116
Chem. Res. Toxicol. 1992,5, 116-123
Release of Iron from Ferritin by Aqueous Extracts of Cigarette Smoke Juan J. Moreno, Mahtab Foroozesh, Daniel F. Church, and William A. Pryor* Department of Chemistry and Biodynamics Institute, Louisiana State University, Baton Rouge, Louisiana 70803 Received M a y 29, 1991 This study demonstrates the ability of aqueous extracts of cigarette smoke to reduce iron and cause its release from ferritin. Superoxide dismutase (SOD) increases the rates of iron release with the less filtered smoke extracts, but has no effect on the rate of iron release caused by aqueous extracts of well-filtered gas-phase cigarette smoke. Faster rates of iron release are observed under anaerobic conditions, and the reducing power of the cigarette smoke extracts is prolonged when incubated in argon. Hydroquinone and catechol, two of the major polyhydroxybenzenes in cigarette smoke, increase in concentration in the smoke extracts as these are subjected to less filtration, and higher concentrations of polyhydroxybenzenes correlate with higher rates of iron release from ferritin. Concomitant with iron release, depletions of amino acids in ferritin are observed. Depletion of histidine is partially prevented by bathophenanthrolinedisulfonate and mannitol, while lysine and arginine depletions remain unaffected. These observations suggest that cigarette smoke components react directly with these amino acid residues in ferritin. Cigarette smoke induced release of iron could alter iron metabolism in the lungs of chronic smokers and contribute to the increase in the total oxidative burden on the lungs of smokers.
Introduction Cigarette smoking has been implicated in a variety of lung diseases, such as emphysema and lung cancer ( 1 , 2 ) , but the basic mechanisms by which tobacco smoke causes disease are still not well understood. Prolonged exposure to cigarette smoke also causes inflammation in the lower respiratory tract, a condition associated with larger numbers of pigment-laden macrophages found in the bronchioles, alveolar ducts, and alveoli of smokers (3). It has been shown that the pulmonary alveolar macrophages (PAM)' of smokers contain increased amounts of iron and ferritin ( 4 ) ;the reason for these increases is not known. McGowan et al. have suggested that the majority of this iron is insoluble and seems not to be associated with the iron storage protein ferritin (5). Also, Qian and Eaton recently showed that cigarette smoke extracts were able to reduce salts containing ferric ions and to bind the resulting ferrous ions (6). Preliminary electron spin resonance studies of lung tissue from smokers suggest that iron accumulation in microdomains is proportional to the dark pigmentation of the lung at that locus (7). These considerations prompted us to study the ability of aqueous extracts of cigarette smoke to release iron from ferritin, the principal iron storage protein in mammals. Cigarette smoke contains high concentrations of polyhydroxybenzenes (1,8, 9 ) with reduction potentials (10) lower than ferritin (11);these compounds could reduce and release iron from ferritin. In the studies reported here we have investigated the ability of aqueous extracts of cigarette smoke to release iron from ferritin, and the effects of SOD and catalase on this release. We also report the effect of aqueous extracts of cigarette smoke on the structure of ferritin. Experimental Procedures Chemicals. Bathophenanthrolinedisulfonic acid, 4,l-diphenyl-1,lO-phenanthrolinedisulfonic acid, o-phthalaldehyde,
* Address correspondence to this author at the Biodynamics Institute, 711 Choppin, Louisiana State University, Baton Rouge, LA 70803-1800.
1,4-benzenediol(hydroquinone),1,2-benzenediol(catechol),diethylenetriaminepentacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)piperazine-N'-2-
ethanesulfonic acid (HEPES), mannitol, amino acid standard solution (2.5 mM, 0.1 N HCl), D-glUCOSe, and 1,4-dithiothreitol were all purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Hydrochloricacid (6 N) was purchased from Pierce (Rockford, IL). Research cigarettes (1R1 and 1R2F) were purchased from the University of Kentucky, Tobacco and Health Research Institute, stored at -20 "C, and humidified in a desiccator over saturated ammonium nitrate at room temperature for at least 24 h prior to use. Chelex 100 resin was purchased from Bio-Rad Laboratories (Richmond,CA). All other chemicals were of reagent grade and used without further purification. Enzymes. Bovine liver catalase (EC 1.11.1.6),bovine erythrocyte copperzinc superoxide dismutase (EC 1.15.1.1),and g l u m oxidase (EC 1.1.3.4) were all purchased from Sigma. In order to remove adventitious iron, glucose oxidase was incubated with 10 mM DTPA for 1h in ice, ultrafitered, and washed with 2 volumes of 250 pL of deionized distilled water in a Millipore Ultrafree-PF filter unit with a molecular weight cutoff of 1OOOO. Ferritin Preparations. Aliquots of type I horse spleen ferritin, purchased from Sigma, were treated by two different procedures to eliminate unconfined iron: (a) by ultrafiltering ferritin twice with an equal volume of deionized distilled water and (b) by incubating ferritin with EDTA (10 mM) for 1h at 4 "C followed by chromatography on Sephadex G-25. After these treatments, ferritin was redissolved in 0.1 M phosphate buffer (pH 7.4, Chelex treated) to give a final concentration of 30 mg/mL. These solutions were stored at 4 "C for periods not longer than 72 h. The rates of iron release (measured as indicated below) obtained upon incubation with smoke extracts were compared for both of these ferritin preparations and found to give similar results. This indicates that the rates of iron release measured with the ultrafiltered ferritin preparation arise from iron being reduced and released from the core of the protein as opposed to reduction of unconfined iron. Due to the simplicity of the ultrafiltration Abbreviations: PAM, pulmonary alveolar macrophages; SOD,superoxide dismutase; BSA, bovine serum albumin; ESR, electron spin resonance; EDTA, ethylenediaminetetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; HPLC-ECD, high-pressure liquid chromatog raphy with electrochemical detection; HEPES, N-(2-hydroxyethy1)piperazine-N'-2-ethanesulfonic acid; QH,, hydroquinone; QH, semiquinone.
0 1992 American Chemical Society
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 117
Cigarette Smoke and Release of Iron from Ferritin technique and since the aqueous extracts of cigarette smoke contain traces of iron themselves, all experiments in this report were conducted with ferritin prepared by ultrafiltration. Total iron content of ferritin, determined as described by Brumby and Massey (12),was found to be 4.69 pmol of Fe/mg of protein. Preparation of Cigarette Smoke Aqueous Extracts. Using the puff protocol described elsewhere (13),the smoke from 1R1 or 1R2F research cigarettes was bubbled through phosphate buffer (0.1 M, pH 7.4, Chelex treated) contained in a test tube immersed in an icewater bath. No fdter was used to prepare extracts termed "wholesmoke extract", while those termed 'fdtered-smoke extract" were prepared by passing the smoke through the filter tip that 1R2F cigarettes contain. Extracts termed 'gas-phase extracts" were prepared by smoking 1R1 cigarettes through two back-toback Cambridge filters. All extracts were prepared by bubbling the smoke of 1cigarette (ten 35-mL puffs) per 100 pL of buffer. All smoke extracts used were prepared immediately before experiments unless otherwise indicated. Gel Electrophoresis. Aliquota (25 pg) of ferritin solutions similar to those used for iron release experiments were subjected to native electrophoresis on a 5.5% acrylamide gel at 30 mA for approximately 3 h at 25 OC. After electrophoresis, gels were removed and stained with Coomassie brilliant blue R-250for 1 h and then allowed to destain for 24 h in destaining solution (5% methanol, 7% glacial acetic acid, and 2% glycerol). Amino Acid Analysis. Ferritin samples (125-pg aliquots) were incubated with extracts of cigarette smoke for 12 h at 37 OC and hydrolyzed with 6 N HCl at 110 O C for 24 h in vacuo. Amino acids were separated and quantitated by HPLC in an AminoQuant column (Hewlett-Packard) using o-phthalaldehyde precolumn derivatization according to Schuster and Apfel(14). Analysis of proline, cysteine, and tryptophan was not performed. Oxidation of methionine to methionine sulfoxide was determined by the method described by Shechter et al. (15). Briefly, lyophilized protein samples were dissolved in 80% formic acid and allowed to react with cyanogen bromide (100 mM) for 24 h at room temperature. The reaction was stopped by addition of an equal volume of water, and the samples were then frozen and lyophilized. The cyanogen bromide peptides were hydrolyzed as described above but in the presence of 13 mM 1,4-dithiothreitol. Iron Release. Rates of iron release were measured spectrophotometrically, using bathophenanthrolinedisulfonateas metal chelator, essentially as described by Thomas et al. (16).Incubation mixtures (1 mL final volume) contained 1 mM bathophenanthrolinedisulfonate and 750 pg of ferritin in 0.1 M phosphate buffer (pH 7.4,15 mh4 NaCl, Chelex treated). Catalase and SOD solutions were added as indicated in figure legends. Reactions were started by the addition of 100 pL of smoke extract or benzenediol solution. The change in absorbance was measured continuously a t 530 nm for the first 20 min using a double-beam spectrophotometer. A cuvette containing ferritin, bathophenanthroline, and added enzymes (catalase or SOD) but lacking cigarette smoke extract or hydroquinones was used as the reference solution. The rate of iron release by extracts of cigarette smoke shows biphasic kinetics (see Figure 2). Both the early and later portions of the curve are essentially linear. We have chosen to calculate the rate of iron release from the nearly linear slope of the curve from 3 to 10 min, using a molar extinction coefficient of 20.66 mM-l cm-' obtained from a calibration curve using ferric sulfate reduced with a 10% solution of thioglycolic acid. For anaerobic measurements all solutions were purged with argon, and before addition of the smoke extract, incubation mixtures were purged again with argon through a capillary glass tube inserted in the UV cell. In order to scavenge remaining oxygen, glucose oxidase and glucose were always added along with catalase to both sample and reference cuvettes. Iron Incorporation. All studies were conducted at pH 7.0 in 0.1 M HEPES buffer according to the method of Collawn et al. (17)with only minor changes. Briefly, 4.5 mL of a solution of native or smoke-exposed ferritin (0.1 mg/mL) which had been previously dialyzed against water was placed in a constant-temperature water bath a t 14 "C and allowed to equilibrate. A solution of ferrous ammonium sulfate (0.5 mL, 10 mM) dissolved in deaerated water was added and mixed vigorously. Aliquots (1mL) were taken a t 1-min intervals and added to Eppendorf tubes containing 0.3 g of Chelex 100. Samples were vortexed for
Table I. Aerobic Release of Iron from Ferritin by Aqueous Extracts of Cigarette Smoke" nmol of Fe reduced/(minmg of ferritin) entry no. smoke extractb additions none 0.25 i 0.01 1 gas 0.27 i 0.02 2 gas catalase SOD 0.27 i 0.01 3 gas none 0.89 & 0.05 4 filtered 5 filtered catalase 1.15 i 0.01 6 filtered SOD 1.88 f 0.07 7 whole none 1.95 i 0.17 a whole catalase 2.56 i 0.27 SOD 4.69 i 0.26 9 whole "eaction mixtures (final volume 1 mL) contained ferritin (750 smoke from one 1R1 or 1R2F cigarette blown into 100 pL of buffer (see Experimental Procedures), bathophenanthrolinedisulfonate (1 mM), SOD (500 units), or catalase (500 units) in 15 mM NaCl, pH 7.4. Reactions were initiated by addition of smoke extracts, and absorbance was measured continuously at 530 nm during the first 20 min. b'Gas" refers to extracts of cigarette smoke from 1R1 cigarettes passed through two Cambridge filters, 'filtered" to smoke extract from 1R2F cigarettes filtered with the self-contained filter tips, and 'whole" to extracts of unfiltered smoke from 1 R l cigarettes. pg), the
4 min, and needle holes were punched in the bottom of the tubes.
The perforated Eppendorfs were placed in larger test tubes and centrifuged in a bench-top centrifuge for 4 min. The eluted femtjn solution, free of Chelex 100, was then collected and its absorbance measured a t 310 nm in a Hewlett Packard 8451A diode array spectrophotometer; HEF'ES buffer was used as reference. A blank solution consisting of ferrous ammonium sulfate without ferritin was used. The time of reaction was taken as the period elapsed between the addition of iron and the addition of the reaction mixture to Chelex 100. HPLC. (A) Electrochemical Detection. Aqueous extracts of cigarette smoke were separated using a Hewlett-Packard ODS (5 pm, 200 X 2.1 mm) (Hewlett-Packard Co., Avondale, PA) reverse-phase column. The electrochemical detector consisted of a Model 5100A Coulochem electrochemical detector (ESA, Inc. Bedford, MA), a dual-electrode analytical cell, Model 5010 (ESA), consisting of two porous graphite in-line working electrodes with associated reference and counter electrodes, and a guard cell, Model 5020 (ESA), placed before the injection port. Constant potentials of +350 and +400 mV were applied to the working electrodes of the analytical and guard cells, respectively. The mobile phase consisted of a binary system of water and methanol; both solvents contained 10 mM NaClO,. With a flow rate of 0.50 mL/min, 5-pL aliquota of smoke extract were injected onto the column and eluted with 10% MeOH. After 6 min the percentage of methanol was linearly increased to 100% over 35 min. Response factors used for quantitative analysis of hydroquinone and catechol were calculated from calibration c w e s obtained using authentic compounds. (B)UV Detection. Aqueous extracts of cigarette smoke were separated in a Hypersil ODS column (200 X 4.6 mm) connected to a Hewlett-Packard HPLC Model 1090M equipped with a diode array detector and an automatic injector. The eluent consisted of water and acetonitrile with an initial composition of 3% acetonitrile which was linearly increased to 50% acetonitrile over 12 min at a constant flow rate of 1.0 mL/min. The column was washed with 100% acetonitrile for 20 min after each injection (15 pL). All solvents used were HPLC grade and filtered through a 0.3-pm filter prior to use.
Results Release of Iron from Ferritin by Aqueous Extracts of Cigarette Smoke. W h e n ferritin is incubated with aqueous extracts of cigarette smoke, iron is reduced and mobilized from the protein core. Table I shows rates of iron release, measured spectrophotometrically under aerobic conditions. I n the absence of ferritin none of t h e cigarette smoke extracts form significant amounts of bathophenanthroline-Fe complex (data not shown). Lines
Moreno et al.
118 Chem. Res. Toxicol., Vol. 5, No. 1, 1992
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.-c
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\ c '
-
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,
-
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300
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700
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900
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Figure 2. Time course for the formation of the Fe(I1)-bathophenanthrolinedisulfonate complex as iron is mobilized from ferritin by (1)filtered-smoke extract under anaerobic conditions, (2) filtered-smokeextract incubated in argon for 1h under aerobic conditions, and (3) filtered-smoke extract incubated in air for 1 h under aerobic conditions. Table 11. Effect of Oxygen on the Release of Iron from Ferritin by Aqueous Extract of Filtered Smoke" smoke extract nmol of Fe incubareduced/(minentrv no. anaerobic svstem tion mn of ferritin) 1 no air 1.18 0.09 2 no argon 1.79 0.08 3 Yes argon 4.23 i 0.16 ~
0.0 0.0
11'00
TIME (sec)
i
m
100
0.4
0.8
1.2
1.6
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FERRITIN (mg/mL soln.)
Figure 1. Panel A shows rates of iron release from ferritin as a function of the concentration of the different cigarette smoke extracts. Reaction mixtures (finalvolume 1mL) contained ferritin (750 g)and bathophenanthrolinedisulfonate (1mM) in 15 mM NaCl, pH 7.4. Reactions were started by addition of different volumes of smoke extract prepared by passing the smoke of 1 cigarette per 100 p L of buffer. Whole-smoke extract (m); filtered-smoke extract (0); gas-phase smoke extract (v). Panel B shows the rates of iron release a function of the concentration of ferritin. Reaction mixtures contained bathophenanthrolinedisulfonate (1mM), 100 p L of whole-smoke extract, and different amounts of ferritin. 1, 4, and 7 suggest the amounts of ferrous iron released increase as the smoke is subjected to less filtration (see Experimental Procedures for smoke extract preparations). The rates of iron release depend on the amounts of all three types of smoke extract (Figure lA), and on the concentration of ferritin (Figure 1B; data only shown for filtered-smoke extract). Figure 1A shows that the rates of iron release for the different extracts of cigarette smoke decrease over the range of smoke extract concentrations used in the order whole > filtered > gas smoke. To determine whether superoxide participates in the reductive mobilization of ferric iron by cigarette smoke extracts, SOD was included in the incubation mixtures. SOD does not affect the rate of iron release when the reaction mixture contains gas-phase extract (line 3, Table I). However, for both filtered- and whole-smoke extracts, rates of iron release increase when SOD is present in the reaction mixture (lines 6 and 9 in Table I). The production of hydrogen peroxide by extracts of cigarette smoke (18,19) could have an inhibitory effect on the observed rates of iron release. Hydrogen peroxide could oxidize Fe(I1) before complexation with bathophenanthrolinedisulfonate (20)but probably not after
~~~
OReaction mxtures (final volume 1 mL) contained ferritin (750 100 pL of filtered-smoke extract (1 cigarette/100 pL), bathophenanthrolinedisulfonate (1 mM), and catalase (500 units) in 15 mM NaC1, pH 7.4. Anaerobic systems were purged with argon and contained glucose oxidase (10 unita) and glucose (5 mM) in addition to the above components. Reactions were initiated by addition of filtered-smoke extracts (incubated for 1 h in either air or argon at room temperature), and absorbance was measured continuously at 530 nm during the first 20 min. pg),
formation of the complex (21,22). In the presence of Fe(II), hydrogen peroxide also could oxidize species that are present in cigarette smoke extracts which could be responsible for iron mobilization such as benzenediols. Both of these effects could cause a reduction in the amount of bathophenanthrolineFe(I1)complex formed. Inclusion of catalase in the reaction mixture with gas-phase smoke extract does not affect the rate of Fe(I1)-bathophenanthroline complex formation (line 2 in Table I), while with filtered and whole smoke (lines 5 and 8 in Table I) catalase causes about 30% higher rates. To verify that these results are due to the catalytic activity of the enzymes, BSA was added to the incubation mixtures in concentrations equal to those of catalase and SOD and shown to have no effect in the rates of iron release (data not shown). To investigate the role of oxygen in the rates of iron release, aerobic and anaerobic conditions were compared. The effect of oxygen on smoke was also investigated by measuring rates of iron release by filtered-smoke extracts incubated for 1h at room temperature in air or in argon. Figure 2 shows typical curves obtained in these experiments with filtered-smoke extracts. It is apparent from these curves that the release of iron is faster in the anaerobic system and that, after 1 h at room temperature, smoke extracts incubated in argon release iron at a faster rate than its analogue incubated in air. Table I1 sum-
Chem. Res. Toxicol., Vol. 5, No.1, 1992 119
Cigarette Smoke and Release of Iron from Ferritin
Table IV. Aerobic Release of Iron from Ferritin by Benzenediolsa nmol of Fe
reduced/
entry no. 1 2 3 4 5 6
benzenediol catechol hydroquinone catechol + hydroquinone catechol + hydroquinone catechol hydroquinone whole smokeb
(mimmg of ferritin) 0.50 f 0.04 0.42 i 0.02 1.61 f 0.02 1.73 & 0.03 1.76 f 0.02 1.95 i 0.17
additions none none none catalase
+
SOD none
"Reaction mixtures (final volume 1 mL) contained ferritin (750 pg), bathophenanthrolinedisulfonate (1 mM), catechol (3.09 mM),
i
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c i
fi
4
6
8
10
TIME (minutes)
12 1'4
I
Figure 3. HPLC with electrochemical detection of aqueous extracts of cigarette smoke. ECD detection was carried out by applying a voltage of +350 mV in the detection cell and +400 mV in the guard cell. Smoke extracts were diluted 1:lOO before injection. Whole-smoke extract (A), filtered-smoke extract (B), and gas-phasesmoke extract (C).
hydroquinone (6.10 mM), catalase (500 units), and SOD (500 units) in 15 mM NaC1, pH 7.4. Reactions were initiated by addition of benzenediols, and absorbance was measured continuously at 530 nm during the first 20 min. Contains 3 mM catechol and 6 mM hydroquinone (Table 111).
catechol" hydroquinoneb molarity, molarity, extract mM pcg/cigarette mM pg/cigarette gas phase 0.51 f 0.04 5.76 i 0.46 0.18 f 0.01 2.01 f 0.12 1.20 f 0.10 13.16 f 1.05 1.86 i 0.11 20.45 f 1.23 filtered whole
3.09 i 0.25 33.97 i 2.72
6.10 f 0.37 67.20 f 4.03
" Concentrations were determined by HPLC with electrochemi-
cal detection applying a constant voltage of +350 mV in the detection cell and +400 mV in the guard cell. Response factors were calculated from calibration curves using pure compounds. Concentrations of benzenediols in the aqueous extacta (1 cigarette 100 pL); expressed as mM (or pg/cigarette) f SD (n = 3).
marizes these results and shows that rates of iron release with filtered-smoke extracts under completely anaerobic conditions (entry 3 buffer and extract in argon) are about 350% faster than in air and that the smoke extract incubated in argon releases iron from ferritin at rates about 150% faster. HPLC Analysis of Aqueous Extracts of Cigarette Smoke. HPLC separation of smoke extract components allowed positive identification of catechol and hydroquinone by spiking the extracts with authentic compounds, and by comparing the UV spectra recorded with a diode array detector in chromatograms obtained from smoke extrads and from authentic compounds (data not shown). Figure 3 shows typical HPLC-ECD chromatograms of all three aqueous extracts of cigarette smoke obtained with oxidative electrochemical detection. These traces show that 1,4-dihydroxybenzene (hydroquinone) and 1,2-dihydroxybenzene (catechol) are the major electrochemically active components in the aqueous extracts of cigarette smoke under the oxidative conditions used for this analysis. Quantitative analysis of both dihydroxybenzenes in the smoke extracts is shown in Table 111. A comparison of these results with previously reported concentrations of catechol and hydroquinone in cigarette smoke condensates is difficult due to differences in the preparation of the condensates and the type of cigarettes used. Nanni et al. (23)recently reported concentrations of 38-58 pg for each catechol and hydroquinone per cigarette in electrostaticprecipitated condensates using commercially available and 1R4F cigarettes. Our determination shows that filtered
-
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-
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Table 111. Concentrations of Hydroquinone and Catechol in Aaueous Extracts of Ciuarette Smoke" -
0.18
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Figure 4. Rates of iron uptake by smoke-treated ferritin. Ferritin samples were incubated with cigarette smoke extracts for 12 h at 37 " C and then exhaustively dialyzed before iron incorporation assay. Blank (no ferritin) (v);untreated ferritin control ( 0 ) ; exposed to gas-phase smoke (0); exposed to filtered smoke (0); exposed to whole smoke ( 0 ) .
and gas-phase smoke extracts contain concentrations of both dihydroxybenzenes below this range and that hydroquinone is the major dihydroxybenzene in wholesmoke extract with a concentration of 67 pg per IRI cigarette. Release of Iron from Ferritin by Hydroquinone and Catechol. Polyhydroxybenzenes (24, 25) and more recently hydroquinone and catechol (26)have been shown to release iron from ferritin. Table IV shows rates of iron release obtained with catechol and hydroquinone under aerobic conditions in the presence of either catalase or SOD. Although the concentrations used for both benzenediols are equal to those found in whole-smoke extract by HPLC-ECD (Table 111),rates of iron release measured for a solution containing both diols (entry 3, Table IV) are about 17% slower than those measured with the smoke extract (entry 6, Table IV). The effects of catalase and SOD (entries 4 and 5, Table IV) on these rates are not nearly as dramatic as are the effects of these enzymes on the rates obtained with filtered- or whole-smoke extracts. Both enzymes slightly increase the rates of iron release by a solution containing both benzenediols. Structure of Smoke-ExposedFerritin. The structure of ferritin after incubation with cigarette smoke extracts was studied by three methods: iron incorporation, native electrophoresis, and amino acid analysis. Figure 4 shows the uptake of ferrous iron by smoketreated ferritin samples. After incubation with smoke
Moreno et al.
120 Chem. Res. Toxicol., Vol. 5, No.1, 1992
1
2
3
4
5
6
Figure 5. Nondenaturing polyacrylamide gel electrophoresis of smoke-treated ferritin. A typical gel obtained after electrophoresis of ferritin samples exposed to different aqueous extracts of cigarette smoke for 12 h at 37 "C, with lanes: 1, ferritin exposed to whole-smoke extract; 2, 4, and 6, native ferritin; 3, ferritin exposed to filtered-smoke extra&, 5, ferritin exposed to gas-phase smoke extract. Table V. Amino Acid Composition of Cigarette Smoke Exmsed Ferritin" residue nativeb control" gas filtered whole His 5.7 f 0.5 5.9 f 0.1 5.8 f 0.1 4.0 f 0.8 1.3 f 0.7 Thr 6.0 f 0.0 5.1 f 0.3 6.0 f 0.3 4.3 f 0.1 2.0 f 0.5 Arg 10.1 f 0.8 10.1 f 0.5 10.4 f 0.4 9.9 f 0.0 8.0 f 0.4 Tyr 5.8 f 0.3 5.0 f 0.5 5.4 f 0.3 5.2 f 0.5 3.5 f 0.2 Met 3.0 f 0.0 3.0 f 0.2 3.5 f 0.5 2.9 f 0.7 2.9 f 0.5 0.0 0.0 0.2 f 0.1 0.0 f 0.0 MetSO Phe 7.6 h 0.4 7.4 f 0.4 6.5 f 0.7 7.2 f 0.4 5.3 f 0.5 Lys 9.4 f 0.4 9.2 f 0.7 9.4 f 0.5 7.4 f 0.4 6.1 f 0.5
~~
"Cysteine, proline, and tryptophan were not determined in this analysis. Amino acids listed are only those which are depleted upon exposure to cigarette smoke! Compositions are given as number of residues per subunit of 22500 Da. Ferritin samples (750pg) were incubated wtih cigarette smoke extracts (100pL) in 100 mM phosphate buffer for 12 h at 37 "C. Results are expressed as the mean f SD (n = 5). bPreviously published amino acid compositions for horse spleen ferritin averaged from refs 27-29. "Amino acid composition of horse spleen ferritin determined in this report.
extracts for 12 h at 37 "C followed by exhaustive dialysis, all three smoke-exposed ferritin preparations exhibit rates of ferrous iron uptake comparable to that of unexposed ferritin. The choice of buffer in the Fe(I1) uptake assay is of importance. When acetate buffer is used in place of HEPES, considerably slower rates of iron incorporation are observed.2 Nonetheless, our results indicate that any damage suffered by the protein during the incubation with smoke extrads does not change its ability to reincorporate iron compared to native ferritin. Smoke-exposed ferritin solutions were also subjected to nondenaturing electrophoresis; these results are shown in Figure 5. Ferritin exposed to gas-phase extract (lane 5) does not exhibit a change in mobility compared to native ferritin (lanes 2,4, and 6). On the other hand, filteredand whole-smoke-treated ferritin samples (lanes 3 and 1, respectively) exhibit small changes in their mobility compared to the native protein, indicative of an increased anionic nature compared to native ferritin. Table V shows the results of amino acid analysis of ferritin samples exposed to all three types of smoke extracts. Amino acid analysis obtained for native ferritin in this study compares very well with previously published min~ acid compositions for horse spleen ferritin (27-29). Table V shows that no significant changes in amino acid composition are detected for ferritin exposed to gas-phase S. D. Aust, personal communication of unpublished results (1991).
Table VI. Amino Acid Composition of Whole Smoke Exposed Ferritin in the Presence of Metal Chelators or Mannitol" bathophenanthrolinedisulforesidue control wholeb nate" mannitold His 5.9 f 0.1 1.3 f 0.7 4.1 f 0.7 3.3 f 0.1 Thr 5.1 f 0.3 2.0 f 0.5 4.4 f 0.0 4.7 f 0.0 7.3 f 0.1 7.5 f 0.2 10.1 f 0.5 8.0 f 0.4 Arg 5.4 f 0.1 4.3 f 0.1 T3n 5.0 f 0.5 3.5 f 0.2 Met 3.0 f 0.2 2.9 f 0.5 3.1 f 0.3 3.1 f 0.0 MetSO 0.0 0.0 0.0 0.0 Phe 7.4 f 0.4 5.3 f 0.5 6.9 f 0.1 7.0 f 0.0 6.5 f 0.1 6.1 f 0.1 9.2 f 0.7 6.1 i 0.5 LYS "Amino acid composition is given as number of residues per subunit of 22 500 DA. Results are expressed as the mean f SD (n = 5). *Ferritin samples (750pg) were incubated with whole-smoke extracts (100pL) in 100 mM phosphate buffer for 12 h a t 37 "C (from Table V). 'In the presence of 1 mM bathophenanthrolinedisulfonate. the presence of 1 mM mannitol.
extract. However, ferritin exposed to filtered- and whole-smoke extracts shows significant changes in the levels of some amino acids. Histidine levels decrease by about 30% for filtered and 75% of whole-smoke-treated ferritin. Lysine depletions of about 20 and 35% are observed for fdtered- and whole-smoke extracts, respectively. Threonine, tyrosine, phenylalanine, and arginine levels decrease by approximately 60, 30, 28, and 20%, respectively, in whole-smoke-exposed samples. Cigarette smoke extracts produce hydrogen peroxide (18, 19,30)which could react with ferrous iron released from ferritin to generate an activated oxygen species such as the hydroxyl radical or ferry1 ion; these species could react with the side chains of amino acids in ferritin. The amino acid composition of ferritin incubated with whole-smoke extract in the presence of bathophenanthrolinedisulfonateor the hydroxyl radical scavenger mannitol was studied in an effort to determine the origin of the damage to the protein, these results are shown in Table VI. Both bathophenanthroline and mannitol give similar results; they afford almost complete protection to threonyl, tyrosyl, and phenylalanyl residues. Histidine is partially protected (ca. 61%)3by bathophenanthroline and is protected less efficiently by mannitol (ca. 43%). Lysine and arginine depletions are not prevented by the metal chelator or by mannitol. Alanine and valine levels remain higher than in the control in the presence of bathophenanthrolinedisulfonate or mannitol, while levels of glycine are increased by 30% compared to an increase of 80% in the absence of bathophenanthroline or mannitol (data not No siflicant methionine oxidation was detected in either the presence or absence of bathophenanthrolinedisulfonate or mannitol.
Dlscussion The data presented above demonstrate that aqueous extracts of cigarette smoke are capable of reducing and mobilizing iron from ferritin. Two possible mechanisms for this release are the following: reduction of ferritin iron by superoxide ion generated during the autoxidation of or polyhydroxybenzenes present in smoke extracts (8,30); direct reduction of iron by polyhydroxybenzenes and/or ~
~~
Percent (9%) protection is defined as 100 ti&& the difference: 100 - [(control - experiment with additive)/(control - smoke experiment)]. ' For a complete list of amino acids analyzed see: Juan J. Moreno (1991)Studies on cigarette smoke: I. Inactivation of a-1-PI. 11. Oxidation of methionine by peroxonitrite. 111. Release of iron from ferritin by smoke solutions, Ph.D. Dissertation, Louisiana State University, pp 127-128.
Cigarette Smoke and Release of Iron from Ferritin
Y
0.00
x 25 50 75 100 0
DIHYDROXYBENZENE CONCENTRATION (pM)
Figure 6. Rates of iron release as a function of the combined concentrations of hydroquinone and catechol found in the three different extracts of cigarette smoke. Abscissa values obtained from Table I, ordinate values obtained from Table 11(R2= 0.992). (A) Gas-phase smoke extract; (B) filtered-smoke extract; (C) whole-smoke extract. other smoke components. Our data on the effect of SOD suggest that the second mechanism is more likely. Superoxide ion and hydrogen peroxide are formed in aqueous tar extracts of cigarette smoke from the autoxidation of smoke components (19,30). The results obtained here with SOD indicate that superoxide ion, which is known to release iron from ferritin (20, 31), does not contribute significantly to the release of iron caused by cigarette smoke. In fact, SOD enhances the rate of formation of the Fe(I1)-bathophenthroline complex in the presence of filtered and whole smoke and has no effect with gas-phase smoke extracts. Addition of catalase gives similar results, enhancing rates of Fe(I1) complex formation with filtered- and whole-smoke extracts but showing no effect in the presence of gas-phase smoke. The effects of these two enzymes indicate that superoxide ion and hydrogen peroxide formation is only relevant in filtered- and whole-smoke preparations and that both species inhibit the rate of formation of the Fe(I1)-bathophenanthroline complex. The effect of catalase is probably due to its preventing the oxidation of Fe(I1) released from ferritin by hydrogen peroxide and consequently causing an increased recovery of reduced iron by bathophenanthroline (20). The effect of SOD is discussed below. Analysis by HPLC using electrochemical detection shows the presence of hydroquinone and catechol in all our smoke extracts, along with other unidentified minor components (Figure 3). Recently Leanderson and Tagesson (32) also detected hydroquinone and catechol, using HPLC-ECD under oxidative conditions, as the major electrochemically active components in aqueous extracts of cigarette smoke. The concentration of these phenols increases as the smoke is subjected to less filtration, and the sum of their amounts in all smoke extracts correlates with the rates of iron release (see Figure 6). The difference in concentration of phenols in fitered and nonfiitered smoke extracts could in part explain the differing rates of iron release observed. Although these benzenediols are abundant in cigarette smoke (23) and probably important contributors in the reduction of ferritin, our data do not allow us to eliminate the possibility that other smoke components also contribute to the observed release of iron. Our results in fact show that cigarette smoke is a more complex system than a mixture of hydroquinone and catechol; the rates of iron release measured with a mixture of both benzenediols, in concentrations comparable to those determined in whole
Chem. Res. Toricol., Vol. 5, No. 1, 1992 121
smoke, are not increased by catalase and SOD as much as with the smoke extracts (Table IV). Protein Damage. Amino acid analysis (Tables V and VI) along with the results obtained from native electrophoresis indicates that both filtered- and whole-smoke extracts are capable of changing the chemical composition of ferritin. These modifications, however, do not seem to undermine the ability of ferritin to reincorporate iron. The changes in the levels of histidine, lysine, and arginine for both filtered- and whole-smoke-exposed ferritin samples could explain the changed electrophoretic mobilities observed for these two treated ferritin samples in native gels. Modification of these basic amino acids could result in a net decrease in positive charge, making the protein more anionic than untreated ferritin. Damage to lysine, arginine, and about 30% of total histidine is not protected by either bathophenanthroline or mannitol, indicating that not all amino acid damage involves ferrous iron or an active oxygen species. These results suggest that cigarette smoke components react with histidyl, arginyl, and lysyl residues in ferritin. We have previously observed depletions of lysyl and histidyl residues in a-1-proteinase inhibitor after incubation with cigarette smoke aqueous extract^.^ Also, the formation of cyanomethyl derivatives of lysyl and arginyl residues upon reaction with cigarette smoke has been reported (33). We have also observed4that cigarette smoke oxidizes methionine residues in a-1-proteinase inhibitor. The lack of oxidation of ferritin methionyl residues by smoke extracts even in the absence of bathophenanthroline or mannitol is unexpected, but our present data do not allow us to afford a proper explanation for this observation. The reductive release of iron from ferritin has been well documented to cause oxidative damage, principally by We suggest initiating lipid peroxidation (20,24,25,34,35). that release of iron from ferritin caused by extracts of cigarette smoke in the presence of hydrogen peroxide, also produced by smoke extracts, could also catalyze the oxidation of proteins. The Effect of SOD. The release of iron is faster under argon than under air, and filtered-smoke extracts release iron faster when incubated in argon than when incubated in air. It is also observed that filtered- and whole-smoke extracts release iron from ferritin more efficiently when SOD is added in the incubation mixture. This suggests that under aerobic conditions autoxidation of species responsible for the release of iron from ferritin occurs, with the effect of diminishing the reducing capability of the smoke extracts, and that SOD prolongs the life of these reducing species. Oxygen uptake experiments with smoke extracts and ferritin (data not shown) suggest that SOD and catalase inhibit rates of oxygen consumption of both filtered- and whole-smoke extracts, confirming this hypothesis. A possible mechanism for the release of iron from ferritin by cigarette smoke extracts which could explain the effect of SOD is shown in eqs 1-4. In eq 1,hydroquinone and QH, + Fe"' QH' + Fe" + H+ (1) QH' QH' QH,
+ Fe"'
-- + + + + Fe"
+ 02 *Q
+ 0,'-+ H+
Q H+ 02'- H+
-
QH'
+ H202
(2)
(3)
(4) catechol (QH2),along with other electrochemically active compounds present in lower concentrations in cigarette smoke extracts, are proposed to be the major contributors in the release of iron from ferritin. A contribution to the release of iron from semiquinone5 (QH'), generated in eqs 1and 4, cannot be eliminated by the data presented here.
122 Chem. Res. Toxicol., Vol. 5, No. 1, 1992
Semiquinones have been shown to effectively release iron from ferritin principally under anaerobic or hypoxic conditions (36). However, Monteiro et al. observed that benzosemiquinone does not cause the release of iron under aerobic conditions and that oxygen causes a marked inhibition in iron release by other semiquinones (37). Other studies (25,35) have shown enhancement of iron release from ferritin in the presence of SOD using dialuric acid, acid-hydrolyzed divicine, and 6-hydroxydopamine as reducing species, and a rationale similar to that proposed here was given.
Conclusion Our in vitro observations indicate that cigarette smoke releases iron from ferritin and that this release is accompanied by damage of the protein, which is prevented in part by a metal chelator and a hydroxyl radical scavenger. We suggest the release of iron from ferritin by cigarette smoke may alter iron metabolism in the lungs of smokers. Iron is known to catalyze the formation of reactive oxygen species (38) which have been implicated in a variety of deleterious oxidative processes (39). Also, cigarette smoke in the presence of biological forms of iron could cause oxidative damage to proteins such as a-1-proteinase inhibitor. These findings provide possible explanations for the observed inflammation of the lower respiratory tract and increased concentrations of iron found in PAM of tobacco users. Acknowledgment. This work was supported in part by a grant from NIH and a contract from the National Foundation for Cancer Research. Helpful discussions with Professors C. C. Winterbourn and S. D.Aust are greatly appreciated.
References (1) U S . Public Health Service (1979) Smoking and Health: A Report of the Surgeon General, U S . Department of Health, Education and Welfare, Washington, DC. (2) U S . Public Health Service (1982) The health consequences of smoking, CANCER: A Report of the Surgeon General, US.Department of Health and Human Services, Washington, DC. (3) Auerbach, O., Hammond, E. C., Garfunkel, L., and Benante, C. (1972) Relation of smoking and age to emphysema. Whole lung study. N . Engl. J. Med. 286, 853-857. (4) McGowan, S. E., Murray, J. J., and Parrish, M. G. (1986) Iron binding, internalization, and fate in human alveolar macrophages. J. Lab. Clin. Med. 108, 587-595. (5) McGowan, S. E., and Henley, S. A. (1988) Iron and ferritin contents and distribution in human alveolar macrophages. J.Lab. Clin. Med. 111, 611-617. (6) Qian, M., and Eaton, J. W. (1989) Tobacco-borne siderophoric activity. Arch. Biochem. Biophys. 275, 280-288. (7) Church, D. F., Burkey, T. J., and Pryor, W. A. (1990) Preparation of human lung tissue from cigarette smokers for analysis by electron spin resonance spectroscopy. Methods Enzymol. 186, 665-669. (8) Hoffmann, D., and Wynder, E. L. (1986) Chemical constituents and bioactivity of tobacco smoke. In Tobacco: a major international health hazard (Zaridze, D. G., and Peto, R., Eds.) Vol. 74, pp 145-165, International Agency for Research of Cancer, IARC, Lyon. (9) Guerin, M. R. (1980) Chemical composition of cigarette smoke. In The Banbury Report 3; a safe cigarette? (Gori, G. B., and Bock, F. G., Eds.) pp 191-204, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (10) Wardman, P. (1989) Reduction potentials of one-electron couples involving free radicals in aqueous solution. J. Phys. Chem. Ref. Data 18, 1637-1755. (11) Watt, G. D., Frankel, R. B., and Papaefthymiou, G. C. (1985) Reduction of mammalian ferritin. Proc. Natl. Acad. Sci. U.S.A. 82, 3640-3643. (12) Brumby, P. E., and Massey, V. (1967) Determination of nonheme iron, total iron, and copper. Methods Enzymol. 10,463-474.
Moreno et al. (13) Pryor, W. A., Dooley, M. M., and Church, D. F. (1984) Inactivation of human alpha-1-proteinase inhibitor by gas-phase cigarette smoke. Biochem. Biophys. Res. Commun. 122, 676-681. (14) Schuster, R., and Apfel, A. (1986) A new technique for the analysis of primary and secondary amino acids. Hewlett Packard HPLC Application Note (Bioscience) Publication No. 12, 5954-6257 (Abstract). (15) Schechter, Y., Bernstein, Y., and Patchornic, A. (1975) Selective oxidation of methionine residues in proteins. Biochemistry 14, 4497-4503. (16) Thomas, C. E., and Aust, S. D. (1986) Reductive release of iron from ferritin by cation free radicals of paraquat and other bipyridyls. J. Biol. Chem. 261, 13064-13070. (17) Collawn, J. F. Jr., Priest, D. G., and Fish, W. W. (1982) A sensitive assay employing a solid-phase metal chelator to estimate rates of iron accumulation by ferritin. Anal. Biochem. 127, 105-1 11. (18) Nakayama, T., Kodama, M., and Nagata, C. (1984) Generation of hydrogen peroxide and superoxide anion radical from cigarette smoke. Gann 75,95-98. (19) Nakayama, T., Church, D. F., and Pryor, W. A. (1989) Quantitative analysis of the hydrogen peroxide formed in aqueous cigarette tar extracts. Free Radical Biol. Med. 7, 9-15. (20) Thomas, C. E., Morehouse, L. A., and Aust, S. D. (1985) Ferritin and superoxide-dependent lipid peroxidation. J.Biol. Chem. 260, 3275-3280. (21) Koppenol, W. H., Maskos, Z., and Rush, J. D. (1991) Catalysis of oxyradical reactions by iron complexes. In Oxidative Damage and Repair: Clinical, Biological and Medical Aspects (Davies, K. J. A., Ed.) Pergamon Press, New York (in press). (22) Gutteridge, J. M. C., Maidt, L., and Poyer, L. (1990)Superoxide dismutase and Fenton chemistry. Reaction of ferric-EDTA complex and ferric-bipyridyl complex with hydrogen peroxide without the apparent formation of iron(I1). Biochem. J. 269, 169-174. (23) Nanni, E. J., Lovette, M. E., Hicks, R. D., Fowler, K. W., and Borgerding, M. F. (1990)Separation and quantitation of phenolic compounds in mainstream cigarette smoke by capillary gas chromatography with mass spectrometry in the selective ion mode. J. Chromatogr. 505, 365-374. (24) Boyer, R., Clark, H. M., and LaRoche, A. P. (1988) Reduction and release of ferritin iron by plant phenolics. J. Znorg. Biochem. 32, 171-181. (25) Monteiro, H. P., and Winterbourn, C. C. (1989) 6-Hydroxydopamine releases iron from ferritin and promotes ferritin-dependent lipid peroxidation. Biochem. Pharmacol. 38,4177-4182. (26) Lode, H. N., Bruchelt, G., Rieth, A. G., and Niethammer, D. (1990)Release of iron from ferritin by 6-hydroxydopamineunder aerobic and anaerobic conditions. Free Radical Res. Commun. 11, 153-158. (27) Bryce, C. F. A., and Crichton, R. R. (1971) The subunit structure of horse spleen apoferritin. J. Biol. Chem. 246, 4198-4205. (28) Arosio, P., Adelman, T. G., and Drysdale, J. W. (1978) On ferritin heterogeneity. J. Biol. Chem. 253, 4451-4458. (29) Heusterspreute, M., and Crichton, R. R. (1981) Amino acid sequence of horse spleen ferritin. FEBS Lett. 129, 322-327. (30) Cosgrove, J. P., Borish, E. T., Church, D. F., and Pryor, W. A. (1985) The metal-mediated formation of hydroxyl radical by aqueous extracts of cigarette tar. Biochem. Biophys. Res. Commun. 132, 390-396. (31) Biemond, P., van Eijk, H. G., Swaak, A. J. G., and Koster, J. F. (1984) Iron mobilization from ferritin by superoxide derived from phagocytosing polymorphonuclear leucocytes. J. Clin. Invest. 73, 1576-1579. (32) Leanderson, P., and Tagesson, C. (1990) Cigarette smoke-induced DNA-damage: role of hydroquinone and catechol in the formation of the DNA-adduct, 8-hydroxydeoxyguanosine. Chem.-Biol. Interact. 75, 71-81. (33) Yu, P. H. (1988) Formation of cyanomethyl derivatives of basic amino acids and proteins with components in cigarette smoke. Life Sci. 43, 1633-1641. (34) O’Connell, M. J., and Peters, T. J. (1987) Ferritin and haemosiderin in free radical generation, lipid peroxidation and protein damage. Chem. Phys. Lipids 45, 241-249. (35) Monteiro, H. P., and Winterbourn, C. C. (1989)Release of iron from ferritin by divince, isouramil, acid-hydrolyzed vicine, and dialuric acid and initiation of lipid peroxidation. Arch. Biochem. B i ~ p h y271, ~ . 536-545. (36) Thomas, C. E., and Aust, S. D. (1986) Release of iron from ferritin by cardiotoxic anthracycline antibiotics. Arch. Biochem. Biophys. 248, 684-689.
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123
of metals in oxygen radical reactions. J. Free Radicals Bid. Med. 1, 3-25. (39) Halliwell, B., Borish, E. T., Pryor, W. A,, Ames, B. N., Saul, R. L., McCord, J. M., and Harman, D. (1987) Oxygen radicals and human disease. Ann. Intern. Med. 107,526-545.
Reactive Intermediates in the Oxidation of Menthofuran by Cytochromes P-450 David Thomassen,t>' Norbert Knebel,§ John T. Slattery,t Robert H. McClanahan,§ and Sidney D. Nelson*g§ Departments of Medicinal Chemistry and Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington 98195 Received August 14,1991
Menthofuran, a naturally occurring hepatotoxin, is metabolically activated to chemically reactive intermediates that are capable of covalent binding to cellular proteins. Studies in vivo and in vitro with inhibitors and inducers of hepatic cytochromes P-450demonstrated an association between hepatocellular damage caused by menthofuran and its metabolic activation and covalent binding to target organ proteins. The same y-ketoenal formed from the metabolic precursor of menthofuran, pulegone, is the major electrophilic metabolite of menthofuran as well. Diastereomeric mintlactones also are formed, and studies with Hz180 and 1802 indicate that the y-ketoenal is a precursor to the mintlactones, as well as other reactive intermediates in the cytochrome P-450mediated oxidation of menthofuran.
Introduction Menthofuran' (2, Figure 1) is a monoterpenoid product present in mint plants and is formed as a mammalian metabolite of the monoterpene pulegone (1,2).Pulegone (1, Figure 1) is found in high concentrations in pennyroyal oil, a mint oil obtained from the leaves of the plants Mentha pulegium or Hedeoma pulegoides (3).The oil is widely used as a fragrance and flavoring agent (4) and has been used, based on folklore, to control menses and terminate pregnancy (5). However, the ingestion of large quantities of the oil has been associated with toxicity (611). Hepatotoxicity has been observed in humans after ingestion of pennyroyal oil for abortion (9-11), and the hepatotoxic effects have been modeled in mice (12,13) and rats (14-16). On the basis of toxicokinetic studies in rats (15),menthofuran is formed as a major metabolite of pulegone and accounts minimally for 50% of the hepatotoxicity. Menthofuran is hepatotoxic in mice (12)and rats (15), and studies with inducers and inhibitors of cytochromes P-450 (1) and stable isotope analogues of pulegone (17) have implicated oxidative metabolites of menthofuran as ultimate toxic products of pulegone biotransformation. One of these products has been tentatively identified as an unsaturated yketoaldehyde (3,Figure 1) on the basis of its reaction with semicarbazide (18,19). Glutathione plays a protective role against pulegone-mediated hepatotoxicity (20),and glutathione conjugates of pulegone and men-
* To whom correspondence should be addressed at the Department of Medicinal Chemistry, BG-20, University of Washington, Seattle, WA 98195. Department of Pharmaceutics. *Present address: Laboratory of Chemical Pharmacology, Building 10, Room 8N110, National Institutes of Health, Bethesda, MD 20892. I Department of Medicinal Chemistry.
thofuran have been partially characterized (21)and are indicative of additional oxidative pathways in pulegone and menthofuran metabolism to toxic products. In this report, we more fully characterize pathways involved in the oxidation of menthofuran to its reactive metabolites. Results of these studies indicate that cytochrome P-450 oxidation of the furan ring plays a major role in the formation of reactive metabolites of menthofuran.
Experimental Procedures Chemicals. Sodium phenobarbital was obtained from Spectrum Chemical Co. (Gardena, CA) and piperonyl butoxide from Matheson, Coleman and Bell (Norwood, OH). Gold label semicarbazide hydrochloride, 4-ethylmorpholine, benzoyl chloride, N-acetyl-L-cysteine, N-a-acetyl-L-lysine, 3-methylcyclohexanone, (MNNG) were all purchased and l-methyl-3-nitronitr&dine from Aldrich Chemical Co. (Milwaukee, WI). Diagnostic kit no. 59-UV for determination of plasma alanine transferase (ALT), &NADPH tetrasodium salt (95-97%), and 1,2-epoxy-3,3,3-trichloropropane (TCPO) were from Sigma Chemical Co. (St. Louis, MO). m-Chloroperoxybenzoic acid (m-CPBA) was supplied by Lancaster Synthesis Ltd. (Windham, NH). Oxygen-1802 (96.5 atom 70l8O) was from MSD Isotopes (Montreal, Canada) and labeled water, H280 ('80,97%), from KOR Isotopes (Cambridge, MA). Scintillation cocktail Aquasol-2 was obtained from New England Nuclear (Boston, MA). The derivatizing reagent bis(trimethylsily1)trifluoroacetmnide (BSTFA) was from Supelco Inc. (Bellefonte, PA). Acetanilide was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ), while CD&N was from ICN Abbreviations: pulegone, (R)-(+)-pulegone;menthofuran, (R)-(+)-
menthofuran; TDC, 5,6,7,&tetrahydro-4,7-dimethyl-7H-cinnoliie; mintlactone, 3,6-dimethyl-5,6,7,7a-tetrahydro-2(4f&benzofuranone; MNNG, 1-methyl-3-nitro-1-nitrosoguanidine; ALT, alanine aminotransferase; m-CPBA, m-chloroperoxybenzoic acid; TCPO, 1,2-epoxy-3,3,3-trichloropropane; BSTFA, N,O-bis(trimethylsily1)trifluoroacetamide; GC-MS, gaa chromatography-mass spectrometry; EI, electron (ionization) impact; SIR, selected ion recording; TMS, trimethylsilyl; 0-TMS, 0-(trimethylsilyl); BSA, bovine serum albumin; GC-FID, gas chromatography with flame ionization detection; NS, not statistically significant.
0893-228x/92/2705-0123$03.00/00 1992 American Chemical Society
