Chem. Res. Toxicol. 1990, 3, 296-300
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Microsomal N-Hydroxylation of the Glycolamide 2-(Glycolylamino)fluorene To Give the Glycolylhydroxamk Acid. A New Xenobiotic Reaction Michael D. Corbett,**tt*Bernadette R. Corbett,t Sergio J. Quintana,? Marie-Helene Hannothiaux,? and Cheng-I Weit Food Science and Human Nutrition Department and Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32611 Received January 17, 1990
The glycolamide 2-(glycolylamino)fluorene was found to be metabolized in part by induced rat liver microsomes to the hydroxamic acid N-hydroxy-2-(glycolylamino)fluorene.This is the first report of the ability of a microsomal system to carry out the N-hydroxylation of a glycolamide. A comparison of the relative rates of metabolism of the acetyl and glycolyl amides of 2-aminofluorene showed that the former gave about twice as much of the hydroxamic acid as did the latter. On the other hand, the overall metabolism of the glycolamide was slightly more rapid than that of the acetyl congener. Both the glycolyl- and acetyl-derived hydroxamic acids were further metabolized to unknown products by microsomal preparations in the presence of
NADPH.
Introduction The genotoxicity of aromatic amines and nitro compounds has been shown in many cases to result from the metabolic conversion of such functional groups to the arylhydroxylamine and/or hydroxamic acid metabolites (1,2). In some cases the arylhydroxylamineis sufficiently reactive, particularly under mildly acidic conditions, to form covalent adducts with nucleic acids (3);however, further bioactivation of arylhydroxylamines is often necessary in order for adduct formation to occur. Esterification to produce highly reactive N-acetoxy ( 4 ) and N(sulfooxy) (5) metabolites as the ultimate genotoxic species has been shown to contribute to this required bioactivation in some cases. On the other hand, it appears that all hydroxamic acids require further bioactivation before reaction with macromolecules can occur (2,3). The bioactivation of most hydroxamic acids is currently thought to prxeed via either enzymatic hydrolysis to the hydroxylamine, 0-sulfation to give the N-0 sulfate ester, or N-0 acyl transfer to give the highly reactive N-acetoxy arylamine (2,3). Hydroxamic acid activation has also been shown to occur through a peroxidative pathway (reviewed in ref 6). Most studies on the bioactivation of hydroxamic acids have been concerned with N-acetylhydroxamic acids, since metabolic acylation of arylamines and arylhydroxylamines occurs almost exclusively with acetate as the acyl group (Figure 1, reactions 5 and 6). Although formylation of arylamines is known to occur (7),it is not thought to be a major metabolic reaction in a quantitative sense, and its toxicological significance is not known (7,8). Several research groups have conducted various metabolic studies with hydroxamic acids and arylamides derived from acyl groups other than acetate (8-14); however, with the exception of the formyl derivatives already noted, there is no basis upon which to expect such hydroxamic acids or *Address correspondence to this author at the University of Florida, IFAS-0163, Gainesville FL 32611-0163. ‘Food Science and Human Nutrition Department. t Department of Pharmacology and Therapeutics.
arylamides to be produced as metabolites of arylhydroxylamines or arylamines. On the other hand, there is recent evidence which suggests that arylyides and arylhydroxamic acids derived from glycolic acid might be important metabolites of arylamines, although they are not produced to the extent that has been observed for the acetate-derived metabolites. The glycolamides are not known to be produced by simple acylation, as occurs in the production of acetamides. Both in vitro and in vivo studies have shown that the acetyl group of certain arylacetamides is metabolized by w-oxidation of the methyl group to yield glycolamides as metabolites (Figure 1, reaction 2) (15, 16). No attempts have been reported to determine if such glycolamides might be further metabolized by N-oxidation to give the analogous glycolylhydroxamic acids (Figure I, reaction 9). In fact, indirect evidence suggests that such N-oxidation of 4-(glycolylamino)biphenyl might not occur, since this glycolamide was not active as a carcinogen in female rats, whereas 4-(acety1amino)biphenylwas quite active (12). King concluded that a glycolamide does not appear to be involved in the tumorigenicity of arylamines (17). However, we have discovered a novel metabolic reaction whereby glycolylhydroxamic acids can be produced without the need for the intermediary formation of the glycolamide (18, 19). This reaction (Figure 1, reaction 1) involves the action of transketolase on aromatic nitroso compounds, which in turn are known to be important and very toxic metabolites of many arylamines and related xenobiotics (Figure 1, reactions 3 and 4). Transketolase is an enzyme of the pentose phosphate cycle and is found in most mammalian cells (20). Such a pathway provides a means by which glycolylhydroxamic acids can be produced as metabolites of the nitroso oxidation state in a wide variety of cell types. We also observed that the glycolylhydroxamic acid (NOH-2-GAF)’ was approximately as mutagenic as the acetohydroxamic acid (N-OH-2-AAF)in several Salmonella Abbreviations: 2-AF, 2-aminofluorene; 2-AAF, 2-(acetyla”o)fluorene; N-OH-2-AAF,N-hydroxy-2-(acetylamino)fluorene;2-GAF, 2(glycoly1amino)fluorene; N-OH-2-GAF, N-hydroxy-2-(glycolylamho)fluorene; N-OH-2-AF,N-hydroxy-2-aminofluorene.
QS93-228~/90/2703-0296$02.50~~ 0 1990 American Chemical Society
Microsomal N-Hydroxylation of Glycolamides HO 0 I II Ar -N- CH
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Figure 1. Pathways for the metabolic production of hydroxamic acids. Known reactions are illustrated with solid-line arrows (-). Reactions: (1)Transketolase-catalyzed conversion of a nitroso aromatic to a glycolylhydroxamic acid; (2) w-oxidation of an acetamide to give a glycolamide; (3) microsomal N-hydroxylation of an arylamine to give an arylhydroxylamine;(4) nonspecific air or peroxide oxidation of an arylhydroxylamine to a nitroso aromatic (e.g., oxyhemoglobin);(5) acetylation of an arylamine to give an arylacetamide; (6) acetylation of an arylhydroxylamine to give an acetohydroxamic acid; (7) microsomal N-hydroxylation of an acetamide to give an acetohydroxamic acid; (9) microsomal N-hydroxylation of a glycolamide to give a glycolylhydroxamic acid; (10) pyruvic acid metabolizing enzymes (see ref 18); (11)the nitrosc-glyoxylatereaction (see ref 27). Reaction 8 w-Oxidation of an acetohydroxamic acid to give a glycolylhydroxamic acid is a potential metabolic reaction for which evidence is lacking. Reactions known to proceed in both directions, although not necessarily catalyzed by the same enzyme, are shown by a double arrow. Ar = aromatic group. strains (Corbett et al., unpublished results). In view of this
observation, we made an effort to determine whether or not N-hydroxylation of a glycolamide might be a metabolic reaction catalyzed by a cytochrome P-450 enzyme.
Materials and Methods Chemicals. 2-AF and 2-AAF were obtained from Sigma Chemical Co. (St. Louis), and 2-nitrofluorenewas obtained from Aldrich Chemical Co (Milwaukee).2-GAF and N-OH-2-GAF were prepared as previously described (21). N-OH-2-AF prepared by the Pd/C-catalyzed reduction of the corresponding nitro compounds with hydrazine hydrate in tetrahydrofuran (22). N-OH2-AAF was prepared by acylation of N-OH-2-AF according to a common procedure (23);mp 149-151 OC [lit (22) mp 149-151 OC]. In Vitro Metabolic Studies. Liver microsomes were obtained from frozen rat liver S-9 fractions that had been prepared from male Sprague-Dawley rats by the standard procedure, including induction with Arochlor 1254 (24).After thawing, the S-9 fraction was diluted and resuspended in 20 mM TriwHCl, pH 7.4, containing 0.154 M KC1 and then centrifuged at lOOOOOg for 60 min a t 4 "C. The pellet was resuspended in the same buffer by use of a Potter-Elvehjem tube and recentrifuged. The microsomal pellets were stored at -70 OC in 10 mM HEPES-HC1, pH 7.6, containing 0.154 M KCl, 1mM EDTA, and 20% glycerol at a concentration of 8.7 mg/mL protein. The microsomal protein concentration was determined by the Bradford method employing bovine serum albumin aa standard (25). In one series of experiments, the microsome fraction was prepared and used on the same day. Incubations of the amide substrates were conducted at 37 OC in 25-mL polycarbonate Erlenmeyer flasks by adding 0.1 mL of the microsomal suspension (4 "C) to 6.4 mL of a buffer solution equilibrated to 37 OC and containingreactants and cofactors such that the final reaction concentrations were 0.10 M KH2P04,pH 7.4, 3 mM MgClz, and 2.5 mM NADPH (Sigma type I). The substrates (generally0.325 pmol) were added to each incubation mixture as a solution in 26 pL of dimethylformamide immediately
Chem. Res. Toxicol., Vol. 3, No. 4, 1990 297 prior to the addition of minosomes. The incubations were agitated at 37 "C, and aliquota of 1.5mL each were taken at various times following the start of the reaction. The aliquota were combined with 2.5 mL of ice-cold ethyl acetate in a 15-mL polypropylene centrifuge tube and vigorously shaken to stop the reaction. Following centrifugation,the aqueous layer was reextracted with 2.5 mL of ethyl acetate, and then the organic extracts were combined and evaporated with a stream of Ar. The residue was dissolved in 150 pL of DMF, and 10-pL portions were analyzed by high-pressure liquid chromatography (HPLC) employing a ClS pBondapak column (Waters Associates) and an isocratic solvent system consisting of 50% MeOH buffered to pH 3.5 with 5 mM KH2P04/H3P04which contained 0.01 % desferal mesylate (26). The productions of N-OH-2-AAF and N-OH-2-GAF from 2-AAF and 2-GAF, respectively, were determined by measurement of peak heights and comparison to those of authentic standards. The consumption of amide substrates was also determined through the measurement of peak heights. The identities and purities of the peaks for N-OH-2-AAF, N-OH-2-GAF, 2-AAF, and 2-GAF were confirmed by examination of the UV spectra generated by a rapid-scanning UV detector (LKB Model 2140 rapid spectral detector), which was used in tandem with a Waters Model 440 UV detector (A 280). To confirm that the chemical structure of the metabolite in question was that of N-OH-2-GAF, the metabolite was isolated in nearly pure form. This was achieved by carrying out the microsomal incubation for 30 min on a 30-mL volume with the concentration of 2-GAF at 0.5 mM. The reaction was extracted twice with ethyl acetate, and then the extract was dried (NafiOJ and evaporated. The residue was dissolved in 400 pL of DMF, and 100-pL portions, one at a time, were chromatographed on a preparative-scale column GLBondapak C18,7.8 mm X 30 cm)with the same buffered solvent as was used for analytical chromatography except with 45% MeOH at a flow rate of 3 mL/min. Those fractions containing the metabolite of interest were combined and extracted three times with CH2C12. After drying (Nafi04), the solvent was evaporated, and the residue was dissolved in 20 pL of ethanol and analyzed by thin layer chromatography along with authentic N-OH-2-GAF. The analyses were achieved on 5 X 10 cm EM silica gel 60 plates (E. Merck) with MeOH/CH2C12(595) as the solvent. Following development, the plates were dried and then sprayed with 1% FeC13to detect hydroxamic acids (191, which had identical R, values of 0.20.
Results and Discussion The production of N-OH-2-GAF via the microsomal oxidation of 2-GAF was determined by HPLC methods. A typical HPLC chromatogram of the products which resulted from the incubation of 2-GAF with rat liver microsomes is presented in Figure 2. The product peak (M), indicated by the mow, was found to have a retention time identical with that of the synthesized standard for N-OH2-GAF. Confirmation of this identity was made through comparison of the UV spectra of both the metabolite and authentic N-OH-2-GAF during chromatography under identical conditions. The UV spectra (Figure 3), which were obtained by use of a rapid-scanning detector, are virtually identical. In addition, the proof that the metabolite, M, possessed the hydroxamic acid functional group was achieved by application of a highly specific colorimetricreaction for this functional group, as has been previously described (19,27). Hydroxamic acids form an intense violet-colored complex with Fe3+,which serves as a sensitive method for their detection on thin-layer chromatograms. The metabolites corresponding to both M and synthetic N-OH-2-GAF chromatographed on thin-layer plates in an identical manner and gave the same colorimetric reaction with Fe3+. The microsomal metabolism of the two amides, including their conversions to the corresponding hydroxamic acid metabolites, are illustrated in Figure 4. Most notable was the production of a higher concentration of the corresponding hydroxamic acid metabolite from 2-AAF than
298 Chem. Res. Toxicol., Vol. 3, No. 4, 1990
Corbett et al.
8-
6 V 0
-
I
10
20
30
0
IO
20
30
Figure 4. Microsomal metabolism of 2-AAF and 2-GAF as a function of time. (a) Conversion of 2-AAF to N-OH-2-AAF ( 0 ) and 2-GAF to N-OH-2-GAF (0).(b) Depletion of the amide substrates from the incubations: (0) 2-AAF and (m) 2-GAF. All points are the average (rtSD) of three time course experiments conducted with the same microsomal preparation.
m I
9 X
40 0
0
2 I
n
n
-
a
2-
IO 20 Time (min)
0
Figure 2. HPLC chromatogram of the microsomal metabolites of 2-GAF. The chromatogram was obtained on a concentrate obtained from the incubation of 2-GAF (50 rM) for 10 min with freshly prepared rat liver microsomes as described under Materials and Methods. The metabolite of interest is indicated as M in the chromatogram and had a retention time of 12.6 min under the HPLC conditions described. The substrate, 2-GAF, eluted as the large peak with a retention time of 15 min. An unknown metabolite eluted at 17 min. Under identical conditions, NOH-2-AAF and 2-AAF had retention times of 15 and 18 min, respectively.
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.
.
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.
.
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Wavelength (nm) Figure 3. UV spectra for authentic N-OH-2-GAF and a microsomal metabolite of 2-GAF. Spectra were obtained through the use of a diode-array spectrophotometer to monitor the elution of components from the HPLC chromatogram presented in Figure 2. (-) UV spectrum for the metabolite, M, indicated in Figure 2; (- - -) UV spectrum for authentic N-OH-2-GAF which was chromatographed immediately after the analysis described in Figure 2. from 2-GAF under identical conditions (Figure 4a). On t h e other hand, t h e rates of total metabolism of the two amides were very similar (Figure 4b). T h e data pesented in Figure 4 suggest t h a t more 2-AAF is N-oxidized than is 2-GAF; however, kinetic differences in subsequent re-
IO
Time
20
30
(min)
Figure 5. Microsomal metabolism of N-OH-2-GAFand N-OH2-AAF with and without NADPH. The hydroxamic acid substrates (10 pM) were incubated with microsomal preparations in a manner identical with that described under Materials and Methods for the amide substrates, both in the presence of 2.5 mM NADPH (solid symbols) and in the absence of NADPH (open symbols). ( 0 ,0)N-OH-2-AAF; (m, 0)N-OH-2-GAF. actions of the two different hydroxamic acids could be major contributors to the shapes of these time course curves. On the other hand, Figure 5 indicates t h a t NOH-2-GAF is metabolized only slightly faster than is N-OH-2-AAF by microsomes. It is interesting to note that both of these hydroxamic acids are metabolized further by microsomal enzymes, but significant metabolism occurred only in the presence of NADPH. T h e products of these reactions have not been determined or previously reported. The same N-oxidation reaction on 2-GAF to give N-OH-2-GAF was observed with microsomes t h a t were freshly prepared, and which were never subjected t o freezing prior to use. Although extensive studies on the effect of substrate concentration were not completed, this reaction was also observed t o occur with concentrations of 2-GAF as high as 1mM. Such high concentrations were employed t o obtain N-OH-2-GAF on a preparative scale from microsomal reactions. This is t h e first report on the ability of a microsomal preparation t o effect the N-oxidation of any glycolamide (Figure 1, reaction 9). Our results suggest t h a t glycolamides are probably less susceptible t o N-oxidation than are the analogous acetamides. The microsomal conversion of 2-AAF t o 2-GAF has previously been inferred on the basis t h a t 2-GAF was observed as a trace urinary metabolite of 2-AAF (15).T h e conversion of arylacetamides t o glycolamides is apparently more important for mononuclear aromatic compounds than for polynuclear systems
Microsomal N-Hydroxylation of Clycolamides
such as 2-AAF (15). Nevertheless, no reports have been made on the potential for subsequent N-oxidation of any glycolamide metabolites to produce glycolylhydroxamic acids. The report on the conversion of 2-AAF to 2-GAF ( E ) , combined with our present results, suggests that the potential exists for the production of a very small amount of N-OH-2-GAF as a microsomal metabolite of 2-AAF. Attempts to detect the microsomal conversion of N-OH2-AAF to N-OH-2-GAF were made; however, only a small amount of an HPLC peak corresponding to N-OH-2-GAF was observed during the microsomal metabolism of NOH-2-AAF. Although this peak cochromatographed with authentic N-OH-2-GAF, and the response ratio for X313/ Xzso was 0.18 for both this metabolite and authentic NOH-2-GAF, confirmation by use of an LKB diode array spectrophotometric detector was not possible due to the very limited sensitivity of this instrument. We previously reported that the in vitro reaction of aromatic nitroso compounds with transketolase is a general pathway (Figure 1, reaction 1)for the production of glycolylhydroxamic acids (18,19,21,28). Our present results show that it is possible that this unusual type of hydroxamic acid metabolite might also be produced by microsomal oxidation reactions. At this point, it is difficult to predict the overall toxicological effect of the metabolic production of glycolylhydroxamic acids. The nitroso functional group in general is highly toxic and is even known to be much more mutagenic than the analogous hydroxamic acid in most cases (29-31).On the other hand, hydroxamic acids are much less reactive and could serve as transport forms of potentially genotoxic metabolites. Research is in progress to determine the relative degree of nucleic acid binding of N-OH-2-AAF and N-OH-2-GAF via enzymatic reactions thought to contribute to the carcinogenic bioactivation of N-OH-2-AAF. Some of these studies indicate substantial differences between the potential genotoxicity of acetyl- and glycolyl-derived hydroxamic acids, both in terms of potency (6) and in terms of the nature of the final bioactivation pathways (32).
Acknowledgment. This research was supported by Grant ES 03631 from the National Institute of Environmental Health Sciences, DHHS. This paper is journal series no. ROO073 from the University of Florida Agricultural Experiment Station. Registry No. 2-GAF, 51480-57-0; N-OH-2-GAF, 111959-989; 2-AAF, 53-96-3; N-OH-2-AAF, 53-95-2.
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