Chem. Res. Toxicol. 1990, 3, 157-161
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Activation of Methanol by Hepatic Postmitochondrial Supernatant: Formation of a Condensation Product with 2,4-Diaminotoluene Michael L. Cunningham,* H. B. Matthews, and Leo T. Burka Experimental Toxicology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received September 15, 1989 2,CDiaminotolucne, an aromatic amine hepatocarcinogen in rats and mice, reacts extensively with formaldehyde produced by the oxidation of methanol t o form a single reaction product. Using 'H and 13C NMR and high-resolution mass spectroscopy, this product was shown to be bis(2,4-diamino-5-tolyl)methane.This reaction product is shown to occur under conditions whereby methanol is metabolized to formaldehyde by rat hepatic postmitochondrial supernatant. These results demonstrate a novel reaction of aromatic amines and formaldehyde in biological samples and indicate that methanol as a solvent may become activated in vitro and produce complex interactions with solutes.
Introduction 2,4-Diaminotoluene (2,4-DAT) is a high production volume chemical used in the synthesis of toluene diisocyanate for polyurethane manufacture. 2,4-DAT is also used for the synthesis of dyes used for textiles, wood stains, and pigments. Annual production of 2,4-DAT is over 100 million lb in the United States alone. 2,4-DAT is a mutagenic in the Ames/Salmonella assay requiring metabolic activation (I) and is carcinogenic in F344 rats and B6C3F1 mice (2). A structural isomer, 2,6-DAT, is also a high production volume aromatic amine, yet it was shown to be noncarcinogenic in F344 rats and B6C3F1 mice, even at doses approaching 5-fold greater than those used for 2,4-DAT (3). Both compounds are equally mutagenic in the AmeslSalmonella assay with metabolic activation (4). Our laboratory is investigating the mechanisms whereby compounds can produce genotoxicity in short-term in vitro assays yet fail to produce carcinogenicity in in vivo bioassays. Such discordance between in vitro and in vivo test results undermines confidence in short-term tests for the prediction of carcinogenicity. Therefore, we are engaged in the studies of the fate of chemicals which demonstrate discordant results between short-term and bioassays with emphasis on describing the different metabolic activation pathways between the in vitro test system and the whole animal. Human exposure to methanol is extensive due to its widespread use as an industrial solvent and as a component in automobile fuels. Methanol is also used as a solvent for organic chemicals in metabolism and mutagenicity assays such as the AmesISalmonella test. Methanol is metabolized by alcohol dehydrogenase or the catalase peroxidative pathway to formaldehyde (5),which is toxic to retinal cells and carcinogenic to rodents (6),and may interact with test chemicals or test organisms, resulting in a false or disproportionate result. In addition, formaldehyde is produced by the metabolism of a variety of substrates such as nicotine, cocaine, diesel exhaust particles, antihistamines, and solvents such as dimethylaniline and hexamethylphosphoramide ( 7 ) . This report is the first demonstration to our knowledge that metabolically generated
* To whom correspondence
should be addressed.
formaldehyde interacts with a xenobiotic, in our studies the aromatic amine 2,4-diaminotoluene, which results in a condensation product consisting of two molecules of the xenobiotic and formaldehyde.
Experimental Section Apparatus. HPLC was performed with a Waters Associates System (Millipore,Waters Division, Milford, MA), consisting of a Model 490E multiwavelength detector, two Model 510 solvent delivery systems, a 712 automatic sampler (WISP),and a 3.9 mm X 30 cm pBondapak C18reverse-phasecolumn (Waters). Detection was at 254 nm. C18 reverse-phase columns were eluted with a linear gradient of 100% water to 100% acetonitrile in 25 min, with a flow rate of 1.5 mL/min. Both analytical and preparative HPLC were conducted on this system. High-resolution mass spectroscopy (direct inlet) was performed with a VG 7070s mass spectrometer operated at mass resolution of 10000. The samples were peak-matched versus perfluorokerosene. 'H NMR and 13C NMR were performed with a General Electric Model GN 500
spectrometer. Materials. 2,4-DAT was purchased from Aldrich Chemical Co. (Milwaukee, WI). Lot no. 0352851' was found to be chromatographically pure by HPLC and UV detection and was exclusively used in this study. Aroclor-1254 was obtained from Monsanto Chemical Co. (St. Louis, MO). [13C]Methanolwas purchased from Aldrich. Solvents were purchased from J. T. Baker, Inc. (Phillipsburg,NJ). Biochemicals were purchased from Sigma Chemical Cc. (St. Louis, MO). Hepatic 90oOg supernatant (S9) was prepared from male F344 rats (225-250 g) which had been given an intraperitoneal injection of Aroclor-1254 in corn oil 5 days earlier. Protein was assayed by the method of Lowry et al. (8). In Vitro Metabolism. Incubations were carried out in 7 mL of an S9 activation system consisting of 8 mM MgCl,, 33 mM KCl, 5 mM glucose 6-phosphate, 4 mM NADP+, 100 mM sodium phosphate, pH 7.4, and 1.0 mg/mL S9 protein. Cofactor generation was allowed to proceed at 37 "C for 5 min in a gyratory water bath, and metabolism was initiated by the addition of 2,4-DAT in varying amounts of methanol. In some studies, [13C]methanolwas used in place of methanol. For kinetic studies, the concentrations of 2,4-DAT or methanol in the incubation mixture were varied. Aliquots were removed and quenched at the indicated times in an equal volume of acetonitrile at 4 "C and centrifuged at 4000g to deproteinate. Supernatants were filtered through a 0.45-pm Millex-HV syringe filter (Millipore Products, Bedford, MA), and a 50-pL aliquot was analyzed by HPLC. Preparative HPLC was preformed in a similar fashion with the exception that a 200-pL aliquot was applied to the column.
This article not subject to U.S. Copyright. Published 1990 by the American Chemical Societv
158 Chem. Res. Toxicol., Vol. 3, No. 2, 1990
Cunningham et al.
, e 0
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Figure 1. Chromatogram obtained by HPLC analysis (C18 column) of an aliquot of 2,4-DAT incubated with Aroclor-induced rat liver S9, glucose 6-phosphate, and NADP+ in the presence of methanol,after quenching with acetonitrile. The retention time of 2,4-DATis 11.50 min, the condensation product elutes at 16.50 min, and NADP+ elutes with the solvent front. Chemical Synthesis of the Putative 2,4-DAT Metabolite. the preChemical synthesis of bis(2,4-diamino-5-tolyl)methane, sumed metabolite resulting from the interaction of 2,4-DAT and a methanol metabolite,was performed following the method of Ullman and Naef (7) as follows: 1.25 g of 2,4-diaminotoluenewas added to a solution of 0.5 g of concentrated sulfuric acid and 4 mL of water of give a thick crystalline mass. To this mixture was added a solution of 400 mg of 37.6% formaldehyde and 1 mL of water. The mixture was heated at 60 "C to dissolve the remaining crystals. Over the next 10-15 min, needles of the sulfate salt of the bis(diaminotoly1)methane crystallized, after which time the mixture was cooled and filtered and the crystals were washed with cold water. The free tetraamine could be obtained by triturating the solid with ammonium hydroxide, filtering, and drying. The following data are consistent with the structure of the expected product: 'H NMR (500 MHz, CD,OD) d 2.00 (s,6 H, CH,), 3.50 (s, 2 H, CH,), 6.21 (s, 2 H, 3- and 3'-CH), and 6.55 (s, 2 H, 6- and 6'-CH); 13C NMR (126 MHz, CD3OD) 6 16.6 (CH,), 33.0 (CH,), 105.5 (c-3),115.3 (C-1or C-5),117.5 (C-5or C-l), 132.5 (C-6),144.7 (C-2 or C-4),and 145.0 (C-4 or C-2); mass spectrum (direct probe, 70 eV) m/e (re1intensity) 256 (62, M'), 135 (100,cleavage of the CH2-Ar bond), and 122 (51, cleavage of the CH2-Ar bond).
Results After incubation of 2,4-DAT in methanol with the S9 activation system, aliquots were removed for HPLC analysis. A single major metabolite was consistently observed. A typical chromatogram identifying 2,4-DAT (retention time 11.5 min) and the single metabolite which was less polar than the parent compound (retention time 16.5 min) is shown in Figure 1. One of the components of the cofactor-generating system, NADP+, elutes a t the solvent front (retention time 3.11 min). This profile was highly reproducible and dependent on the presence of methanol. When 2,4-DAT was incubated with other solvents in the absence of methanol, e.g., DMSO, acetonitrile, or dimethylformamide, the peak a t 16.50 min was not observed (data not shown). The formation of the metabolite of 2,4-DAT increased over a 60-min period, and the concentration of 2,4-DAT decreased a t approximately inverse rates in the presence of saturating methanol concentrations, as shown in Figure 2. A double-reciprocal plot of the formation of the metabolite is presented in the inset, for 2,4-DAT concentra-
i ib
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Figure 2. Formation over time of the primary metabolite (open circles) and decrease of 2,4-DAT (closed circles) with saturating concentrations of methanol in Aroclor-inducedrat liver S9. The initial concentration of 2,4-DAT was 5 mM. Inset: Double-reciprocal plot for the formation of the metabolite at various concentrations of 2,4-DAT in this system. tions of 2.5-10 mM. The apparent K , and V,, for the formation of the metabolite were 5.28 mM and 18.1 nmol/ (mg of proteimmin), respectively, with S9 from Aroclor 1254 induced rats, calculated from data obtained from the linear portion of the curve. A similar analysis of metabolite formation in the presence of 10 mM 2,4-DAT and varying amounts of methanol demonstrated a similar apparent V,, [16.6 nmol/(mg of proteimmin)] but a much lower apparent K , (5.0 pM). Following semipreparative HPLC and repeated purification as described under Experimental Section, the metabolite of 2,4-DAT was examined by high-resolution mass spectroscopy and nuclear magnetic resonance spectroscopy. The data from the high-resolution mass spectrum shown in Figure 3 indicated an exact measured mass of 256.16698, which is consistent with a molecular formula of C12HmN4. The theoretical mass for this formula is 256.1688; therefore, the measured/theoretical mass measurements agreed to 5.2 ppm. Two major fragments were observed a t 135.0924 and 122.0846, which are assigned the formulas C8HllN2 and C7HI0N2,with measured/ theoretical mass measurements of 6.1 and 4.2 ppm, respectively. Figure 4 presents the 'H NMR (CD,OD) spectrum of the metabolite with 6 2.00 (s, 6 H, CH,), 3.51 (5, 2 H, CH,), 6.23 (s, 2 H, A r m , and 6.55 (s, 2 H, ArH). In studies with [13C]methanolin place of methanol, the I3C NMR presented in Figure 5 showed enhancement of one peak corresponding to a methylene carbon a t 6 33.0 (CH,). On the basis of the mass spectral and NMR data, the metabolite was identified as bis(2,4-diamino-5-tolyl)methane(see Figure 6 for structure). The chemical synthesis of bis(2,4-diamino-5-tolyl)methane was performed as described under Experimental Section. The mass spectral and NMR data are consistent with the structure of this product. In addition, the metabolite isolated from S9 and the chemically synthesized product comigrated on reverse-phase HPLC (data not shown).
Discussion The results of this study demonstrated that methanol is activated by hepatic postmitochondrial supernatant to an intermediate capable of reacting with the aromatic
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 159
Methanol Interactions with Aromatic Amines
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by HPLC from the Aroclor-induced rat liver S9 metabolism of 2,4-DAT (A) and of chemically synthesized bis(2,4-diamino-5toly1)methane(B); the peak marked X is due to dioxane. Shown also is a 13C NMR of the metabolite produced by S9 in the presence of 13C-enrichedmethanol, demonstrating the methylene bridge at 33 ppm (C). formation of the metabolite was time-dependent and followed Michaelis-Menten kinetics (Figure 2), consistent with its formation being due to the activity of enzymes in the activation mixture. The reaction did not proceed in the absence of oxygen, NADPH, or an intact enzyme preparation. The reaction product was not observed when the solvent was changed to DMSO, THF, or acetonitrile. Characterization of this metabolite by high-resolution mass spectroscopy and 'H NMR demonstrated that it had a molecular weight of 256.16698 and proton assignments consistent with the structure of bis(2,4-diamino-5-tolyl)methane (see Figure 6). The synthesis of bis(2,4-diamino-5-tolyl)methanewas accomplished by using formaldehyde to cross-link the 2,4-diaminotoluene constituents. The synthetic product was demonstrated to be identical with the product isolated from S9 incubations of 2,4-DAT and methanol on the basis
160 Chem. Res. Toxicol., Vol. 3, No. 2, 1990 0
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Figure 6. Mechanism proposed for the formation of bis(2,4diamino-5-toly1)methane by interaction of 2,4-diaminotoluene and methanol in an S9 activation system.
of HPLC retention time, mass spectroscopy (Figure 31, and 'H NMR (Figure 4). It was suspected that the carbon forming the methylene bridge in this unusual metabolite arose from methanol. By substituting the methanol with 13C-enriched methanol, it was demonstrated that methanol was indeed the source of the bridging carbon. That is, the 13C NMR of the product of the S9-mediated reaction between 2,4-DAT and 13C-enrichedmethanol shows enhancement of the peak at 33 ppm, the chemical shift of the methylene carbon in the metabolite (Figure 5). These data and the observation that the reaction product did not appear when other solvents were used indicated that the product was a result of activation of methanol and its subsequent interactions with two molecules of 2,4-DAT. The condensation of formaldehyde with electron-rich aromatic systems is a well-known reaction. The reaction of formaldehyde with phenol to form Bakelite plastics is a familiar example. Figure 6 shows one pathway by which the condensation of formaldehyde and 2,4-DAT might occur. Hydroxymethylation of C-5 of 2,4-DAT is the first step. This step would be facilitated by protonation of the carbonyl oxygen of formaldehyde. Produds from the other likely site of hydroxymethylation, (2-3, were not found either in S9 products or in products from chemical syn-
Cunningham et al. thesis. The next step requires dehydration of the hydroxymethyl compound to form either a quinone iminium ion intermediate, as depicted, or possibly a benzyl carbenium ion (these two intermediates are essentially equivalent). Reaction of this intermediate with a second molecule of 2,4-DAT at C-5, followed by proton loss, would give the observed product. The cross-linked metabolite of 2,4-DAT, bis(2,4-diamino-5-tolyl)methane, was purified by HPLC for characterization of its mutagenicity. In the standard Ames/ Salmonella assay, this condensation product was shown to be approximately 5 times more mutagenic than the parent 2,4-DAT, in the presence of an S9 activation system (10). In the absence of this methanol interaction, 2,4-DAT is metabolized to an ultimate mutagenic metabolite, 4(acetoxyamino)-2-aminotoluene, by N-hydroxylation and acetylation (11). This observation demonstrates the complexities encountered in conducting mutagenicity studies without an appreciation of the metabolism of the test chemical as well as possible interactions of the solvents and the metabolic activation systems used to activate the test chemicals. That is, when the test substrate interacts with the solvent, an activated metabolite of the solvent, or possibly even an endogenous compound in the S9 mix, the result could be a false or disproportionate response in the AmeslSalmonella assay. Depending on the reaction, results could be either falsely positive or negative. Additional research to determine the active species that accounts for the in vitro and in vivo toxicity should enhance the utility of in vitro assays such as the AmeslSalmonella assay to predict in vivo toxicity. The kinetics of the formation of the cross-linked product in S9 indicate that the reaction is primarily dependent upon methanol concentration. The apparent K , for the reaction is 5 pM for methanol at saturating 2,4-DAT concentrations and 5.3 mM for 2,4-DAT at saturating methanol concentrations, indicating that the initial reaction rate is more sensitive to the methanol concentration and the rate-limiting step in the reaction is the oxidation of methanol to formaldehyde. Additionally, we have prepared this reaction product by substituting CH,O for methanol in the presence and absence of NADP+ cofactors and an activation system, indicating that the reactions following the generation of CHzO are spontaneous (data not shown). The formation of formaldehyde as a metabolic product of methanol by cytochrome P-450 is well-known (5). It is also formed as a consequence of demethylation reactions, especially N-demethylation, and the peroxidation of methanol by catalase, hydrogen peroxide, and the reaction of Fenton-derived hydroxyl radicals (12). In addition, formaldehyde is a significant pollutant due to the use of formaldehyde resins in building materials such as plywood and as a result of the combustion of gasoline containing methanol. Formaldehyde has recently been demonstrated to interact with the hepatocarcinogen inorganic hydrazine (H,NNH,), producing a polymer metabolite consisting of 4 equiv of formaldehyde to 3 equiv of hydrazine (13). This polymer is thought to decompose to produce a reactive intermediate capable of alkylating DNA. The results of our study demonstrate that formaldehyde generated from the metabolism of methanol alkylates the aromatic amine 2,4-DAT to produce a novel cross-reactive intermediate. Recent studies in our laboratory indicate that formaldehyde can also cross-link the 2,6-DAT isomer, resulting in an as yet unidentified reaction product, which indicates that the condensation reaction described in this report may be a general one for other aromatic amines such as ben-
Methanol Interactions with Aromatic Amines zidine, 2-(acetylamino)fluorene, naphthylamines, etc. It is, therefore, conceivable that methanol converted to formaldehyde should be capable of alkylating the aromatic residues in protein, resulting in protein-protein cross-links, or, of more serious consequence to the cell, resulting in protein-DNA cross-links. We are investigating this phenomenon in greater detail. In summary, the data from this study demonstrate that methanol can be oxidized by rat liver S9 to formaldehyde, which interacts with two molecules of the primary aromatic amine 2,4-DAT to produce a mutagenic product, bis(2,4diamino-5-toly1)methane. These results should be considered when one uses methanol as a solvent in mutation and metabolism studies and may also be significant in understanding the moleulcar basis for methanol toxicity.
Acknowledgment. Critical review of the manuscript by Dr. James McKinney, NIEHS, and Dr. Greg Kedderis, CIIT, Research Triangle Park, NC, is gratefully acknowledged.
References (1) Ames, B. N., Kammen, H. O., and Yamasaki, E. (1975) Hair dyes
are mutagenic: Identification of a variety of mutagenic ingredients. Proc. Natl. Acad. Sci. U.S.A. 72, 2423-2427. (2) U S . Department of Health, Education and Welfare, Public Health Service, National Institutes of Health (1979) Bioassay of 2,4Diaminotoluene for Possible Carcinogenicity, NCI Carcinogenesis Technical Report Series 162, National Technical Information Service (NTIS), Springfield, VA [DHEW Publication No. (NIH) 79-1718 (NTIS Accession No. PB293593)I. (3) U.S. Department of Health, Education and Welfare, Public Health Service, National Institutes of Health (1980) Bioassay of 2,6-Diaminotoluene Hydrochloride for Possible Carcinogenicity, NCI Carcinogenesis Technical Report Series 200, National Tech-
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 161 nical Information Service (NTIS), Springfield, VA [DHEW Publication No. (NIH) 80-1756 (NTIS Accession No. PB80217912)]. (4) Cunningham, M. L., Burka, L. T., and Matthews, H. B. (1989) Metabolism, disposition, and mutagenicity of 2,6-diaminotoluene-A mutagenic noncarcinogen. Drug Metab. Dispos. 17, 6 12-6 17. (5) Teschke, R., Hasumura, Y., and Lieber, C. S. (1974) NADPHdependent oxidation of methanol, ethanol, propanol and butanol by hepatic microsomes. Biochem. Biophys. Res. Commun. 60, 851-857. (6) Swenberg, J. A., Kerns, W. D., Mitchell, R. I., Gralla, E. J., and Pavkov, K. L. (1980) Induction of squamous cell carcinoma of the rat nasal cavity by inhalation exposure to formaldehyde vapor. Cancer Res. 40, 3398-3402. (7) Dahl, A. R., and Hadley, W. M. (1983) Formaldehyde production promoted by rat nasal cytochrome P-450-dependent monooxygenases with nasal decongestants, essences, solvents, air pollutants, nicotine, and cocaine as substrates. Toxicol. Appl Pharmacol. 67, 200-205. (8) Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265-275. (9) Ullman, F., and Naef, E. (1900) Ueber synthesen in der naphtacridine-reihe. I. 2'-methyl-1,2-naphtacridin. Chem. Ber. 33, 905-919. (10) Cunningham, M. L., Burka, L. T., and Matthews, H. B. The interaction of methanol, rat liver S9, and the aromatic amine 2,4-diaminotoluene produces a new mutagenic compound. Mutat. Res. (in press). (11) Cunningham, M. L., and Matthews, H. B. Evidence for an acetoxyarylamine as the ultimate mutagenic reactive intermediate of the carcinogenic aromatic amine 2,4-diaminotoluene. Mutat. Res. (in press). (12) Cederbaum, A. I., and Qureshi, A. (1982) Role of catalase and hydroxyl radicals in the oxidation of methanol by rat liver microsomes. Biochem. Pharmacol. 31, 329-335. (13) Bosan, W. S., Lambert, C. E., and Shank, R. C. (1986) The role of formaldehyde in hydrazine-induced methylation of liver DNA guanine. Carcinogenesis 7, 413-418.
