Chem. Res. Toxicol. 1995, 8, 903-906
903
Communications Identification of cis-2-Butene-1,4=dialas a Microsomal Metabolite of Furan Ling-Jen Chen, Stephen S. Hecht, and Lisa A. Peterson* Division of Chemical Carcinogenesis, American Health Foundation, Valhalla, New York 10595 Received June 23, 1995@ The hepatocarcinogen furan is believed to be activated to the reactive aldehyde, cis-2-butene1,6dial, by microsomal enzymes. The rat liver microsomal metabolism of furan was examined in the presence of NADPH and semicarbazide. HPLC analysis of incubation mixtures revealed the formation of a metabolite that coeluted with standards for the bis-semicarbazone adduct of cis-2-butene-1,4-dial. The formation of this compound required the presence of NADPH, semicarbazide, and microsomes. Preparative isolation and chemical characterization of this metabolite confirmed the structural assignment. These data provide evidence that the reactive aldehyde, &-2-butene-1,4-dial, is a major metabolic product of furan.
Introduction The industrial chemical furan (1)is used primarily as a solvent or as an intermediate in the synthesis of commercial compounds. It has also been detected in many foods and beverages (11. The widespread occurrence of furan and potential for human exposure warrant investigations into the toxicological properties of this compound. In laboratory animals, furan causes liver and kidney toxicity (2, 3 ) and induces hepatocellular carcinomas and cholangiocarcinomas (3-5). Furan is extensively metabolized in vivo; COZis the major metabolite (6). Kinetic analysis of furan metabolism in gas uptake studies indicated that there is a single saturable uptake process that can be blocked by pyrazole, a cytochrome P-450 inhibitor (7). Furan is converted to protein binding metabolites both in vivo and in vitro (6, 8). Glutathione and, to a lesser extent, semicarbazide blocked this binding. Furan depletes cellular glutathione levels and reduces cell viability at biologically relevant concentrations in freshly isolated hepatocytes (9). These effects can be inhibited by 1-phenylimidazole (inhibition of cytochrome P-450) and enhanced by acetone pretreatment of rats (induction of cytochrome P-450 2EI), paralleling the effects of inducers and inhibitors on furan metabolism and protein binding in vitro (7). These studies support the role of cytochrome P-450 in the activation of furan to a reactive metabolite. The postulated mechanism for furan hepatocarcinogenesis does not involve a DNA reactive intermediate since furan is negative in Salmonella typhimurium mutagenicity tests (10) and was unable to induce unscheduled DNA synthesis in livers of rodents (11). In addition, no radioactivity was associated with DNA isolated from livers of [14Clfuran-treatedrats (6). However, cell proliferation follows a toxic response in the livers of furantreated animals (11). Therefore, the current hypothesis follows a sequence in which furan is activated to a reactive and cytotoxic intermediate by cytochrome P-450. * T o whom correspondence and reprint requests should be addressed. Abstract published in Advance ACS Abstracts, September 15, 1995.
The resulting toxicity stimulates cell replication which increases the likelihood of tumor induction. The postulated reactive intermediate is cis-2-butene1,4-dial (41, which is formed either directly or via an epoxide intermediate (Figure 1).Analogous unsaturated aldehydes-4-0~0-2-pentenaland 3-methyl-2-butene-l,4dial-are microsomal metabolites of 2-methylfuran and 3-methylfuraq respectively (12). We have observed 4-oxo-2-pentenal in the solvolysis of a-acetoxy-N-nitrosopiperidine and found that it reacts with deoxyguanosine to form a 7-(2-oxopropyl)-substitutedlp-ethenodeoxyguanosine adduct (13). cis-2-Butene-l,4-dial is probably responsible for the reactivity of furan with proteins in biological systems and is also likely to react with DNA. However, this postulated reactive intermediate has not been previously characterized as a metabolite of furan.
Experimental Procedures1 Materials. Male F344 rats (200-400 g) were purchased from Charles River Laboratories (Kingston, NY). Glucose 6-phosphate, glucose-6-phosphate dehydrogenase, and NADP+ were obtained from Sigma Chemical Co. (St. Louis, MO). All other compounds were purchased from Aldrich Chemical Co. (Milwaukee, WI). Furan was freshly distilled before use. Instrumental Analyses. NMR spectra were acquired with a Bruker Model AM 360 WB spectrometer and are reported in ppm relative to an internal standard (solvent or tetramethylsilane). Melting points were obtained on a Hoover Unimelt capillary apparatus and are uncorrected. W spectra were collected on a Hewlett Packard 8425A diode array spectrophotometer, which was computer controlled by H P 89530 MS-DOS W - v i s operating software. HPLC analyses were carried out with a Waters 510 system (Millipore, Waters Division, Milford, MA) with either a Waters Model 990 photodiode array detector o r a Shimadzu SPD-1OA UV-vis detector. MS was performed on a Hewlett Packard Model 5988A instrument. Synthesis. cis-2-Butene-l,4-dialBis-Semicarbazone (6). Compound 6 was prepared by two methods: (1)2,5-Dimethoxy2,5-dihydrofuran (1.75 g, 13.5mmol) was added dropwise to a stirred solution of semicarbazide hydrochloride (3.0 g, 26.9 'Abbreviations: CI-MS, chemical ionization-mass spectrometry; DCI-MS, desorption chemical ionization-mass spectrometry; NADP', nicotinamide adenosine dinucleotide phosphate.
0893-228x/95/2708-0903$09.00/0 0 1995 American Chemical Society
Communications
904 Chem. Res. Toxicol., Vol. 8, No. 7, 1995
HzNCONHN~NNHCONH~
-
H2NCONHN
7
6
Figure 1. Proposed activation pathway of furan. mmol) in H2O (30 mL) a t room temperature. After 30 min, the red solution contained a precipitate. The pale yellow solid was collected by filtration, washed with HzO, and air-dried to yield 6 (0.64 g, 3.24 mmol, 24% yield), mp 226-227 "C (darkens at 217 "C). (2) cis-2-Butene-1,4-dial was generated via the method of Adger et al. (14). Briefly, furan (6 pL, 5.6 mg, 0.08 mmol) was added to a stirred solution of dimethyldioxirane in acetone (1.6 mL, 0.05 M, 0.08 mmol) (15, 16). After 30 min a t room temperature, the solvent was removed at reduced pressure to yield a yellow oil. Concentration of the product increased the impurities present, presumably due to polymerization. Subsequent preparations were stored and used as acetone solutions. When the reaction was monitored by 'H NMR, dimethyldioxirane-ds was prepared from acetone-&. Spectral properties: 'H NMR of the concentrated product (CDC13)6 10.45 (dd, J~,z= 4.4, J1,3= 2.7 Hz, 2H, CHO), 6.65 (dd, J 2 , l = 4.4, J 2 , 4 = 2.7 Hz, 2H, CH-CH) (lit. (17) 6 10.25, m, aldehydic H, 6.48, m, olefinic H); positive ion DCI-MS m l z (re1 intensity) 85 (M + 1, 100). A freshly prepared cis-2-butene-l,4-dialsolution (1.6 mL as above) was added dropwise to a 1mL aqueous solution of semicarbazide hydrochloride (30 mg, 0.27 mmol) and sodium acetate (44 mg, 0.54 mmol). A yellow solid precipitated from the solution after 3 h at room temperature. The precipitate was collected by filtration, washed with HzO, and air-dried to yield 6 (5.4 mg, 0.027 mmol, 34% yield). Spectral properties: UV (HzO) Amax ( E , M-' cm-l) 238 nm (5950), 310 nm (39 100); 'H NMR (DMSO-&) 6 10.4 (s,2H, NHCONHz), 7.94 (dd, J 1 , 2 = 6.74, J1,3= 2.26 Hz, 2H, CH=N), 6.45 (br s, 4H, NHCONHz), 6.25 (dd, J z ,=~6.74, J 2 , 4 = 2.26 Hz, 2H, CH=CH); positive ion DCI-MS m l z (re1 intensity) 199 (M 1, 201, 139 (M - NHCONH2, 901, 126 (M - NNHCONHz, 80), 81 [M 1 - ~(NHCONHZ), 1001; high resolution positive ion CI-MS, calcd 199.09349, found 199.09517. truns-2-Butene-1,4-dial(5, 18). 1,1,4,4-Tetramethoxytrans-2-butene was shaken with 3 mL of 0.36 N H2S04 saturated with Na2S04 for 20 min a t room temperature. The solution was extracted with ether (2 x 25 mL). The combined ether layers were dried over MgS04 and filtered. A light yellow oil was obtained upon evaporation of the ether. The IH NMR spectrum of this oil demonstrated that hydrolysis was incomplete. An additional 2 mL of 0.36 N HzS04 saturated with NazS04 was added to the oil. After 20 min, the mixture was extracted with ether as above. trans-2-Butene-1,4-dial was purified by silica gel column chromatography with elution by 30% ethyl acetate in hexane to give a light yellow solid (71,mp 40-41 "C (lit. (18) 42-44 "C). Spectral properties: 'H NMR (CDC13) 6 9.89 (dd, J,,*= 4.55,51,3= 2.8 Hz, 2H, CHO), 6.83 (dd, Jz,i = 4.55, J z , ~= 2.8 Hz, 2H, CH=CH), positive ion DCI-MS m l z (re1 intensity) 85 (M + 1, 100). trans-2-Butene-l,4-dialBis-Semicarbazone (7). Water (10 mL) was added to a solution of trans-2-butene-l,4-dial(O.l g, 1.2 mmol) in ethanol (3 mL). Then sodium acetate (0.44 g, 5.4 mmol) and semicarbazide hydrochloride (0.30 g, 2.7 mmol) were added. The solution was briefly heated to 100 "C in an H2O bath and then cooled to 0 "C. A pale yellow precipitate formed. The yellow solid was filtered and air-dried to yield 7
+
+
(0.12 g , 0.61 mmol, 11%yield), mp '240 "C. Spectral properties: lH NMR (DMSO-&) 6 10.27 (s,2H, NHCONHz), 7.61 (dd, J 1 , 2 = 5.8, 51,s = 2.8 Hz, 2H, CHN), 6.47 (dd, J 2 , i = 5.8, J 2 , 4 = 2.8 Hz, 2H, CH=CH), 6.33 (br s, 4H, NHCONHz); positive ion DCI-MS m l z (re1 intensity) 199 (M 1, 41), 139 (M N H C O N H Z , ~81 ~ )[M , 1- 2(NHCONH2) 1001; high resolution positive ion CI-MS, calcd 199.09349, found 199.09468. Stability of cis-2-Butene-1,4-dialin Aqueous Solutions. cis-2-Butene-l,4-dial was prepared from furan and dimethyldioxirane in acetone as described above. The acetone solution was added to 20 mM deuterated phosphate buffer (pD 7.4). The acetone was removed under reduced pressure (5-10 min), and lH NMR spectra were obtained at various time points between 0 and 24 h. Only a 1 : l ratio of the cis- and trans-isomers of the cyclic cis-2-butene-1,4-dialhydrate (3)was observed (a 2:l ratio of cis- to trans-hydrate has been reported (19)): 'H NMR (DzO) 6 6.16, 6.15, 6.14 (s, cis- and trans-vinylic and trans-methine protons), 5.86 (s, cis-methine proton). Microsomal Assays. F-344 rat liver microsomal fractions were prepared by the method of Guengerich et al. (20). Triplicate incubations of furan (0.025, 0.05, 0.1, 0.2, 2, and 20 mM) with rat liver microsomes (1mg/mL) were conducted in 2 mL of 400 mM potassium phosphate buffer (pH 6.8) in the presence of 25 mM glucose 6-phosphate, glucose-6-phosphate dehydrogenase (2 unitdml), 4 mM NADP+,3 mM MgC12,l mM EDTA, and 60 mM semicarbazide hydrochloride. Furan was added as an ethanol solution (20 pL). Controls were performed in the absence of NADP+ or microsomes. After 30 min incubation at 37 "C in capped vials, reactions were terminated by addition of 0.3 N Ba(0H)z and 0.3 N ZnSO4 (0.2 mL each). Following centrifugation, the supernatant was filtered through an Acrodisc (Gelman, Ann Arbor, MI; 0.45 pm, 3 mm) and analyzed directly by reverse-phase HPLC with UV a t 300 nm. The mixtures were separated on a Phenomenex Bondaclone C18 column (300 x 3.9 mm) with 5.5% acetonitrile-H2O as the mobile phase and a flow rate of 1mumin. trans-2-Butene-1,4dial bis-semicarbazone (7)and cis-2-butene-l,4-dial-bis-semicarbazone (6)eluted with retention times of 13 and 22.5 min, respectively. Metabolite concentrations were estimated using standard curves prepared for each analysis. The 20 mM furan incubations were scaled up to 20 mL for further characterization of the furan metabolite. It was collected from HPLC using the system described above. The acetonitrile was removed under reduced pressure and HzO was lyophilized. Spectral properties: UV (HzO)Amax 238 nm, 310 nm; 'H NMR (DMSO-&) 6 10.4 (s,2H, NHCONHz), 7.94 (dd, J ~= ,z 6.77,51,3= 2.05 Hz, 2H, CH=N), 6.37 (br s, 4H, NHCONHd, 6.25 (dd, JZJ = 6.77, J 2 , 4 = 2.05 Hz, 2H, CH=CH), positive ion DCI-MS m l z (re1 intensity) 199 (M 1, 201, 139 (M - NHCONH2, 901, 126 (go), 81 [M + 1 - 2(NHCONH2), 1001.
+
+
+
Results and Discussion In these studies, cis-a-butene-l,4-dial (4) was generated by oxidizing furan with dimethyldioxirane (14). Reaction of this compound with semicarbazide under
Chem. Res. Toxicol., Vol. 8, No. 7, 1995 905
Communications
Table 1. Chemical Shifts (ppm) of Protons in cis- and trans-2-Butene-1,I-dialand Their Bis-Semicarbazones compound &-2-butene-1,4-dia1(4) trans-2-butene-1,4-dial(5) 4-oxo-2-pentenal cis-2-butene-l,4-dialbis-semicarbazone ( 6 ) trans-2-butene-1,4-dial bis-semicarbazone (7) 4-oxo-2-pentenalbis-semicarbazone
'_I
CHO 10.45 10.25 9.89 10.2
CH=CH 6.65 6.48 6.83 6.96, 6.15 6.25 6.47 6.52
9:
5
L
r3
>
30
10
20
30
10
20
30
D
0
10 20 30 l i m e (min)
Jd 10
20
30
l i m e (min)
Figure 2. Representative HPLC traces from incubation mixtures of rat liver microsomes with furan in the presence of 60 mM semicarbazide. (A)cis-2-Butene-1,4-dialbis-semicarbazone standard; (B) furan (20 mM) incubated with rat liver microsomes (1 mg of proteidml) and required cofactors; (C) furan (20 mM) incubated in the absence of rat liver microsomes; (D)furan incubated with rat liver microsomes in the absence of NADP+. All incubations were performed in 400 mM potassium phosphate buffer (pH 6.8) in the presence of 25 mM glucose 6-phosphate, glucose-6-phosphate dehydrogenase (2 units/mL), 4 mM NADP+, 3 mM MgC12, 1 mM EDTA, and 60 mM semicarbazide for 30 min a t 37 "C.
aqueous conditions produced a yellow solid that was identified as cis-2-butene-lj4-dia1bis-semicarbazone (6). This compound was also synthesized by cleaving 2,5dimethoxydihydrofuran to cis-2-butene-1,4-dial in situ with semicarbazide hydrochloride. The identity of the product was confirmed by IH NMR and MS analysis. Four signals were detected in the 'H NMR spectra. The assignments, listed in Table 1, are consistent with our assigned structures, with singlets a t 10.4 and 6.45 ppm for the amide protons and doublets of doublets at 7.94 and 6.25 ppm for the CH=N and CH-CH protons, respectively. The positive ion DCI-MS displayed a molecular ion at m l z 199. The W spectrum had ;Imax at 310 nm. There were indications in the literature that isomerization around the double bond might occur during the reaction with semicarbazide (28, 21, 22). Therefore,
CH-N
N=NH
CONH2
CH3
ref ~~
17 7.94 7.61 6.5
10.40 10.27 10.15, 9.42
6.45 6.33 not reported
2.38
12
2.02
12
trans-2-butene-1,4-dial bis-semicarbazone (7) was independently prepared to confirm our stereochemical assignment. It was obtained upon reaction of semicarbathe product of acid zide with trans-2-butene-1,4-dial(5), The hydrolysis of 1,1,4,4-tetramethoxy-trans-2-butene. 'H NMR spectrum of this compound was similar to that of the cis compound; however, the CH=N protons were shifted upfield and the CH-CH protons were shifted downfield relative to the corresponding protons in the cisbis-semicarbazone (Table 1). Therefore, in the reaction of semicarbazide with cis-2-butene-1,4-dial (4)the cis orientation around the double bond was retained. Further experiments indicated that isomerization of cis-2-butene-l,4-dial(4) under neutral aqueous conditions is slow. The isomerization in deuterated phosphate buffer was monitored by lH NMR. The IH NMR spectrum revealed loss of the aldehydic and vinylic proton signals and appearance of new signals at 6.16,6.15,6.14, and 5.86 ppm. These singlets were assigned to the cisand trans-isomers 3 (19). Isomerization to trans-2butene-194-dial(5) was minimal in 24 h at room temperature. Therefore, 4 exists as hydrates 3 under physiological conditions (19). We took advantage of the bis-semicarbazone's strong absorbance at 310 nm to develop a selective HPLC method for the detection of this compound. Furan was incubated with rat liver microsomal preparations in the presence of semicarbazide and the required cytochrome P-450 cofactors. HPLC analysis of the reaction mixtures demonstrated the presence of a compound that coeluted with synthetic cis-2-butene-l,4-dial bis-semicarbazone (6, retention time: 22.5 min, Figure 2). The formation of this metabolite required NADPH, microsomes, and semicarbazide. Only trace amounts of a compound that eluted with a similar retention time as trans-2-butene-1,4-dial bis-semicarbazone (7, retention time: 13 min) were observed, demonstrating that little if any isomerization of the aldehyde had occurred. When the incubations were conducted in the presence of 6 mM semicarbazide, insufficient trapping of the metabolite occurred (data not shown). Metabolite formation was concentration dependent (Table 2). The bis-semicarbazone levels are likely underestimations of the amount of cis-2-butene-1,4-dial formed in these incubations since semicarbazide and the carrier solvent ethanol are potentially inhibitory against the oxidation of furan. In addition, further studies are required to determine the recovery of the dialdehyde metabolite as the bis-semicarbazone. A preparative scale incubation was conducted, and the metabolite was collected for chemical characterization. The IH NMR, W, and MS of this metabolically generated compound were identical to those of synthetic cis-2-butene-1,4-dial bis-semicarbazone (6).These results demonstrate that furan is oxidized by rat liver microsomes to cis-2-butene-1,4-dial (4). It has been proposed that furan metabolism by liver microsomes involves successive electron and oxygen
906 Chem. Res. Toxicol., Vol. 8, No. 7, 1995 Table 2. Concentration Dependence of Furan Metabolism to cis-2-Butene-l,4-diala furan (mM) 20 20 20 2 2 2 0.2 0.1 0.1 0.1 0.05 0.25
cis-2-butene-1,4-dia1 bis-semicarbazone ( 6 ) formed (nmolimL)
31.8 f 1.7 ndb 1.8 i 0.3 15.5 f 2.1 nd 0.5 i 0.1 4.6 f 0.4 2.3 f 0.2 nd
