Chem. Res. Toricol. 1988,1, 101-107
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Mitochondrial Reduction of the Carcinogen Chromate: Formation of Chromium(V) Susan C. Rossi,? Nadia Gorman,t and Karen E. Wetterhahn* Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755 Received January 11, 1988
Incubation of chromate with isolated rat liver mitochondria in vitro resulted in the uptake and reduction of chromium(VI), as well as the formation of chromium(V) species. Chromate was rapidly taken up and reduced by intact mitochondria. T h e rate of reduction of chromate by intact mitochondria was increased upon addition of succinate or malate plus glutamate, substrates for the electron-transport chain, but was decreased upon addition of cyanide, an inhibitor of the electron-transport chain. Incubation of chromate with mitochondria in the presence or absence of malate, glutamate, and succinate resulted in a steady increase in the level of chromium(V) over time. The extent of chromium(V) formation was increased upon addition of malate, glutamate, and succinate but was inhibited upon addition of the electron-transport chain inhibitors, antimycin, cyanide, or rotenone, to whole mitochondria. High levels of glutamate plus malate inhibited chromium(V) formation; however, high concentrations of succinate or sulfate had no effect. These studies suggest that the chromate-reductase activity in mitochondria is due to the electron-transport chain as well as other mitochondrial reducing systems which are insensitive to inhibitors of the electron-transport chain. Since chromium(V1) is effectively metabolized by mitochondria in vitro and chromium(V) “reactive intermediates” are formed in the process, mitochondria may play a role in chromium(V1) carcinogenesis.
Introduction The carcinogenicity of chromium(V1) compounds has been well established in epidemiological studies and in animal studies (I). Chromium(V1) compounds are known to cause chromosomal abberations (sister chromatid exchanges, gaps and breaks) in cellular systems in vitro, whereas chromium(III) compounds produce either very low levels of chromosomal damage or no detectable damage (2). Chromium(V1) but not chromium(II1)compounds are mutagens in bacterial cell systems (2). The uptake-reduction model has been proposed to account for the correlation between chromium carcinogenesis and the oxidation state of chromium (3). Chromium(V1) compounds as chromate readily cross cellular membranes, whereas the majority of chromium(II1) compounds only slowly enter cells. The inability of chromium(II1) to penetrate the cell, coupled with the relative kinetic inertness of chromium(II1) compounds, prevents reactions of chromium(II1) with nucleophilic sites within cells. Upon entering the cell chromium(V1) compounds encounter a number of cellular components potentially capable of reducing chromium(V1) to species capable of reacting with critical cellular targets, such as DNA, RNA, and protein. Several cellular components including cytochrome P-450 ( 4 , 5 ) ,glutathione and other cellular thiols ( 3 ) ,ascorbate (31, hydrogen peroxide (6),aldehyde oxidase (7), and DT diaphorase (8)have been shown to reduce chromium(V1) compounds in vitro. The ultimate form of chromium which is responsible for DNA damage has not yet been identified; however, the possibilities include reactive chromium intermediates produced upon reduction of chromium(VI), such as chromium(V) or chromium(1V) (6,
* To whom correspondence should be addressed.
Present address: Lineberger Cancer Research Center, University of North Carolina, Chapel Hill, NC 27514. Present address: Veterans Administration Medical Center, Research Service, White River Junction, VT 05001.
*
9 ) , stable chromium(II1) complexes, and reactive radical species formed during reduction of chromium(VI), such as sulfhydryl or oxygen radicals (6, 10). Mitochondria are an excellent model system for examining the proposed uptake-reduction model of chromium carcinogenesis. Mitochondria contain membranes with anion carrier systems ( l l ) ,a cytochrome P-450-like protein (12, 13), high levels of glutathione (5-10 mM) (24, 15), redox-active enzymes in the electron-transport chain (16), and potential macromolecular targets, including mitochondrial DNA, RNA, and proteins. Chromium distribution studies in rats have shown that the liver accumulates chromium after injection of chromium(VI), and within the liver chromium accumulates in the mitochondria ( 1 7 ) . Isolated rat liver mitochondria have been shown to rapidly take up chromium from the medium when incubated with chromate and inhibitors of the dicarboxylate and phosphate carriers have been shown to decrease chromate transport in isolated mitochondria (18). Respiration in isolated rat liver mitochondria was found to be inhibited by sodium chromate, whereas chromium(111) chloride had no effect on the respiratory rate (19,20). Isolated rat liver mitochondria have also been shown to be capable of reducing chromium(VI), and the chromatereductase activity was attributed to NADH ubiquinoneoxidoreductase (complex I), although free thiols were implicated in causing a later, slow reduction of chromate (19). The following study was undertaken in order to determine whether reactive intermediates such as chromium(V) are formed during the reduction of chromate by isolated rat liver mitochondria. We have found that intact mitochondria have the ability to take up and reduce chromium(V1) and to produce chromium(V) species in vitro.
Experlmental Procedures Materials. Male Sprague-Dawley [CRL:CD(SD)BR] rats weighing 15C-200 g were obtained from Charles River Breeding Laboratories, Wilmington, MA. L-Glutamic acid (monosodium
0893-228~/88/2701-0101$01.50/0 0 1988 American Chemical Society
102 Chem. Res. Toxicol., Vol. 1, No. 2, 1988 salt), succinic acid (disodium salt hexahydrate), L-malic acid (monosodium salt), antimycin (type Al/A3), rotenone, and bovine serum albumin (BSA)l were obtained from Sigma Chemical Co., St. Louis, MO. Atomic absorptionreference potassium dichromate solution (1mg of Cr/mL, 0.0192 M chromium), sodium cyanide, sodium dithionite, and disodium ethylenediaminetetraacetic acid (EDTA) were obtained from Fisher Chemical Co., Pittsburgh, PA. [S1Cr]Na2Cr04 (5 mCi/mL, 12.6 pg of Cr/mL) was obtained from Amersham, Arlington Heights, IL. N-(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonicacid (HEPES) was obtained from Research Organics Inc., Cleveland, OH. Tris(hydroxymethy1)aminomethane hydrochloride (TriwHC1, ultrapure) and sucrose (ultrapure) were obtained from Bethesda Research Laboratories, Gaithersburg, MD. PMannitol was obtained from Mallinckrodt, Paris, KY. Aquasol was obtained from New England Nuclear, Boston, MA. AU other chemicals were of reagent quality and were commercially available. Methods. 1. Isolation of Mitochondria. Mitochondriawere isolated from rat liver according to the procedure of Pederson et al. with slight modifications (21). Male Sprague-Dawley rats were sacrificed by decapitation and the livers removed and placed in 2 mM H medium (2.0 mM HEPES, 220 mM D-mannitOl, 70 mM sucrose, 0.5 g/L BSA, pH 7.4 at 4 "C). AU remaining isolation procedures were carried out at 0-4 "C. The liver was then homogenized with a Dounce glass homogenizer by using a loose pestle (B) at a ratio of 1part liver to 10 parts 2 mM H medium. Nuclei were removed from the homogenate by centrifuging at 930g for 10 min, removing the supernatant, and centrifuging it at 930g for 10 min. The mitochondria were pelleted by centrifugation of the supernatant at 9700g for 10 min. The post-mitochondrial supernatant was discarded, the mitochondria were gently resuspended with a blunt glass cold finger and diluted (1:25, v/v) with 2 mM H medium and again centrifuged at 9700g for 10 min. The resuspension and centrifugation procedures were repeated until the supernatant was clear (2-4 times). The mitochondria were further purified of cytosolic and nuclear contaminants by layering the mitochondria on top of a discontinuous sucrose (1.8 M/1.6 M/0.8 M) gradient in Tris-EDTA medium (10 mM TriwHC1,l mM EDTA, pH 7.4, at 4 "C) and centrifuging at 60000g for 2 h a t 0-4 "C with a Sorvall AH-627 swinging bucket rotor. The mitochondria, which banded a t the 1.8-1.6 M sucrose interface, were gently removed with a blunt-tipped serological pipet. The mitochondria were slowly diluted 1:20 (v/v) with 2 mM H medium and pelleted by centrifugation at 13000g for 15 min. The purified mitochondria (-0.7-1.0 mg of mitochondrial protein/g of rat liver) were kept on ice and used within 2-3 h. This method of preparation resulted in mitochondria which completely lacked detectable NADH oxidase activity and, therefore, were essentially free of microsomal contamination. It was important to have mitochondrial preparations free of microsomal contamination, since previous studies had shown that rat liver microsomes possess substantial chromate-reductase activity and ability to produce chromium(V) species (4, 9). 2. Protein Determinations. The protein content of mitochondria used for the chromate uptake and chromate-reductase studies was determined by a modified Lowry procedure (22)using BSA as a standard. Rapid spectrophotometric measurement of the protein content of whole mitochondria was achieved by determining the absorbance at 280 nm of a solution of mitochondria in 1% sodium dodecyl sulfate (SDS) and using the extinction coefficient em 10 mg-' mL cm-I, which was determined experimentally by using the modified Lowry procedure (22). 3. Uptake of Chromate by Intact Mitochondria in Vitro. The ability of isolated rat liver mitochondria to take up chromate in vitro was determined by incubating mitochondria (final concentration 3.7 mg of protein/mL) with [S1Cr]Na2Cr04 (3.46 X lo4 nmol of Cr/dpm, 1 mM chromate final concentration) in the presence of malate plus glutamate (final concentrations 5 mM each) in 50 mM H medium (50 mM HEPES, 220 mM D-n-"itd, 70 mM sucrose, 0.5 g/L BSA, pH 7.4 at 25 "C). Aliquots were removed every 10 min up to 100 min total incubation time. The Abbreviations: BSA, bovine serum albumin; EDTA, ethylenediaminetetraaceticacid; EPR, electron paramagnetic resonance; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid Tris.HC1, tris(hydroxymethy1)aminomethanehydrochloride.
Rossi et al. sample aliquots were immediately diluted 20-fold with ice-cold 50 mM H medium, and the mitochondria were pelleted by centrifugation at loooOg for 10 min. The supernatant was removed and the samples were analyzed for by scintillation counting at 23-25% efficiency on a Mark I1 Model 6847 liquid scintillation counter. 4. Spectrophotometric Determination of Chromate-Reductase Activity in Mitochondria. Chromate-reductase activity was monitored by following the decrease in chromium(V1) concentration by its absorbance at 372 nm 4600 M-' cm-', pH 7.4). A Varian Cary 219 UV-vis spectrophotometerequipped with a thermostated cell holder held at 25 "C was used for absorbance measurements. Mitochondria were suspended in 50 mM H medium (pH 7.4,25 "C) to a final concentration of 0.9-2.5 mg of protein/mL. The mitochondria were preequilibrated for 5 min with either glutamateplus malate (5 mM each final concentration) or succinate (10 mM f i a l concentration). The reaction was started by addition of chromate to a final concentration of 0.10 mM. The reaction was monitored spectrophotometricallyat 372 nm for 8.5 , calculated from min at 25 "C. Observed rate constants, k o ~were the slopes of plots of In A372 vs time. 5. Electron Paramagnetic Resonance (EPR) Studies of Chromium(V) Formation upon Incubation of Chromate with Mitochondria. a. EPR Spectroscopy Parameters. All EPR spectra were run on a Varian V4500 spectrometer at 77 K, 100-kHz modulation frequency, 8.32-G modulation amplitude, 1.5-mW microwave power, 9.2-GHz microwave frequency, and 630-3200 gain. Signal intensities were calculated by determining the peak to trough height of first derivative spectra and dividing by the gain. Line widths were measured between the maximum and minimum of the first derivative spectral peak. b. Time Course Studies with Various Substrates. Mitochondria were suspended at a final concentration of 0.66 mg of protein/mL in 50 mM H medium containing substrates as follows: (1) 25 mM glutamate and 25 mM malate; (2) 50 mM succinate; (3) 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate; or (4) no substrates. Mitochondria were preincubated with substrates for 2 min prior to the addition of chromate to a final concentration of 1.0 mM, and the reactions were sampled every 5 min for a total of 15-30 min of incubation at 25 "C. Sample aliquots (0.5 mL) were rapidly transferred to EPR tubes, and the reactions were stopped by freezing the samples in liquid nitrogen. c. Protein Dependence of Chromium(V) Formation. Mitochondria were suspended in 50 mM H medium (pH 7.4,25 "C) to final concentrations of 0.33,0.66, 1.0, and 1.3 mg of protein/mL and were preequilibrated for 2 min with 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate. The reaction was started by addition of chromate to a final concentration of 1.0 mM and was stopped after 15 min of incubation at 25 "C. d. Effect of Electron-Transport Chain Inhibitors on Chromium(V) Formation. Mitochondria were suspended to a final concentration of 0.66 mg of protein/mL in 50 mM H medium containing 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate. Mitochondria were preincubated with substrates for 2 min prior to addition of inhibitors and chromate. Immediately prior to chromate addition, the inhibitors were added as follows: (1)sodium cyanide (1.25 M stock solution in 50 mM HEPES, pH 7.4, 25 "C), 4500 nmol/mg of protein final concentration; (2) antimycin (3.7 X M stock solution in absolute ethanol), 45 nmol/mg of protein final concentration; and (3) rotenone (1.1X M stock solution in absolute ethanol), 13.5 nmol/mg of protein final concentration. Absolute ethanol (0.5%,v/v, final concentration) added to mitochondria (0.66 mg of protein/mL final concentration) preincubated with substrates as described above had no effect on chromate-reductase activity relative to identical incubations in the absence of absolute ethanol. The reaction was started by addition of chromate to a final concentration of 1.0 mM and was stopped after 5,10, and 15 min of incubation at 25 "C. e. Effect of Sulfate on Chromium(V) Formation. Mitochondria were suspended to a final concentration of 0.22 mg of protein/mL in 50 mM H medium containing 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate. The mitochondria were preincubated with substrates for 1.5 min prior to addition of sodium sulfate to a final concentration of 0-90 mM. Thirty
Chem. Res. Toxicol., Vol. 1, No. 2, 1988 103
Mitochondrial Reduction of Chromate to Chromium(V) 0.07
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Time (minutes) Figure 1. The uptake of 51Cr-labeledsodium chromate by rat liver mitochondria in vitro. Mitochondria (3.7 mg of protein/mL) in 50 mM H medium, pH 7.4, at 25 "C, were incubated with [51Cr]NazCr04 (1mM chromate, 3.46 X lo4 nmol of Cr/dpm) in the presence of malate (5 mM) and glutamate (5 mM). The samples were analyzed for 51Crand protein content aa described in Experimental Procedures. Values are the mean S D of duplicate determinations. seconds after sulfate was added, chromate was added to a final concentration of 1.0 mM and the reaction was stopped after 15 min of incubation at 25 "C.
Results Uptake of Chromate by Mitochondria in Vitro. Intact mitochondria isolated from rat liver were shown to effectively take up chromate in vitro in the presence of substrates (Figure 1). Approximately 70% of maximal uptake occurred within 10 min of the start of incubation. Chromium uptake reached a maximum level of 0.04 nmol of Cr/pg of protein (15% of the initial 0.27 nmol of Cr/pg of protein) within 20-30 min of incubation and remained at that level for up to 100 min of incubation. Therefore, as has been reported by other investigators (17,18), chromate is rapidly taken up by isolated mitochondria, saturation occurs, and the chromium taken up is stably incorporated into the mitochondria. Chromate-Reductase Activity of Mitochondria. Incubation of chromate with intact mitochondria resulted in reduction of chromium(VI), as shown by the decrease in the intensity of the charge-transfer absorption band of chromate at 372 nm (Figure 2). The observed rate constant for reduction of chromate by mitochondria, kom,was decreased 34% upon addition of cyanide, an inhibitor of the electron-transport chain, whereas the rate of chromate reduction increased -35% upon addition of malate plus glutamate or succinate, substrates for the electron-transport chain (Figure 2, Table I). Plots of the In A372vs time were linear except in the case where malate plus glutamate were used as substrates (Figure 2). No reduction of chromate was observed by substrates in the absence of mitochondria. Thus, it appears that the chromate-reductase activity of mitochondria is due at least in part to the activity of the electron-transport chain of the inner membrane. Formation of Chromium(V) Species upon Incubation of Mitochondria with Chromate. An EPR signal was observed upon incubation of chromate with intact mitochondria which increased in intensity in the presence of substrates (Figure 3). The signal was approximately isotropic, had g = 1.98 and AH 5 G, and, therefore, was characterized as chromium(V). The EPR signal also exhibited a small shoulder at g = 1.99. Incubation of chro-
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Figure 2. Spectrophotometricdetermination of the chromatereductase activity of intact mitochondria in the presence and absence of substrates or inhibitors. Chromate (0.1 mM) was incubated in 50 mM H medium, pH 7.4, at 25 O C , with mitochondria (2.5 mg of protein/mL) (a)as isolated or preequilibrated for 5 min with (b) 10 mM succinate, (c) 5 mM malate plus 5 mM glutamate, or (d) 10 mM cyanide. Solid lines represent results of linear regression analyses of the In A372vs time data. Table I. Observed Rate Constants for Chromate Reduction by Intact Mitochondriaa condition mitochondria mitochondria mitochondria mitochondria (5 mM)
+ cyanide (10 mM) + succinate (10 mM) + malate (5 mM) + glutamate
k o w , m i d mg-' mLb (7.3 2.2) x 10-3 (4.8f 0.4) X 10" (9.9 3.9) x 10-3 (9.8 f 1.9) X
a Isolated mitochondria (0.9-2.5 mg of protein/mL) were incubated with chromate (0.1 mM) in the absence and presence of substrates or inhibitors in 50 mM H medium at 25 "C. bValues represent mean f SD of three to four determinations.
mate with glutamate, malate, and succinate in the absence of mitochondria gave a barely detectable EPR signal (Figure 3). The time course for formation of chromium(V) was studied by incubating chromate with mitochondria in the presence and absence of glutamate, malate, and succinate for a period of 15 min (Figure 4). The intensity of the chromium(V) signal increased steadily over the 15-min incubation period both in the presence and absence of substrates; however, a higher level of chromium(V) was observed in the presence of substrates throughout the 15-min time period. Inclusion of moderate levels of substrates (2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate) resulted in a 30% increase in the intensity of the chromium(V) signal after 15 min of incubation, compared to mitochondria in the absence of exogenous substrates (Figure 4). Glutamate, malate, and succinate, but not NADH, are readily transported into mitochondria by
104 Chem. Res. Toxicol., Vol. 1, No. 2, 1988
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Figure 3. Typical chromium(V) EPR signals observed after a 15-min incubation of chromate (1.0 mM) with mitochondria (1 mg/mL) in the presence and absence of substrates. (a) Chromate incubated with mitochondria preequilibrated for 2 min with 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate. (b) Chromate incubated with mitochondria in the absence of substrates. (c) Chromate incubated with 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate in the absence of mitochondria. All reactions were run in 50 mM H medium, pH 7.4, at 25 O C . EPR spectra were run at 800 gain, h
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Time (minutes) Figure 4. Time course of chromium(V) formation in mitochondria incubated with chromate in the presence and absence of Substrates. Mitochondria (0.66 mg of protein/mL) were either (m)preequilibrated for 2 min in the presence of 2.5 mM glutamate, 2.5 mM malate, and 5.0 mM succinate as substratesor (a)with no substrate added. A control sample (+) of chromate incubated with 2.5 mM glutamate, 2.5 mM malate, and 5.0 mM succinate in the absence of mitochondria was also run. All reactions were run in 50 mM H medium, pH 7.4, at 25 “C. Chromate (final concentration 1 mM) was added to start the reaction and the reaction was stopped at the indicated times. Values are the mean *SD of duplicate determinations. EPR spectra were run at 800-2000 gain. anion carrier systems (11). Glutamate and malate are Substrates for glutamate and malate dehydrogenases which reduce NAD+ to NADH in the mitochondrial matrix; the resulting NADH donates electrons at the level of complex I in the electron-transport chain, whereas succinate donates electrons directly to the electron-transport chain at complex I1 (23). Since inclusion of moderate levels of substrates for the electron-transport chain resulted in an in-
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Protein (mg/ml) Figure 5. Dependence of the intensity of the chromium(V)EPR signal upon protein concentrationin incubationsof chromate with mitochondria in the presence of substrates. Mitochondria (0-1.3 mg of protein/mL) were preequilibrated for 2 min with 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate in 50 mM H medium, pH 7.4, at 25 OC. Chromate (final concentration 1mM) was added to start the reactions and the reactions were stopped after 15-minincubation. Linear regression analysis gave y = 0.22 + 0.006. Values are the mean *SD of duplicate determinations. EPR spectra were run at 630 gain. crease in chromate-reductase activity in whole mitochondria and since levels of endogenous substrates may vary between preparations of isolated mitochondria, later studies with mitochondria were run in the presence of 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate to maximize the observable chromium(V) signal. Under these conditions the level of chromium(V) increased steadily over a 30-min time period (data not shown). Incubation of chromate with increasing concentrations of mitochondrial protein in the presence of glutamate, malate and succinate as substrates resulted in an increased level of chromium(V) (Figure 5). The dependence of the intensity of the chromium(V) signal on mitochondrial protein concentration was linear in the range of 0-1.3 mg of mitochondrial protein/mL. Effects of Electron-Transport Chain Inhibitors on Reduction of Chromium(V1) to Chromium(V). Since a 30% increase in the level of chromium(V) was observed in the presence of electron-transport chain substrates, the effects of the electron chain transport inhibitors cyanide, antimycin, and rotenone on chromium(V) formation in mitochondria was examined (Figure 6). The effects of the inhibitors on chromium(V) formation were quite dependent upon time. At 5 min of incubation, there was no apparent effect for any of the inhibitors studied. However, after 15 min of incubation rotenone caused a 7% decrease in the intensity of the chromium(V) signal, cyanide caused a 13% decrease, and antimycin caused a 25% decrease in the level of chromium(V) compared to that observed in the absence of inhibitor (Figure 6). Therefore, at least 75% of the chromate-reductase activity in mitochondria, as measured by chromium(V) formation, appears to be insensitive to electron-transport chain inhibitors. Effect of Anions on Chromium(V) Formation. Since previous work had indicated that chromate is transported by the dicarboxylate carrier and to a lesser degree the phosphate carrier in mitochondria (18),the effects of high concentrations of anions, including exogenous substrates and sulfate, on production of chromium(V) upon incubation of chromate with mitochondria was examined. High concentrations of glutamate (25 mM) and malate (25 mM) inhibited production of chromium(V) by 40% with respect
Chem. Res. Toricol., Vol. 1, No.2, 1988 105
Mitochondrial Reduction of Chromate to Chromium(V) 0.06
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Figure 6. Effects of electron-transport chain inhibitors on reduction of chromate to chromium(V) by mitochondria in the presence of substrates. Mitochondria (0.66 mg of protein/mL) were preequilibrated for 2 min with 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate. Inhibitors were added immediately prior to chromate as follows: (4) sodium cyanide (4500 nmol/mg of protein), (u) antimycin (45nmol/mg of protein), ( 0 ) rotenone (13.5 nmol/mg of protein), and (m) no inhibitor added. All reactions were run in 50 mM H medium, pH 7.4, at 25 "C. Chromate (final concentration 1 mM) was added to start the reaction. The reaction was stopped at the indicated times. Values are the mean i S D of duplicate determinations except antimycin results which result from a single time course determination. Spectra were run at 800-2000 gain. u)
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Figure 8. Effect of sulfate on time course of chromium(V) formation in mitochondria incubated with chromate in the presence of substrates. Mitochondria (0.22 mg of protein/mL) were preequilibrated for 2 min with 2.5 mM glutamate, 2.5 mM malate, and 5 mM succinate. Sodium sulfate was added after preequilibration and immediately prior to chromate addition to the following final concentrations: (m) 0 mM, (+) 30 mM,).( 60 mM, or ( 0 ) 90 mM. All reactions were run in 50 mM H medium, pH 7.4, at 25 O C . Chromate (finalconcentration 1mM) was added to start the reaction and the reaction was stopped at the indicated times. Values are the mean i S D of duplicate determinations. Spectra were run at 800-2000 gain. systems that are unaffected by succinate and sulfate.
Discussion
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Figure 7. Effect of high concentrationsof substrates on the time course of chromium(V)formation in mitochondria incubated with chromate. Mitochondria (0.66 mg of protein/mL) were preequilibrated for 2 min in the presence of substrate as follows: (+) 25 mM glutamate plus 25 mM malate or).( 50 mM succinate or (a)with no substrate added. All reactions were run in 50 mM H medium, pH 7.4, at 25 O C . Chromate (final concentration 1 mM) was added to start the reaction and the reaction was stopped at the indicated times. Values are the mean i S D of duplicate determinations. Spectra were run at 800-2000 gain.
to incubation of mitochondria in the absence of exogenous substrates (Figure 7). High concentrations of succinate (50 mM) did not have a statistically significant effect on the production of chromium(V) (Figure 7). Addition of sodium sulfate (finalconcentrations of 30,60,and 90 mM) to incubation mixtures had no effect on the rate or level of chromium(V) production by isolated mitochondria in the presence of substrates (Figure 8). Thus it appears that uptake of chromate by the dicarboxylate carrier is not rate-limiting in the reduction of chromium(V1) to chromium(V) or that chromate is transported by other carrier
The role of mitochondria in chromium(V1)metabolism and subsequently in chromium carcinogenesis has been unclear. The mitochondrial matrix contains DNA which may be damaged upon metabolism of chromate in vivo. Mitochondria have been shown to readily take up chromate in vitro and in vivo (17,lB). The present study shows that our mitochondrial isolation method results in active mitochondria capable of efficient chromate transport when incubated in the presence of malate and glutamate as substrates in vitro. In agreement with the previous suggestion of carrier-mediated chromate uptake (18), saturation of chromate uptake was observed with maximal uptake occurring within 20-30 min of incubation with whole mitochondria. No loss of chromium from mitochondria was observed over the 100-min period of incubation, suggesting that chromate is metabolized by mitochondrial components to species which are incapable of being released from the intact mitochondria. This is consistent with the "uptake-reduction"model of chromium carcinogenesis, since the chromate taken up was very likely reduced to chromium(III), which forms stable complexes with mitochondrial components including macromolecules. Chromate-reductase activity in mitochondria has been previously reported (19); however, the site or sites of chromate-reductase activity have remained unidentified. Potential sites for reduction of chromate within mitochondria include the following: inner membrane electron-transport chain complexes (16); superoxide anion and hydrogen peroxide (24);cytochrome P-450(12,13); and glutathione (14, 15). In the present study we have investigated the chromate-reductase activity of whote mitochondria by UV-vis spectroscopy which allows the detection of disappearance of chromium(V1) and EPR which allows direct detection of the chromium(V) species produced upon reduction of chromium(V1). Intact mitochondria were found to be capable of reducing chromate as isolated; however, addition of sub-
106 Chem. Res. Toxicol., Vol. 1, No. 2, 1988
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strates for the electron-transport chain increased the rate of reduction by -35%, whereas cyanide, an inhibitor of ferrocytochrome c-oxygen oxidoreductase (complex IV of the electron-transport chain), decreased the rate of reduction by -35%. Additional evidence for the involvement of the electron-transport chain in mitochondrial reduction of chromate was obtained from EPR studies of chromium(V) formation. The level of chromium(V) produced upon incubation with mitochondria increased 10-30% upon addition of substrates. The electrontransport chain inhibitors cyanide and antimycin decreased the level of chromium(V) -15-25%, whereas rotenone had little or no effect upon chromium(V) formation. Thus, it appears that 1535% of the chromate-redudase activity of mitochondria is affected by the activity of the electron-transport chain. The ability of antimycin and cyanide to produce significant inhibition of chromium(V) formation implicates ferrocytochrome c-oxygen oxidoreductase (complex IV)in the chromate-reductase activity of whole mitochondria. The inhibitor-insensitive chromate-reductase activity in mitochondria may be due to NADHubiquinone oxidoreductase (complex I) and/or succinate-ubiquinone oxidoreductase (complex 11)of the electron-transport chain, which are rotenone-insensitive. Previous studies have shown that chromate reduction by mitochondria and chromate inhibition of mitochondrial respiration occur predominantly at complex I (19). Preliminary experiments with purified mitochondrial inner membrane vesicles (submitochondrial particles) indicate that chromium(V) is produced by complex I, complex 11, and complex IV.* It is also possible that the inhibitor-insensitive chromate-reductase activity in mitochondria may be due to non-electron-transport chain components of mitochondria, such as glutathione and/or cytochrome P-450. Preliminary experiments indicate that the intact mitochondria possess approximately twice the chromate-reductase activity (as measured by the ability to form chromium(V)) of mitochondrial inner membrane preparations when normalized for protein contenta2The chromium(V) EPR signal produced in isolated mitochondria incubated with chromate possesses a shoulder at g = 1.99 in addition to the strong g = 1.98 signal. Preliminary experiments indicate that only an isotropic chromium(V) EPR signal at g = 1.98 is produced by mitochondrial inner membrane preparations.2 Therefore, it appears that at least two chromium(V) species are produced in mitochondria incubated with chromate and the chromium(V) species produced by the electron-transport chain complexes is only one component of the chromium(V) signal at g = 1.98. The cytochrome P-450 chromate-reductase activity in rat liver microsomes has been shown to produce a major chromium(V) EPR signal with g = 1.979 and AH = 8.5 G and a minor signal (shoulder) at lower field upon reduction of chromate in the presence of NADPH (9). Therefore, it is possible that mitochondrial cytochrome P-450 may be involved in chromate reduction. Glutathione has also been shown to reduce chromium(V1) in vitro to EPR detectable chromium(V) species having g 1.99 and A H 1-9 G (6, 25-27). I t was suggested that thiol groups were involved in the mitochondrial chromate-reductase activity, since pretreatment of mitochondria with N-ethylmaleimide, a thiol-blocking reagent, resulted in a decrease in the slow phase of chromate reduction and chromate treatment resulted in a decrease in the number of free thiols (19). Since high concentrations (5-10 mM) of glutathione have been
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-
* Rossi, 5. R., and Wetterhahn, K. E., unpublished results.
observed in the mitochondrialmatrix (14,15),it is possible that glutathione participates in the reduction of chromium(V1) to chromium(V) in whole mitochondria. The effort to locate the site or sites of chromate-reductase activity in mitochondria is complicated by the uptake effects. Chromate, substrates, and inhibitors will all be subject to the kinetics of mitochondrial uptake. Mitochondria also contain unknown quantities of endogenous substrates, reduced and oxidized glutathione, and hydrogen peroxide in the matrix. The levels of these componentsin the mitochondria are likely to be dependent upon the isolation method and the length of time betwen isolation and the experiment. Although a substantial chromium(V) signal was produced in the absence of exogenous substrates, addition of substrates (2.5 mM malate, 2.5 mM glutamate plus 5 mM succinate, or 50 mM succinate) caused an increase in chromium(V) signal relative to mitochondria in the absence of substrate. This increase in chromium(V) formation is probably due to increased electron-transport chain activity; however, these substrates are linked to the tricarboxylic acid cycle and/or to production of NADH which is involved in many enzymatic processes in the mitochondrion including activation of transport systems. In contrast, high concentrations of substrate (25 mM malate plus 25 mM glutamate) produced a 40-50% inhibition of chromium(V) production. The substantial inhibition of chromate-reductase activity by high levels of glutamate and malate is probably due to the inhibition of chromate uptake. Previous studies have shown that malate and glutamate dehydrogenases do not reduce chromate in vitro (3),and therefore, glutamate and malate are not competing with chromate for active sites on these enzymes. The dicarboxylate carrier has been proposed to be the main transporter of chromate in mitochondria in vitro based upon transport protein inhibitor studies and the competitive inhibition of mitochondrial chromate uptake by sulfate (18). Sulfate, malate, and succinate are transported mainly by the dicarboxylate carrier; however, glutamate is not (11). Glutamate and malate are transported by a wide variety of carrier proteins and "shuttles". Succinate and sulfate had no effect upon the level of chromium(V) formation even at high concentrations (50 and 90 mM, respectively). Therefore, the inhibition of chromium(V) formation by 25 mM malate plus 25 mM glutamate is unlikely to be due to competitive inhibition between chromate and malate for the dicarboxylate carrier, since sulfate should have shown a similar inhibitory effect, unless the rates of transport are vastly different for sulfate and malate on the dicarboxylate carrier. The lack of inhibition of chromate-reductase activity by high concentrations of sulfate and succinate could indicate that uptake of chromate by the dicarboxylate carrier is not the ratelimiting step of chromate reduction or that chromate is primarily taken up by another carrier which is unaffected by sulfate or succinate. The inhibition of chromate-reductase activity by high concentrations of malate plus glutamate suggests that at least one other carrier system besides the dicarboxylate carrier is likely to be involved in chromate uptake. In summary, chromium(V1) is taken up by intact mitochondria and reduced to EPR-detectable chromium(V) species in vitro. The mitochondrial electron-transport chain is involved in reducing chromate and producing chromium(V). However, there appears to be more than one site of chromate-reductase activity in mitochondria, and it is possible that glutathione and/or mitochondrial cytochrome P-450 may also be involved in chromium(V)
Chem. Res. Toxicol., Vol. 1, No. 2, 1988 107
Mitochondrial Reduction of Chromate to Chromium(V)
formation. Since treatment of rats with 51Cr-labeled chromate leads to uptake of chromium into mitochondria it is likely that chromium(V1) will be taken up in mitochondria and reduced in vivo. Thus, mitochondria are likely to be important participants in cellular metabolism of chromium(V1) and production of reactive intermediates in vivo, and may contribute to the carcinogenicity of chromium(V1) compounds.
(12) Niranjan, B. G., Wilson, N. M., Jefcoate, C. R., and Avadhani, N. G. (1984) “Hepatic mitochondrial cytochrome P-450 system: distinctive features of cytochrome P-450 involved in the activation of aflatoxin B, and benzo(a)pyrene”. J . Biol. Chem. 259, 12495-12501. (13) Sato, R., Atsuta, Y., Imai, Y . , Taniguchi, S., and Okuda, K. (1977) “Hepatic mitochondrial cytochrome P-450 isolation and functional characterization”. Proc. Natl. Acad. Sci. U.S.A. 74, 5477-5481. (14) Meredith, M. J., and Reed, D. J. (1982) “Status of the mito-
Acknowledgment. This investigation was supported by PHS Grant CA34869 awarded by the National Cancer Institute, DHHS and by the donors of the Petroleum Research Fund, administered by the American Chemical Society.
chondrial pool of glutathione in the isolated hepatocyte”. J. Bid. Chem. 257,3747-3753. (15) Griffith, 0. W., and Meister, A. (1985) “Origin and turnover of mitochondrial glutathione”. Proc. Natl. Acad. Sci. U.S.A. 82,
(In,
Registry No. Cr042-, 13907-45-4; Cr, 7440-47-3; chromate reductase, 113321-67-8.
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