of Carcinogenesis Research, National Center for Toxicological Research. Department of Health and Human Services, Jefferson, Arkansas 72079 Received July 29, 198 1 A new chromatographic detection method for oxidized metabolites has been developed based on the reaction of eluted compounds with an Fe+3- bathophenanthroline calorimetric reagent in a postcolumn reactor. The method is sensitive to N-hydroxyarylamines, aryldiamines, phenolic amines, and ascorbic acid. It has been applied to the analysis of toxic N-oxidized metabolites in rhesus monkey urine after the animals were dosed with the bladder carcinogens, l- and 2-napthylamine. These compounds are oxidized to the corresponding N-hydroxyarylamines in the liver, conjugated as the N-glucuronide, and excreted in the urine. The Ngmcuronide has been shown to undergo acidic hydrolysis in the urine to release the free Nhydroxya~iamine, an ultimate carcinogen for the induction of bladder tumors. In this study, the N-hydroxy-N-glucuronide of 2-naphthylamine was found to be excreted at a rate that was 6.8 times that of the l-naphthylamine isomer. This is consistent with the much higher carcinogenic potency of 2naphthylamine in a variety of species.
A classic problem in toxicology has involved an explanation of the differing carcinogenic potentials of l- and 2-naphthylamine (l- and 2-NA).’ The data, as recently reviewed by Radomski ( I), indicates that 2NA is a bladder carcinogen for humans, monkeys, dogs, rats, rabbits, guinea pigs, and hamsters; whereas mice develop liver tumors only. The closely related structural isomer, 1-NA, has been tested in dogs, hamsters, and mice with negative results ( 1,2). However, the epidemiological evidence in man is equivocal with a significant incidence of bladder cancer reported in an industrial group exposed to l-NA which was contaminated with approximately 4% 2-NA (3). In contrast, the primary oxidation products and presumably the ultimate carcinogenic forms of 1-NA and 2-NA, i.e., N-OH’ Abbreviations used: HPLC, high-performance liquid chromatography; I-NA, I-naphthylamine; 2-NA, 2-naphthylamine; N-OH-I-NA, N-hydroxy-l-naphthylamine; N-OH-2-NA, N-hydroxy-2-naphthylamine. 0003-2697/81,‘170120-06$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of nprcduction in any form nswved.
I-NA and N-OH-2-NA, are highly carcinogenic (4-7) and react with DNA, RNA, and protein under slightly acidic conditions (8). In fact, N-OH- 1-NA appears to be significantly more carcinogenic than N-OH-2NA (4,6,7), and N-OH- 1-NA binds to DNA in vitro to give a lo-fold higher level of covalent carcinogen-DNA adducts (8). Previous studies have suffered from the formidable analytical problems associated with the quantitation of these N-hydroxyamines. In contrast to their N-acetyl derivatives, these primary ~-hydroxyaryIamines are highly reactive and decompose to the nitrosoarene and the corresponding amine, This degradation is facilitated by heat, light, oxygen, and metal ions. These problems have forced some investigators to oxidize the hydroxylamines to the presumably more stable nitroso derivative (also suggested to be metabolically produced in vivo) and to report the total N-oxidation products (9,lO). Nitrosonaphthalenes react rapidly with protein, but not with DNA and RNA (1 I), and 120
DETECTION
OF
N-HYDROXYARYLAMINES
have not been implicated as ultimate carcinogens for the urinary bladder (6). We have developed a new analytical method to quantitate the N-hydroxylation of aromatic amines. This technique utilizes HPLC to separate the complex mixture of amine metabolites. The specificity of the analysis is introduced by means of a postcolumn reactor which allows a calorimetric determination for N- and ring-hydroxylated arylamines. We have used this technique to analyze the urine of rhesus monkeys dosed with l- and 2-NA, and to quantitate specific metabolites. Conzelman and his co-workers have demonstrated that 2-NA is carcinogenic in this species (12) and the monkey’s ability to N-acetylate aromatic amines may make it a more representative model for human arylamine metabolism than the dog which cannot acetylate these compounds ( 13). MATERIALS
AND METHODS
Animals and dosage. Two adult male rhesus monkeys (Macaca mulatta) from the National Center for Toxicological Research primate colony weighing 11.2 and 11.3 kg were given 100 mg/kg doses of [ 3-3H]- 1-NA (1.28 &i/mM) and [ 5,6,7,8-3H]-2-NA (1.28 pCi/mlvr), respectively. The animals were lightly anesthetized with 8.9 mg/kg ketamine hydrochloride (Bristol Laboratories) im, and the animals were given the amine dissolved in 25 ml trioctanoin by oral gavage. The experiment was repeated with the reversed treatment regimen (i.e., the 11.2-kg animal receiving 2-NA and the 11.3-kg animal receiving I-NA) 1 month after the original dosing and gave similar results. The monkeys were housed in stainlesssteel metabolism cages fitted with a fine mesh screen to separate urine and feces. The urine was collected in a flask chilled with dry ice, but sealed in such a manner as to minimize exposure of the urine to CO2 and light. Standard Purina monkey diet was provided twice daily, fresh fruit was provided several
121
times a week, and water intake limited to 1 liter per day. Extraction of the urine. The urine was thawed under a stream of argon gas and extracted immediately as previously outlined (14). Briefly, the method involved an initial ethyl acetate extraction of unconjugated metabolites (Fraction I). The aqueous phase was then acidified to pH 4 to hydrolyze Nglucuronides and the aglycones were extracted into ethyl acetate (Fraction II). Analysis of extracts. A lOO-~1 aliquot of each fraction was analyzed on an Altex Instruments 322 MP gradient HPLC system using a O-100% methanol-water linear gradient for a duration of 20 min with a flow rate of 2 ml/min. The solvent reservoirs were continuously flushed with argon. Separations were conducted on a Whatman Partisil PXS lo/25 ODS-3 reversed-phase column.2 Detection of urinary metabolites was accomplished by two complementary detectors in tandem as detailed in Fig. 1. The column effluent was directed to a Waters Associates 440 UV-Vis detector operated at 254 nm. The effluent from this detector then proceeded to the first port of a three-port mixing chamber (Waters Associates No. 55318) which served as a postcolumn reactor. The second port on this chamber was connected to a Waters Model 6000 HPLC pump which delivered, at 2 ml/min, a calorimetric reagent which consisted of: (1) 6.2 mM bathophenanthrolinedisulfonic acid, disodium salt hydrate (Aldrich Chemical Co.) dissolved in 1.0 M sodium acetate buffer at pH 4.6; (2) 0.01 M ferric nitrate dissolved in 0.1 M acetic acid; and (3) methanol, combined (v/ v) in a 1:0.25:9 ratio, respectively. The contents of the reactor then passed from the third port into a l-ml stainless-steel mixing loop and finally into a Tracer 970A UV-Vis 2 We have recently found that conditioning these columns thoroughly with 0.01 M acetohydroxamic acid (Aldrich Chemical Co.) in methanol significantly improves the sensitivity of the columns for the unstable Nhydroxyarylamines, presumably by removing trace metal contamination.
FREDERICK,
122
COLUMN REACTOR
MAYS, AND KADLUBAR
1 WASTE
FIG. 1. A schematic diagram of a gradient HPLC with a uv-detector modified with a postcolumn reactor. The reagent pump provides an Fe+3-bathophenant~roline reagent that reacts rapidly with N-hydroxyarylamines to produce a highly colored Fe” chelate that is monitored on the second detector.
detector set at 535 nm. The output from each detector was plotted on a Linear 595 threepen recorder and peak areas were determined with two Hewlett-Packard 3380A integrators. The composition of the calorimetric reagent was based on an assay for tocopherols developed by Tsen (15). The use of the water-soluble sulfonate of bathophenanthroline (a chelating agent which forms an intensely colored complex with the ferrous ion produced in the reaction) prevents precipitation of the ligand in the aqueous medium normally employed in reversed-phase HPLC. Similarly, the addition of methanol facilitates the mixing of the reagent and the effluent stream. Safety considerations limited the amount of radioactivity that could be used in our animal care facility and only the major metabolites could be quantitated by collecting and counting fractions from the HPLC. The source of other compounds and enzymes is as previously described ( 14). RESULTS
Calibration curves for N-OH-l-NA and N-OH-2-NA are presented in Fig. 2. The peak areas were directly proportional to the
amount of N-hydroxyarylamine, each with a limit of detection (twice background) of approximately 0.1 pg (0.6 nmol) per injection. This corresponded to 0.017% of the arylamine dose excreted in the usual 500-ml urine sample, and this level of sensitivity proved to be adequate for the determination of oxidized urinary metabolites used in this study. The cumulative excretions of I-NA and 2-NA metabolites were as follows. In the first 24 h after dosing, 70% of the radioactivity was excreted into the urine. The first extraction (Fraction I) removed 21% of INA metabolites and 7% of the 2-NA metabolites into the organic phase. The unextracted products in the aqueous fraction consisted of highly water-soluble conjugates.
FIG. 2. Calibration curves for N-OH-I-NA and NOH-2-NA. An ethyl acetate solution of a synthetic standard of N-OH-I-NA was eluted through the HPLC apparatus described under Materials and Methods with 50% methanol-water and a flow rate of 2 ml/min. The mean peak areas derived from the integrator connected to the postcolumn reactor (detector 2) are recorded ( + SD) with the detector on 0.02 absorbance units for full scale deflection with the smallest injection.
DETECTION
123
OF N-HYDROXYARYLAMINES
.
Incubation of this fraction at pH 4 hydrolyzed N-glucuronides and allowed extraction of the aglycones into the organic phase (Fraction II), accounting for an additional 18 and 3.4% of the total urinary metabolites of l-NA and 2-NA, respectively. A typical chromatogram for Fraction II is shown in Fig. 3. The uv trace is quite complex, and the proximity of the small N-OH2-NA peak to other dominant uv-absorbing peaks makes quantitation of the N-hydroxy metabolite by peak area (or radioactivity) quite difficult. In contrast, the plot of absorption at 535 nm from the calorimetric reaction in the postcolumn reactor is much less complex, and the small but reproducible peak for JV-OH-2-NA is well resolved and easily quantitated. Using these methods, the urinary amounts of l-NA and 2-NA and their nitroso and nitro derivatives were quantitated from integration of ultraviolet-absorbing peak areas; and urinary N-OH-lNA and N-OH-2-NA levels were determined by integration of visible-absorbing peak areas, as mediated by the postcolumn reactor. Urinary Metabolites in the Monkey
N-OH-2-l 2-NitroroNophtholene
of 1 -NA and 2-NA
Analysis of the urine of monkeys given lNA (Table IA) indicated that the parent compound was a major excretory product (2 1.1% of the dose) and that it was present in both the free form (Fraction I) and as the glucuronide (Fraction II). Only minimal amounts of N-OH- l-NA (0.15%) and trace amounts (
1
I I IO 15 Time (Min)
I 20
FIG. 3. The simultaneous HPLC chromatograms from detectors 1 (uv; top) and 2 (visible; bottom) following elution of Fraction II from monkey urine after 2-NA dosing. The IV-OH-2-NA peak is well resolved in the bottom chromatogram from the postcolumn reactor, but is not well-defined from other uv-absorbing (but nonreactive) components in the top chromatogram. Peaks C,,z are components of control urine samples, and peak M, is a metabolite (structure unknown) of 2-NA.
observed. To validate the extraction techniques, control urine samples were spiked with the free N-hydroxyarylamines and their iV-glucuronides. More than 95% of the Nhydroxyarylamines were recovered in each case as determined by HPLC analysis.
124
FREDERICK,
MAYS, AND KADLUBAR TABLE
EXCRETION
Expt
OF I-NA
Compound
AND 2-NA
1
METABOLITES
O-24
h AFTER DOSING
Retention time (min)
Free
IV-Glucuronide
Percentage of dose excreted
A
I-NA N-OH-I-NA I-Nitrosonaphthalene I-Nitronaphthalene
15.8 14.9 19.0 18.3
21.1 f 0.1
17.7 + 0.5 0.15 + 0.02
B
2-NA N-OH-2-NA 2-Nitrosonaphthalene 2-Nitronaphthalene
15.7 14.8 18.8 18.6
2.85 + 0.23
1.20 + 0.20 1.03 + 0.18 0.10 + 0.05
’ The data are expressed as the mean f SD (n = 3).
Applicability
to Other Compounds
Preliminary experiments were conducted with a variety of reducing agents of biological and toxicological interest. The technique appears to be suitable for N-hydroxyarylamines, phenolic amines (2-amino- 1-naphthol), ascorbic acid, and aryldiamines (benzidine). The compound for analysis need only have an oxidation potential such that it will efficiently reduce Fe+3 under the reaction conditions at a rate (~30 s) that is adequate for detection. DISCUSSION
We have developed a unique and reasonably specific analytical technique for the quantitation of carcinogenic N-hydroxyarylamines. The values reported for total Noxidized naphthylamine metabolites in rhesus monkey urine approximate those observed previously in dogs and monkeys by a less specific gas chromatographic technique (9,13). This HPLC technique, supplemented with the postcolumn reactor, is suitable for the analysis of a variety of biological reducing agents and oxidized metabolites, e.g., ascorbic acid, aryldiamines, and phenolic amines. The rate of reaction of these compounds with the reagent in the mixing coil determines the intensity of the colori-
metric response and will vary somewhat depending upon the compound (cf. Fig. 2). By this method, we found that N-hydroxylation and subsequent N-glucuronidation of l- and 2-NA is a significant, though relatively minor metabolic pathway (x2%), in the rhesus monkey. Thus, the urinary excretion of the N-hydroxy metabolite should be a reasonable indication of the carcinogenic potency of these compounds in the test species, as their formation is considered to be a necessary prerequisite for the initiation of bladder carcinogenesis ( 1,8,16). This urinary analysis did indicate that N-OH-2-NA accumulates in the urine as the N-glucuronide at a level that is 6.8 times that of NOH- l-NA N-glucuronide. Furthermore, the unoxidized l-NA is excreted at a level 10 times that of 2-NA, indicating that it may be a poor substrate for the oxidative metabolic enzymes of the monkey. These results correlate with the carcinogenicity of the parent amines in a variety of species ( 1). In contrast to previous studies on the urinary excretion of l- and 2-NA in dogs (9, 13), we did not detect free N-OH- I-NA or N-OH-2-NA. This is consistent with our recent study involving the bladder instillation of radioactive N-OH-l-NA and N-OH-2NA into rats (18). These compounds were
DETECTION
OF N-HYDROXYARYLAMINES
absorbed from the bladder at a rate (>90%/ 30 min) that was independent of urine acidity; consequently only trace amounts of the free N-hydroxyarylamine should be detectable in those species retaining their urine for extended periods of time. Previously, we and others (1,8,16) have proposed that acid-catalyzed hydrolysis of N-hydroxyarylamine iV-glucuronides in the urine (pH 5-6) is necessary for their conversion to the N-hydroxyarylamine, and that this step may be a necessary prerequisite for arylamine-induced bladder carcinogenesis. However, the urinary pH of the monkeys used in this study ranged from 6.7 to 7:5 which suggests that they would be relatively resistant to bladder carcinogenesis. Conzelman et al. noted that monkeys were much less susceptible to bladder tumors from 2NA than beagle dogs which were treated similarly (12). They calculated that to induce a 75% tumor incidence, a dose of 200 mg/kg, 6 days/week, for 36-60 months was necessary in their monkey colony, versus a dose regimen of only 6.25 mg/kg, 6 days/ week, for 24 months in their dog colony. It should also be noted that dog and human urine typically has a pH range of 5 to 6 (8,17), and these species may conform to a model in which an acidic urine enhances oncogenic susceptibility. ACKNOWLEDGMENTS The authors would like to express their appreciation to John Bailey and Skeeter Georgeson for their skilled contribution in animal handling and dosing.
REFERENCES 1. Radomski, J. L. ( 1979) in Annual Reviews of Pharmacology and Toxicology (George, R., Okun, R.,
2.
3.
4. 5. 6. 7.
8. 9. 10.
125
and Cho, A. K., eds.), Vol. 19, pp. 129-156, Annual Reviews Inc., Palo Alto, Calif. Radomski, J. L., Diechmann, W. B., Altman, N. H., and Radomski, T. (1980) Cancer Res., 40,3537-3539. Case, R. A. M., Hosker, M. E., McDonald, D. B., and Pearson, J. T. (1954) Brit. J. Ind. Med. 11, 75-104. Belman, S., Troll, W., Teebor, G., and Mukai, F. (1968) Cancer Res. 28, 535-542. Boyland, E., Dukes, C. E., and Grover, P. L. (1963) Brit. J. Cancer 17, 79-84. Radomski, J. L., Brill, E., Deichmann, W. B., and Glass, E. M. (1971) Cancer Res. 31.1461-1467. Dooley, K. L., Beland, F. A., Heath, J. E., and Kadlubar, F. F. (1981) Proc. Amer. Assoc. Cancer Res. 21, 85. Kadlubar, F. F., Miller, J. A., and Miller, E. C. (1977) Cancer Rex 37, 805-814. Radomski, J. L., and Brill, E. (1970) Science 167, 992-993. Uehleke, H. (1966) Life Sci. 5, 1489-1494.
Il. Kadlubar, F. F., Unruh, L. E., Beland, F. A., Straub, K. M., and Evans, F. E. (1980) Curcinogenesis 1, 139-150. 12. Conzelman, G. M., Jr., Moulton, J. E., Flanders, L. E., III, Springer, K., and Crout, D. W. (1969) J. Nat. Cancer Inst. 42, 825-836. 13. Radomski, J. L., Conzelman, G. M., Jr., Rey, A. A., and Brill, E. (1973) J. Nat. Cancer Inst. 50,989-995. 14. Kadlubar, F. F., Unruh, L. E., Flammang, T. J., Spark, D., Mitchum, R. K., and Mulder, G. J. (1981) Chem. Biof. Interact. 33, 129-147. 15. Tsen, C. C. (1961) Anal. Chem. 33, 849-851. 16. Lotlikar, P. D. (1981) J. Cancer Res. C/in. Oncol. 99, 125-136. 17. Osbourne, C. A., Low, D. G., and Finco, D. R. (1972) in Canine and Feline Urology, p. 39, Saunders, Philadelphia. 18. Gglesby, L. A., Flammang, T. J., Tullis, D. L., and Kadlubar, F. F. (1981) Carcinogenesis 2, 15-20.
ANALYTI~ALBI~HEMISTRY
118,
A Chromatographic Application
Technique for the Analysis of Oxidized Metabolites: to Carcinogenic ~~ydroxyarylamines in Urine
CLAY B. FREDERICK,
JOANN B. MAYS, AND FRED F.
KADLUBAR
Division of Carcinogenesis Research, National Center for Toxicological Research. Department of Health and Human Services, Jefferson, Arkansas 72079 Received July 29, 198 1 A new chromatographic detection method for oxidized metabolites has been developed based on the reaction of eluted compounds with an Fe+3- bathophenanthroline calorimetric reagent in a postcolumn reactor. The method is sensitive to N-hydroxyarylamines, aryldiamines, phenolic amines, and ascorbic acid. It has been applied to the analysis of toxic N-oxidized metabolites in rhesus monkey urine after the animals were dosed with the bladder carcinogens, l- and 2-napthylamine. These compounds are oxidized to the corresponding N-hydroxyarylamines in the liver, conjugated as the N-glucuronide, and excreted in the urine. The Ngmcuronide has been shown to undergo acidic hydrolysis in the urine to release the free Nhydroxya~iamine, an ultimate carcinogen for the induction of bladder tumors. In this study, the N-hydroxy-N-glucuronide of 2-naphthylamine was found to be excreted at a rate that was 6.8 times that of the l-naphthylamine isomer. This is consistent with the much higher carcinogenic potency of 2naphthylamine in a variety of species.
A classic problem in toxicology has involved an explanation of the differing carcinogenic potentials of l- and 2-naphthylamine (l- and 2-NA).’ The data, as recently reviewed by Radomski ( I), indicates that 2NA is a bladder carcinogen for humans, monkeys, dogs, rats, rabbits, guinea pigs, and hamsters; whereas mice develop liver tumors only. The closely related structural isomer, 1-NA, has been tested in dogs, hamsters, and mice with negative results ( 1,2). However, the epidemiological evidence in man is equivocal with a significant incidence of bladder cancer reported in an industrial group exposed to l-NA which was contaminated with approximately 4% 2-NA (3). In contrast, the primary oxidation products and presumably the ultimate carcinogenic forms of 1-NA and 2-NA, i.e., N-OH’ Abbreviations used: HPLC, high-performance liquid chromatography; I-NA, I-naphthylamine; 2-NA, 2-naphthylamine; N-OH-I-NA, N-hydroxy-l-naphthylamine; N-OH-2-NA, N-hydroxy-2-naphthylamine. 0003-2697/81,‘170120-06$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of nprcduction in any form nswved.
I-NA and N-OH-2-NA, are highly carcinogenic (4-7) and react with DNA, RNA, and protein under slightly acidic conditions (8). In fact, N-OH- 1-NA appears to be significantly more carcinogenic than N-OH-2NA (4,6,7), and N-OH- 1-NA binds to DNA in vitro to give a lo-fold higher level of covalent carcinogen-DNA adducts (8). Previous studies have suffered from the formidable analytical problems associated with the quantitation of these N-hydroxyamines. In contrast to their N-acetyl derivatives, these primary ~-hydroxyaryIamines are highly reactive and decompose to the nitrosoarene and the corresponding amine, This degradation is facilitated by heat, light, oxygen, and metal ions. These problems have forced some investigators to oxidize the hydroxylamines to the presumably more stable nitroso derivative (also suggested to be metabolically produced in vivo) and to report the total N-oxidation products (9,lO). Nitrosonaphthalenes react rapidly with protein, but not with DNA and RNA (1 I), and 120
DETECTION
OF
N-HYDROXYARYLAMINES
have not been implicated as ultimate carcinogens for the urinary bladder (6). We have developed a new analytical method to quantitate the N-hydroxylation of aromatic amines. This technique utilizes HPLC to separate the complex mixture of amine metabolites. The specificity of the analysis is introduced by means of a postcolumn reactor which allows a calorimetric determination for N- and ring-hydroxylated arylamines. We have used this technique to analyze the urine of rhesus monkeys dosed with l- and 2-NA, and to quantitate specific metabolites. Conzelman and his co-workers have demonstrated that 2-NA is carcinogenic in this species (12) and the monkey’s ability to N-acetylate aromatic amines may make it a more representative model for human arylamine metabolism than the dog which cannot acetylate these compounds ( 13). MATERIALS
AND METHODS
Animals and dosage. Two adult male rhesus monkeys (Macaca mulatta) from the National Center for Toxicological Research primate colony weighing 11.2 and 11.3 kg were given 100 mg/kg doses of [ 3-3H]- 1-NA (1.28 &i/mM) and [ 5,6,7,8-3H]-2-NA (1.28 pCi/mlvr), respectively. The animals were lightly anesthetized with 8.9 mg/kg ketamine hydrochloride (Bristol Laboratories) im, and the animals were given the amine dissolved in 25 ml trioctanoin by oral gavage. The experiment was repeated with the reversed treatment regimen (i.e., the 11.2-kg animal receiving 2-NA and the 11.3-kg animal receiving I-NA) 1 month after the original dosing and gave similar results. The monkeys were housed in stainlesssteel metabolism cages fitted with a fine mesh screen to separate urine and feces. The urine was collected in a flask chilled with dry ice, but sealed in such a manner as to minimize exposure of the urine to CO2 and light. Standard Purina monkey diet was provided twice daily, fresh fruit was provided several
121
times a week, and water intake limited to 1 liter per day. Extraction of the urine. The urine was thawed under a stream of argon gas and extracted immediately as previously outlined (14). Briefly, the method involved an initial ethyl acetate extraction of unconjugated metabolites (Fraction I). The aqueous phase was then acidified to pH 4 to hydrolyze Nglucuronides and the aglycones were extracted into ethyl acetate (Fraction II). Analysis of extracts. A lOO-~1 aliquot of each fraction was analyzed on an Altex Instruments 322 MP gradient HPLC system using a O-100% methanol-water linear gradient for a duration of 20 min with a flow rate of 2 ml/min. The solvent reservoirs were continuously flushed with argon. Separations were conducted on a Whatman Partisil PXS lo/25 ODS-3 reversed-phase column.2 Detection of urinary metabolites was accomplished by two complementary detectors in tandem as detailed in Fig. 1. The column effluent was directed to a Waters Associates 440 UV-Vis detector operated at 254 nm. The effluent from this detector then proceeded to the first port of a three-port mixing chamber (Waters Associates No. 55318) which served as a postcolumn reactor. The second port on this chamber was connected to a Waters Model 6000 HPLC pump which delivered, at 2 ml/min, a calorimetric reagent which consisted of: (1) 6.2 mM bathophenanthrolinedisulfonic acid, disodium salt hydrate (Aldrich Chemical Co.) dissolved in 1.0 M sodium acetate buffer at pH 4.6; (2) 0.01 M ferric nitrate dissolved in 0.1 M acetic acid; and (3) methanol, combined (v/ v) in a 1:0.25:9 ratio, respectively. The contents of the reactor then passed from the third port into a l-ml stainless-steel mixing loop and finally into a Tracer 970A UV-Vis 2 We have recently found that conditioning these columns thoroughly with 0.01 M acetohydroxamic acid (Aldrich Chemical Co.) in methanol significantly improves the sensitivity of the columns for the unstable Nhydroxyarylamines, presumably by removing trace metal contamination.
FREDERICK,
122
COLUMN REACTOR
MAYS, AND KADLUBAR
1 WASTE
FIG. 1. A schematic diagram of a gradient HPLC with a uv-detector modified with a postcolumn reactor. The reagent pump provides an Fe+3-bathophenant~roline reagent that reacts rapidly with N-hydroxyarylamines to produce a highly colored Fe” chelate that is monitored on the second detector.
detector set at 535 nm. The output from each detector was plotted on a Linear 595 threepen recorder and peak areas were determined with two Hewlett-Packard 3380A integrators. The composition of the calorimetric reagent was based on an assay for tocopherols developed by Tsen (15). The use of the water-soluble sulfonate of bathophenanthroline (a chelating agent which forms an intensely colored complex with the ferrous ion produced in the reaction) prevents precipitation of the ligand in the aqueous medium normally employed in reversed-phase HPLC. Similarly, the addition of methanol facilitates the mixing of the reagent and the effluent stream. Safety considerations limited the amount of radioactivity that could be used in our animal care facility and only the major metabolites could be quantitated by collecting and counting fractions from the HPLC. The source of other compounds and enzymes is as previously described ( 14). RESULTS
Calibration curves for N-OH-l-NA and N-OH-2-NA are presented in Fig. 2. The peak areas were directly proportional to the
amount of N-hydroxyarylamine, each with a limit of detection (twice background) of approximately 0.1 pg (0.6 nmol) per injection. This corresponded to 0.017% of the arylamine dose excreted in the usual 500-ml urine sample, and this level of sensitivity proved to be adequate for the determination of oxidized urinary metabolites used in this study. The cumulative excretions of I-NA and 2-NA metabolites were as follows. In the first 24 h after dosing, 70% of the radioactivity was excreted into the urine. The first extraction (Fraction I) removed 21% of INA metabolites and 7% of the 2-NA metabolites into the organic phase. The unextracted products in the aqueous fraction consisted of highly water-soluble conjugates.
FIG. 2. Calibration curves for N-OH-I-NA and NOH-2-NA. An ethyl acetate solution of a synthetic standard of N-OH-I-NA was eluted through the HPLC apparatus described under Materials and Methods with 50% methanol-water and a flow rate of 2 ml/min. The mean peak areas derived from the integrator connected to the postcolumn reactor (detector 2) are recorded ( + SD) with the detector on 0.02 absorbance units for full scale deflection with the smallest injection.
DETECTION
123
OF N-HYDROXYARYLAMINES
.
Incubation of this fraction at pH 4 hydrolyzed N-glucuronides and allowed extraction of the aglycones into the organic phase (Fraction II), accounting for an additional 18 and 3.4% of the total urinary metabolites of l-NA and 2-NA, respectively. A typical chromatogram for Fraction II is shown in Fig. 3. The uv trace is quite complex, and the proximity of the small N-OH2-NA peak to other dominant uv-absorbing peaks makes quantitation of the N-hydroxy metabolite by peak area (or radioactivity) quite difficult. In contrast, the plot of absorption at 535 nm from the calorimetric reaction in the postcolumn reactor is much less complex, and the small but reproducible peak for JV-OH-2-NA is well resolved and easily quantitated. Using these methods, the urinary amounts of l-NA and 2-NA and their nitroso and nitro derivatives were quantitated from integration of ultraviolet-absorbing peak areas; and urinary N-OH-lNA and N-OH-2-NA levels were determined by integration of visible-absorbing peak areas, as mediated by the postcolumn reactor. Urinary Metabolites in the Monkey
N-OH-2-l 2-NitroroNophtholene
of 1 -NA and 2-NA
Analysis of the urine of monkeys given lNA (Table IA) indicated that the parent compound was a major excretory product (2 1.1% of the dose) and that it was present in both the free form (Fraction I) and as the glucuronide (Fraction II). Only minimal amounts of N-OH- l-NA (0.15%) and trace amounts (
1
I I IO 15 Time (Min)
I 20
FIG. 3. The simultaneous HPLC chromatograms from detectors 1 (uv; top) and 2 (visible; bottom) following elution of Fraction II from monkey urine after 2-NA dosing. The IV-OH-2-NA peak is well resolved in the bottom chromatogram from the postcolumn reactor, but is not well-defined from other uv-absorbing (but nonreactive) components in the top chromatogram. Peaks C,,z are components of control urine samples, and peak M, is a metabolite (structure unknown) of 2-NA.
observed. To validate the extraction techniques, control urine samples were spiked with the free N-hydroxyarylamines and their iV-glucuronides. More than 95% of the Nhydroxyarylamines were recovered in each case as determined by HPLC analysis.
124
FREDERICK,
MAYS, AND KADLUBAR TABLE
EXCRETION
Expt
OF I-NA
Compound
AND 2-NA
1
METABOLITES
O-24
h AFTER DOSING
Retention time (min)
Free
IV-Glucuronide
Percentage of dose excreted
A
I-NA N-OH-I-NA I-Nitrosonaphthalene I-Nitronaphthalene
15.8 14.9 19.0 18.3
21.1 f 0.1
17.7 + 0.5 0.15 + 0.02
B
2-NA N-OH-2-NA 2-Nitrosonaphthalene 2-Nitronaphthalene
15.7 14.8 18.8 18.6
2.85 + 0.23
1.20 + 0.20 1.03 + 0.18 0.10 + 0.05
’ The data are expressed as the mean f SD (n = 3).
Applicability
to Other Compounds
Preliminary experiments were conducted with a variety of reducing agents of biological and toxicological interest. The technique appears to be suitable for N-hydroxyarylamines, phenolic amines (2-amino- 1-naphthol), ascorbic acid, and aryldiamines (benzidine). The compound for analysis need only have an oxidation potential such that it will efficiently reduce Fe+3 under the reaction conditions at a rate (~30 s) that is adequate for detection. DISCUSSION
We have developed a unique and reasonably specific analytical technique for the quantitation of carcinogenic N-hydroxyarylamines. The values reported for total Noxidized naphthylamine metabolites in rhesus monkey urine approximate those observed previously in dogs and monkeys by a less specific gas chromatographic technique (9,13). This HPLC technique, supplemented with the postcolumn reactor, is suitable for the analysis of a variety of biological reducing agents and oxidized metabolites, e.g., ascorbic acid, aryldiamines, and phenolic amines. The rate of reaction of these compounds with the reagent in the mixing coil determines the intensity of the colori-
metric response and will vary somewhat depending upon the compound (cf. Fig. 2). By this method, we found that N-hydroxylation and subsequent N-glucuronidation of l- and 2-NA is a significant, though relatively minor metabolic pathway (x2%), in the rhesus monkey. Thus, the urinary excretion of the N-hydroxy metabolite should be a reasonable indication of the carcinogenic potency of these compounds in the test species, as their formation is considered to be a necessary prerequisite for the initiation of bladder carcinogenesis ( 1,8,16). This urinary analysis did indicate that N-OH-2-NA accumulates in the urine as the N-glucuronide at a level that is 6.8 times that of NOH- l-NA N-glucuronide. Furthermore, the unoxidized l-NA is excreted at a level 10 times that of 2-NA, indicating that it may be a poor substrate for the oxidative metabolic enzymes of the monkey. These results correlate with the carcinogenicity of the parent amines in a variety of species ( 1). In contrast to previous studies on the urinary excretion of l- and 2-NA in dogs (9, 13), we did not detect free N-OH- I-NA or N-OH-2-NA. This is consistent with our recent study involving the bladder instillation of radioactive N-OH-l-NA and N-OH-2NA into rats (18). These compounds were
DETECTION
OF N-HYDROXYARYLAMINES
absorbed from the bladder at a rate (>90%/ 30 min) that was independent of urine acidity; consequently only trace amounts of the free N-hydroxyarylamine should be detectable in those species retaining their urine for extended periods of time. Previously, we and others (1,8,16) have proposed that acid-catalyzed hydrolysis of N-hydroxyarylamine iV-glucuronides in the urine (pH 5-6) is necessary for their conversion to the N-hydroxyarylamine, and that this step may be a necessary prerequisite for arylamine-induced bladder carcinogenesis. However, the urinary pH of the monkeys used in this study ranged from 6.7 to 7:5 which suggests that they would be relatively resistant to bladder carcinogenesis. Conzelman et al. noted that monkeys were much less susceptible to bladder tumors from 2NA than beagle dogs which were treated similarly (12). They calculated that to induce a 75% tumor incidence, a dose of 200 mg/kg, 6 days/week, for 36-60 months was necessary in their monkey colony, versus a dose regimen of only 6.25 mg/kg, 6 days/ week, for 24 months in their dog colony. It should also be noted that dog and human urine typically has a pH range of 5 to 6 (8,17), and these species may conform to a model in which an acidic urine enhances oncogenic susceptibility. ACKNOWLEDGMENTS The authors would like to express their appreciation to John Bailey and Skeeter Georgeson for their skilled contribution in animal handling and dosing.
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