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Chem. Res. Toxicol. 1988, 1, 108-114
Identification of a Novel Dihydroxy Metabolite of Aflatoxin B, Produced in Vitro and in Vivo in Rats and Micet David L. Eaton,* David H. Monroe, Garland Bellamy, and David A. Kalman Department of Environmental Health and Institute for Environmental Studies, SC-34, University of Washington, Seattle, Washington 98195 Received November 30, 1987
HPLC analysis of bile obtained from rata given aflatoxin B1 (AFB) demonstrates the presence of numerous polar metabolites. The glutathione conjugate derived from AFB 8,g-epoxide and the glucuronide conjugate of aflatoxin P1(AFP) have previously been identified as the two major polar metabolites. The most polar peak present in bile from AFB-treated rats is converted to a less polar peak upon incubation with P-glucuronidase, which has a parent ion m / e of 314 amu. Treatment of this aglycon with diazomethane produced a product which cochromatographs with aflatoxin MI (AFM). From these data it is concluded that the most polar peak in bile from AFB-treated rats is the glucuronide conjugate of 4,9a-dihydroxyaflatoxin B1. This dihydroxy AFB metabolite was produced in vitro in mouse microsomal incubations, and time-course studies of its production suggest that it is largely formed by 9a-hydroxylation of AFP, although some may be formed by 4-O-demethylation of AFM. Direct incubation of AFP and AFM with mouse microsomes confirmed that this metabolite can be formed from both AFP and AFM. An HPLC method is described which is capable of base line resolution of this novel dihydroxyaflatoxin metabolite and eight other hydroxylated metabolites of AFB, as well as the glutathione conjugate of AFB 8,g-epoxide.
Introduction The highly toxic and carcinogenic mycotoxin aflatoxin B1 (AFB) undergoes oxidative biotransformation to three established monohydroxy derivatives (Figure l),which are commonly referred to as aflatoxin M1 (9a-hydroxyaflatoxiq AFM), aflatoxin Pl (4-hydroxyaflatoxin, AFP), and aflatoxin Q1 (3-hydroxyaflatoxin, AFQ) (1,2). These mixed function oxygenase-mediated reactions in general lead to less toxic and less carcinogenic metabolites (2, 3). The microsomal oxidation of the 8,9-unsaturated carbons of AFB leads to the putative carcinogenic intermediate, aflatoxin 8,9-epoxide, which readily binds to nucleophilic macromolecules, including DNA (4). This epoxide can undergo nucleophilic substitution with glutathione at the 9-carbon position to yield the 8,9-dihydro-8-S-glutathionyl-9-hydroxyaflatoxin B1 (AFB-GSH), or may be hydrolyzed to the corresponding 8,9-dihydrodiol, which may form Schiff bases with endogenous or exogenous amines (5). Aflatoxin Bk (&hydroxy-8,9-dihydroaflatoxin,AFB2,) may also be formed by oxidation at the 8,9-unsaturated carbons (1,2), but the importance of this pathway in the disposition of AFBl is unclear. In addition t o these oxidative pathways, reduction by a cytosolic enzyme of the l-carbonyl to a hydroxyl group may occur to yield aflatoxicol (l-hydroxyaflatoxin), which may be an important route of metabolism in some species, especially avians (6, 7) and fish (8). Aflatoxicol may undergo further oxidation in the 3-position to yield aflatoxin H1(l,&dihydroxyaflatoxin, AFH) (1). Aflatoxicol MI (1,9a-dihydroxyaflatoxin) has been identified as a metabolite of aflatoxicol in dogs (9) and rainbow trout (8). Glucuronide conjugates of various monohydroxy AFB metabolites have been iden*To whom correspondence should be addressed. 'Presented in part at the 1987 annual meeting of the Society of Toxicology.
tified, including AFP in rats (10) and chickens ( I I ) , AFQ in rata (1.21, and aflatoxicol and aflatoxicol M1 in rainbow trout (13). Wong et al. (14) found that treatment of aqueous urinary metabolites of AFB obtained from rhesus monkeys with @-glucuronidaseand arylsulfatase released AFM, indicating that glucuronide and sulfate conjugates of AFM are also formed in vivo. The glutathione conjugate of AFB has been identified as the major aflatoxin-related peak excreted in bile of rats, and accounts for approximately 30-40% of total biliary metabolites (10, 15, 16). Moss et al. (17) have demonstrated that AFB-GSH is a substrate for y-glutamyl transpeptidase in vivo and that the cysteinylglycine conjugate of AFB (AF'B-Cys-Gly) is present in bile of Fisher 344 rats, particularly when y-glutamyl transpeptidase activity has been induced. These investigators reported that AFB-Cys-Gly was present in bile of male Fisher rats at approximately 20% of the concentration of AFB-GSH, whereas in rats with induced y-glutamyl transpeptidase activity, equal amounts of AFB-GSH and AFB-Cys-Gly were found in bile. Except for the 8,g-dihydrodiol of AFB that is formed in vitro in microsomal incubations (5,18,19) and the oxidative products of aflatoxicol metabolism, AFH and aflatoxicol M1, no dihydroxy metabolites of AFB have previously been characterized. In the present study, we have identified and characterized the glucuronide conjugate of a dihydroxy metabolite of AFB as a substantial biliary metabolite of AFB in rats. The formation of this metabolite is largely produced as a secondary metabolite of AFP in mouse microsomal incubations.
Materials and Methods Chemicals. Aflatoxins B1,Ql, MI, P,, and Bz, and aflatoxicol were obtained from Sigma Chemical Co. (St Louis, MO). Purity of the aflatoxins, determined by absorbance monitoring a t 365
0893-228~/88/2701-0108$01.50/0 0 1988 American Chemical Society
Dihydroxyaflatoxin Metabolite Identification
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Figure 1. Basic structure of aflatoxin and major monohydroxy metabolites, with positions numbered according to IUPAC nomenclature. nm with high-pressure liquid chromatography (HPLC), was greater than 98%, with the exception of AFQ. Commercial sources of AFQ, which are made by chemical oxidation of AFB, contain two apparent isomers of AFQ in an approximately equimolar ratio, as evidenced by the presence of two resolved HPLC peaks with identical mass spectra, neither of which coelute with AFM. The second of these two peaks coelutes with AFQ generated in microsomal incubations, whereas the first is not formed in in vitro systems. Aflatoxin 8,9-dihydrodiol and the Tris-diol Schiff base were synthesized according to the methods of Swenson et al. (18) and Neal and Colley (19). @-Glucuronidase(E.C. 3.2.1.31, from E. coli) was obtained from Sigma Chemical Company. All other chemicals were of reagent grade and available through commercial chemical supply houses. Safety Precautions. Procedures for handling and decontamination of AFB materials used in this laboratory were adopted from recommendations published by the World Health Organization and the International Agency for Research on Cancer (20). Animals and Treatments. Adult male Sprague-Dawley rats (200-300 g) were purchased from Tyler Labs (Bellevue, WA) and housed in a controlled environment (12-h day-night light cycle). Food (Wayne Lab-Blox) and water were provided ad libitum. Male Swiss-Webster mice (28-32 g, Tyler Labs, Bellevue, WA) were housed with a 12-h light cycle and provided food and water ad libitum. Mice were fed a standard rodent diet (Wayne LabBlox) containing 0.75% BHA for 13 days, as described previously (22). For in vivo biliary excretion studies, rata were anesthetized with urethane (1 g/kg, ip) and body temperatures were maintained at 37 OC through the use of a rectal probe coupled to a thermoregulator and a heat lamp. The bile duct was cannulated with PE-10 tubing, the wound was closed by using surgical autoclips, and bile was collected for 15 min prior to dosing. Animals were given AFB (5 mg/kg AFB, 1mL/kg in DMSO, ip), and bile was collected in 30-min intervals, in preweighed collecting tubes, and stored on ice away from light. Bile volume was determined gravimetrically. HPLC Analysis of Hydroxylated AFB Metabolites. This procedure will be referred to as the phase I HPLC procedure. A 150 mm X 4.6 mm Alltech 5-pm C-18 column was used, with a flow rate of 1.5 mL/min and column temperature of 40 OC. The three mobile phases used were as follows: A, 50 mM ammonium acetate, pH 3.5; B, 95% methanol, 5% tetrahydrofuran; C, HPLC grade water. Initial conditions were 90% A, 10% B for 0.5 min, with linear solvent gradients to obtain 24% B, 76% C a t 2 min, then 38% B, 62% C at 13 min, and 90% B, 10% C a t 16 min. HPLC Analysis of Bile. A 250-pL aliquot of bile was combined with 25 pL of 0.5 M sodium phosphate buffer, pH 3.5, and was then filtered through a 0.45 pm filter. Reversed-phase HPLC analysis of 1WpL bile samples was conducted on an IBM LC9953 HPLC, equipped with a 30 X 0.5 cm C-18 preparative HPLC column, at 40 "C, as described previously (10). This procedure
Chem. Res. Toxicol., Vol. I, No. 2, 1988 109 will be referred to as the phase I1 procedure. Water, methanol, and 0.05 M sodium phosphate, pH 3.50, were the mobile phases used with the following gradient: 10% methanol, 90% buffer at 0 min, with linear gradients to 24% methanol, 76% buffer at 1 min, 26% methanol, 74% buffer at 12 min, 30% methanol, 70% buffer a t 20 min, 50% methanol, 50% water at 22 min, 65% methanol, 35% water at 35 min, and 98% methanol, 2% water a t 36 to 40 min. The column was reequilibrated a t starting conditions for a t least 10 min prior to sample injection. UV absorbance at 365 nm was monitored for the presence of AFBrelated peaks. Peak areas were determined with a Shimadzu C-R3A Chromatopac integrator and converted to nanomoles of AFB equivalents with a standard curve constructed by analysis of reference bile spiked with authentic AFB and assuming an equivalent extinction coefficient at 365 nm. Bile samples collected from each animal prior to AFB administration were chromatographed to identify endogenous compounds which absorb at 365 nm. Good correlations between AFB metabolite quantification determined by peak integration and by distribution of 3H radioactivity have been obtained in previous studies, indicating that the assumption of equivalent extinction coefficients for metabolites and authentic AFB is appropriate (10, 21). Incubation with 8-Glucuronidase. The peak of interest was collected from repeated HPLC analyses of bile, pooled and reduced in volume under nitrogen. The pH was adjusted to 6.8 with 0.5 M K2HP04,and a 100-pL aliquot of p-glucuronidase (1mg/mL in 0.05 M K,HPOS was added to 200 p L of sample. Samples were incubated for 1 h at 37 "C. Small aliquots were removed and added to an equal volume of ice cold methanol at 15-min intervals to monitor the progress of the reaction. The pH was adjusted to approximately 3 with a small volume of glacial acetic acid, and samples were analyzed by HPLC as described above. Methylation of the Phenolic Hydroxyl Group of Isolated Aglycon. The aglycon obtained from incubation with p-glucuronidase was isolated by HPLC and evaporated to dryness under vacuum. Diazomethane was generated from 1-methyl-3-nitro1-nitrosoguanidine (MNNG) by dropwise addition of 5 N NaOH in a MNNGdiazomethane apparatus (Aldrich Chemical Co., Milwaukee, WI). The freshly generated diazomethane was trapped in diethyl ether, and a 500-pL aliquot was mixed with the residue. After 30 min, the ether/diazomethane was removed under nitrogen, and 100 pL of methanol was added. An equal volume of 0.05 M ammonium acetate, pH 3.5, was added, and an aliquot was analyzed by the phase I HPLC procedure. Mass Spectral Analysis of t h e Isolated Aglycon. An aliquot of the hydrolyzed peak resulting from p-glucuronidase treatment was evaporated to dryness under vacuum, and the residue (approximately 10 pg, estimatd from peak area integration and assuming an extinction coefficient equivalent to AFB) was dissolved in 10 pL of methanol. The total sample was analyzed by desorption chemical ionization using a direct chemical ionization probe and negative chemical ionization mass spectroscopy with methane reagent gas on a Finnigan 4023 GC/MS. Spectra were acquired between 80 and 650 amu. Mass spectra of known standards (AFM, AFQ, AFP, and AFB) were analyzed in a similar manner and were used to establish optimum temperature gradients for direct probe ionization. Incubation of AFB with Mouse Microsomal Preparations. Livers were obtained from 26 mice which had been treated with 0.75% BHA in the diet for 13 days. The livers were pooled, and microsomal preparations were made as described previously (22). In vitro biotransformation of AFB by mouse hepatic microsomes (0.8-1.6 mg of protein/mL) was conducted as described previously (22). Incubations contained 1 mM GSH and mouse cytosol equivalent to approximately 3 mg of protein/mL to scavenge the epoxide as the glutathione conjugate. Aliquots (100 pL) were removed a t 5-min intervals (up to 45 min) and added to an equal volume of ice-cold methanol. After standing on ice for at least 4 h, samples were centrifuged to remove precipitated proteins and analyzed for metabolites by the phase I HPLC program.
Results I d e n t i f i c a t i o n o f the D i h y d r o x y Metabolite Produced in Vivo. Chromatography of bile which was collected from rats prior to AFB administration shows several
110 Chem. Res. Toxicol., Vol. 1, No. 2, 1988 I A
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following AFB administration (phase I1 HPLC program).
endogenous compounds which have absorbance at 365 nm (Figure 2A). Chromatography of bile obtained from the same rat after AFB administration shows multiple polar metabolites distinct from endogenous compounds, only a few of which have been fully characterized (Figure 2B). The peak at 14.2 min has been identified previously as the glucuronide conjugate of AFP, and the major peak at 25.3 min is the AFB-GSH conjugate (10, 21). Incubation of peak 1 ( t R = 8.4 min) with /3-glucuronidase resulted in a less polar peak ( t R = 28.3) (Figure 3A). When the aglycon of peak 1 was added to bile from AFB-treated rats, the small peak which eluted at 28.4 min (Figure 2B) was increased in magnitude (Figure 3B). These results suggest that a small amount of the aglycon of peak 1is excreted in bile. Peak 2 also was hydrolyzed by @glucuronidase, but peak 3 was not affected by either 0-glucuronidase or arylsulfatase. However, preliminary results indicate that peak 3 is a secondary metabolite of AFB-GSH, possibly the mercapturic acid, as elimination of AFB-GSH formation by reduction of hepatic GSH with a combination of buthionine sulfoximine and diethyl maleate also results in complete loss of peak 3 (23). Direct probe negative ionization mass spectroscopy of the isolated aglycons of both peak 1 and peak 2 revealed a parent ion mass of 314 amu for both. This mass is consistent with a metabolite which has undergone both demethylation and hydroxylation a t a saturated carbon. Based on the known hydroxylation sites of AFB, it could be expected that the metabolites are either 3,4-dihydroxy-AFB (demethylated AFQ) or 4,9a-dihydroxy- AFB (demethylated AFM). It is also possible that the metabolite could be 2,4-dihydroxy-AFB, 4,6a-dihydroxy-AFB, or 4,5-dihydroxy-AFB,although there are no reports of any of these hydroxylation products being formed in vitro in microsomal incubations. Hydroxylation at either the 2or 6a-positions would result in products which are highly chemically unstable and therefore are unlikely candidates. Hydroxylation at the 5-carbon to yield a 4,5-catecholwould
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Figure 3. Effects of treatment of bile peak 1with @-glucuronidase. Panel A shows the production of a nonpolar peak following 10 min of incubation with P-glucuronidase. Complete conversion occurred after 1 h of incubation. No conversion of peak 1 to a nonpolar metabolite was found in parallel incubations containing buffer in place of &glucuronidase. Panel B shows the position of the aglycon of peak 1 in bile, which coelutes with a minor metabolite in bile (see Figure 2B for comparison to nonspiked sample).
also be possible, and this metabolite would be expected to be chemically stable. As insufficient material could be obtained for NMR analysis, the position of the alcoholic hydroxyl group on the dihydroxy metabolite was determined by treatment of the aglycons with diazomethane in ether. Because of the greater acidity of the phenolic hydroxyl group relative to the alcoholic hydroxyl group, selective methylation of the phenolic group should occur. Incubation of the aglycon of peak 1 with diethyl ether alone had no effect on the retention time of the peak (Figure 4A) determined with the phase I HPLC procedure. However, addition of freshly generated diazomethane in diethyl ether to the aglycon of peak 1resulted in the appearance of a less polar peak with a retention time of 11.93, similar to that of AFM (Figure 4B). When authentic AFM was added to the mixture, it coeluted with the methylated peak (Figure 4C). This supports the identity of the aglycon of peak 1 as 4,9a-dihydroxyaflatoxin B1. The same procedure was attempted several times with the aglycon of peak 2, which has a retention time in our phase I HPLC system of 0.5 min later than the aglycon of peak 1. No change in retention time of the aglycon of bile peak 2 was ever observed after incubation with diazomethane. As this is a very minor metabolite in bile and only small amounts of material were available to work with, no further attempts to identify this metabolite were made. Chromatographic Behavior of Aflatoxin Metabolites. The ternary gradient reversed-phase HPLC program developed in our laboratory is capable of base line resolution all of the known oxidative metabolites of AFB, as well as the glutathione conjugate of AFB (Figure 5 ) . Although the ammonium acetate buffer system is used for only a short time in the program, it was found necessary
Chem. Res. Toxicol., Vol. 1, No. 2, 1988 111
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Figure 5. Chromatogram of standards of AFB and metabolites. Base line resolution of eight hydroxylated metabolites of AFB,
and the AFB-GSH conjugate, is achieved with this ternary gradient program (phase I procedure). AFG1, which is not a metabolite of AFB, can conveniently be used as an internal standard. Note that the 4,ga-dihydroxy metabolite (tR= 9.12) would coelute between AFB dihydrodiol and AFB2a. With this procedure, a detection limit of approximately 0.5 pmol (about 150 ng) is achievable. teriorated, presumably because of the spectral shift that occurs when the hemiacetal is converted to the dialdehydic phenolate ion (5). The phenolate ion formed at alkaline pH will readily react with the free amine of Tris to form a Schiff base, which elutes considerably later than the AFB-Tris dihydrodiol (5).
Identification of the Dihydroxy-AFB Metabolite Produced in Vitro. The aglycon of peak 1 from bile
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Figure 4. Remethylation of the phenolic hydroxyl group with diazomethane. Treatment of the aglycon of peak 1with diazomethane (generated from M " G and trapped in ether) resulted in the formation of a less polar peak (B) that is not present when the peak is incubated with ether alone (A). The less polar peak coeluted with AFM, (C).
to retain reproducible retention times and symmetrical peak shapes, especially for the glutathione conjugate. The addition of a small amount of T H F to the methanol was essential to obtain resolution of AFM from AFP and AFG. AFG may be conveniently used as an internal standard. Incubation of AFBz, with Tris buffer at pH 7.4 for 1h resulted in an 80% conversion of the AFBb peak ( t R= 10.1 min) to an apparently less polar peak ( t R = 13.8) (Figure 5). Total conversion occurred within 2 min at pH 10. This peak is presumably the Schiff base of AFBk, as no change in retention time occurs if Tris buffer is substituted with phosphate buffer, although peak shape is noticeably de-
chromatographs with a retention time of 9.10 (Figure 4A). It elutes prior to all of the known monohydroxy AFB metabolites, and slightly later than the 8,9-dihydrodiol of AFB (Figure 5 ) . Thus, this peak can be resolved and identified from other oxidative metabolites produced during in vitro microsomal incubations. Incubation of AFB with microsomes obtained from BHA-treated mice results in extensive metabolism of AFB, relative to that produced by microsomes from rats or untreated mice (22). Thus, to determine if dihydroxy metabolites of AFB are formed in vitro, we utilized BHA mouse liver microsomes. A representative chromatogram of the incubate after 20 min of incubation is shown in Figure 6, With this system, 78% of the added AFB (80 pM initial concentration) was metabolized in 20 min, and greater than 99.9% was metabolized in 45 min. Nearly 90% of the total oxidative metabolism of AFB was to the epoxide (Figure 7A), which is efficiently scavenged as the glutathione conjugate in the presence of cytosolic glutathione S-transferase (22). Numerous hydroxylated metabolites, including AFM, AFP, and AFQ were formed during the initial 15 min of incubation (Figure 7B). The amount of AFQ remained constant after 10 min, and accounted for about 1% of total metabolites. The amount of AFP present in the incubate accounted for 2.3% of total metabolites at 15 min but then declined to less than 0.1% at 45 min. AFM accounted for 5 % of total metabolites at 20 min and then declined slightly to 3.4% at 45 min. The decrease in AFP noted after 15 min was associated with a concomitant increase in 4,9a-dihydroxy-AFB(Figure 7B), which quantitatively accounted for the loss in AFP noted at longer incubation times (Figure 7C). Three ad-
112 Chem. Res. Toxicol., Vol. 1, No. 2, 1988
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ditional hydroxylated metabolites were apparently produced. Two of these eluted after AFB-GSH and before 4,9a-dihydroxy-AFB (Figure 6). One of these coeluted with the Tris dihydrodiol of AFB, which, in the presence of GSH and cytosol, was usually not found in these incubations (22). Another less polar metabolite, which accounted for 1.0% of total metabolism, eluted immediately before AFM (Figure 6), and thus may be an as yet unidentified monohydroxy AFB metabolite. To verify that AFP was indeed converted to the dihydroxy metabolite in microsomal incubations, AFP was added directly to BHA mouse liver microsomes. After 20 min of incubation of 4 pM AFP with mouse liver microsomes, 20% was metabolized to the 4,9a-dihydroxy metabolite (data not shown). AFP can apparently undergo further metabolism to the AFP epoxide, as a small quantity of glutathione conjugate (7%) was also formed. However, in parallel incubations we found that AFM also underwent oxidative demethylation to yield the same 4,9a-dihydroxy-AFB metabolite and also formed a GSH conjugate. The two small peaks which elute just prior to AFB-GSH (Figure 6) may be the GSH conjugates of AFM and AFP, as relative retention times were the same for each of these peaks when incubations with AFP or AFM alone are compared with incubations of AFB. Neither of these peaks are present when GSH and cytosol are absent from the incubation, and neither were present during the first 10 min of incubation with AFB but increased with time with longer incubation.
Discussion The HPLC methods described here offer superior resolution of both conjugated and hydroxylated metabolites of AFB, relative to previously published studies utilizing HPLC for metabolite separation (19,24,25). A significant advantage of this HPLC method is that the rate of for-
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Figure 7. Time course of production of various metabolites of AFB incubated with BHA-treated mouse liver microsomes and cytosol. Panel A shows the disappearance of the added AF'B, and the production of the AFB-GSH conjugate,which in the presence of excess mouse liver cytosol and GSH is indicative of the amount of AFB 8,g-epoxideformed. Panel B shows the formation and disappearance of hydroxylated metabolites of AFB. Panel C demonstrates the quantitative conversion of AFP to 4,9a-dihydroxy-AFB. mation of various hydroxylated metabolites can be determined, including the rate of formation of the putative AFB 8,g-epoxide (22). This improvement in methodology allowed us to identify the presence of previously unidentified oxidative metabolites of AFB in bile and in in vitro microsomal incubations. It is of interest to note that AFB2, is apparently not formed in these microsomal incubations. Initial studies on AFB metabolism suggested that AFB2, was formed in vitro in chicken (6),duck ( 7 ) , and rat (19,26) liver fractions. However, Roebuck and Wogan (27) and Lin et al. (28) were not able to find AFB2, production in liver fractions from a variety of species and suggested either that AFB% is formed but binds to proteins, that it is unresolved from AFM, or that AFB 8,9-dihydrodiol was mistakenly identified in previous studies as AFB2a. Because our HPLC system completely resolves both AFB, and the Tris base of the phenolate ion of AFB2, from all other oxidative metabolites, including the dihydrodiol and the Schiff base of the dihydrodiol, the failure to detect either of these metabolites suggests that AFB2, is not formed in significant quantities in mouse microsomal fractions. The results of this study demonstrate that AFP can undergo secondary microsomal oxidation in the Sa-position to yield a dihydroxyaflatoxin metabolite. This metabolite is formed in vivo in rats and is excreted in bile both directly and as the glucuronide conjugate. In vivo, the gluc-
Dihydroxyaflatoxin Metabolite Identification
uronidation of AFP must effectively compete with the microsomal oxygenases responsible for secondary metabolism of AFP, as the glucuronide conjugate of AFP is excreted in bile in greater amounts than the glucuronide conjugate of 4,9a-dihydroxy- AFB. Detailed analysis of bile from several animals given AFB revealed that AFP-glucuronide represented 14% of the total biliary metabolites found in bile excreted for 6 h following AFB administration, whereas the glucuronide conjugate of 4,9a-dihydroxy-AFB represented 7% of total biliary metabolites (unpublished observation). However, because AFM can undergo oxidative demethylation in vitro to form the same 4,9a-dihydroxy-AFB,it cannot be assumed that all of this dihydroxy metabolite is formed from secondary oxidation of AFP. However, examination of the mass balance of AFP, AFM, and 4,ga-dihydroxy-AFB in microsomal incubations in which AFB metabolism is complete clearly indicates that AFP is the preferred substrate for secondary oxidation to the 4,9a-dihydroxy metabolite, at least in BHA-treated mouse liver microsomes. Secondary oxidation of monohydroxy AFB metabolites may have varying toxicologic importance, depending upon the relative toxicity of the monohydroxy metabolite. AFP is generally consider a detoxification product (2), in part because it is efficiently conjugated with glucuronic acid (IO). Thus, secondary oxidation of AFP is likely of little toxicologic significance. However, AFM is mutagenic and carcinogenic, albeit considerably less so than AFB (2,3). Thus, it is possible that secondary oxidative demethylation of AFM and subsequent glucuronidation of the phenolic hydroxyl group could be a significant route of detoxification of AFM in vivo, although in vitro results in liver microsomes from BHA-treated mice suggests that AFM is a poor substrate for secondary oxidation, relative to AFP. These data also provide some indirect evidence that both AFP and AFM can undergo oxidation to the respective 8,9-epoxides, as GSH conjugates of AFP and AFM are apparently formed in small quantities when AFP or AFM are incubated with microsomes and cytosol. However, in non-enzyme-inducedmice (Figure 7) (22) and rats (29,30), both AFM and AFP are formed in small quantities relative to AFB 8,9-epoxide, and only a small fraction of the AFP and AFM formed appear to be metabolized to their corresponding epoxides. Thus, secondary oxidative of monohydroxy metabolites of AFB to the corresponding 8,9epoxides is probably of little importance in the overall toxicity and hepatocarcinogenicity of AFB in rodents. However, nonhuman primates form large quantities of AFQ, relative to the AFB epoxide (unpublished observation), and induction of cytochrome P-448 results in a large increase in the formation of AFM relative to the AFB epoxide (25). Thus in some circumstances secondary oxidation of the monohydroxy-AFB metabolite could be of toxicologic significance.
Acknowledgment. We thank Patricia Stapleton for her excellent technical assistance. Support for this work was provided by NIH Grants T32 ES-07032 and ES-03415 and Grant IN-26Y from the American Cancer Society. Registry No. AFBl, 1162-65-8; AFPl glucuronide, 50280-92-7; AFBl trisdiol, 113568-93-7; AFBl dihydrodiol, 50668-79-6; AFB&, 1787854-5; AFQ,52819-96-2; AFM1, 679523-9; AFP1, 32215-02-4; AFG1, 1165-39-5; AFBZetris, 113568-94-8; aflatoxicol, 29611-03-8.
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