One-Electron Reduction of Chromate by NADPH-dependent Glutathione Reductase Xianglin Shi and Nar S. Dalal Department of Chemistry,
West Virginia University, Morgantown,
West Virginia
ABSTRACT Electron spin resonance (ESR) measurements provide evidence for the formation of Cr(V) intermediates in the enzymatic reduction of Cr(V1) by glutathione reductase (GSSG-R) in the presence of NADPH, indicating an initial single-electron transfer step in the reduction mechanism. Depending on the pH, at least two different Cr(V) species are generated which are relatively long-lived. In addition, we have detected the hydroxyl ( *OH) radical formation during the GSSG-R catalyzed reduction of Cr(V1) by spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and a-(4-pyridyl-l-oxide)-N-tert-butylnitrone (CPOBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the *OH radical and Cr(V) formation, indicating that the Oz- is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the -OH radical formation, indicating the involvement of hydrogen peroxide (H20z) in the reaction mechanism. Addition of HZ02 suppresses Cr(V) and enhances the *OH radical formation. Measurements involving N-ethylmaleimide show that the Cr(V) species, produced enzymatically by the reduction of Cr(V1) by GSSG-R, react with H20z to generate .OH radicals, which might participate in the initiation of Cr(V1) carcinogenicity.
INTRODUCTION C(V1) compounds have been found to exert serious toxic and carcinogenic effects on humans and animals [l-4] and to cause mutations in bacteria and transformation of mammalian cells [5-81. In contrast, most Cr(II1) compounds are relatively nontoxic, noncarcinogenic, and nonmutagenic [9, lo]. Cr(V1) and Cr(II1) oxidation states are different in their metabolic pathways: Cr(V1) ions are rapidly transported across cellular membranes [2, 1 l-131, while Cr(II1) moieties do not easily penetrate cells and are not oxidized by cellular constituents [3]. Since it is known that the reduction of Cr(VI) is required for its reaction with DNA [2], the molecular mechanisms for the
Address reprint requests to: Professor Morgantown, WV 26506. Journal of Inorganic Biochemistry 0 1990 Elsevier Science Publishing
N. S. Dalal, Department
of Chemistry,
40, 1-12 (1990) Co., Inc., 655 Avenue of the Americas,
West Virginia University,
1 NY, NY 10010
0162-0134/90/$3.50
2 Xianglin Shi and Nar S. Dalal
intracellular Cr(V1) reduction have been the focus of studies during the past decade but the details are still not understood. Earlier studies on the mechanism of Cr(V1) reduction include those involving glutathione [ 1, 14-241, ascorbic acid [25], glucose [26], lactose [26], galacturonic acid [27], microsomes 128-3 11, rat liver DT-diaphorase [32], and aldehyde oxidase [33]. Glutathione reductase (GSSG-R) has been shown to be selectively inhibited by Cr(V1) in erythrocytes and also in liver in a NADPH dependent manner (34-361. Recently, Sugiyama et al. [37] reported that treatment of Chinese hamster V-79 cells with Cr(VI) results in a decrease of GSSG-R activity. They suggested that the inhibition of GSSGR may be involved in the toxic effect of Cr(VI). While the inhibition of GSSG-R by Cr(V1) was hypothesized to be linked to the reduction of chromium from Cr(V1) to Cr(III), the mechanism remains unclear 134, 35, 371. Since GSSG-II is ubiquitously present in cellular systems and has important biological functions f38], studies on the possible reduction of Cr(V1) by this enzyme and its mechanism should be of fundamental interest. Thus we carried out a detailed electron spin resonance (ESR) spectroscopic investigation of the reduction of Cr(V1) by CJSSG-R. It is shown that the reduction process involves a single electron transfer step, as well as the formation of hydroxyl (‘OH) radical, in this well defined enzymatic system.
MATERIALS
AND METHODS
Dimethyl sulfoxide (DMSO), 1,3-dimethyl-2-thiourea (DMTU), ethanol, H>Oz, and potassium dichromate were purchased from Fisher (Pittsburgh, PA). Glutathione reductase (GSSG-R) from bovine intestinal mucosa, superoxide dismutase (SOD) from bovine blood, catalase from bovine liver. sodium formate. NADPH. and Nethylmaleimide were purchased from Sigma Chemical Co. (St. Louis, MO) and used as received. Spin traps, S,5-dimethyl- 1-pyrroline-N-oxide (DMPO) a a-(4-.pyridyl- I oxide)-N-tert-butylnitrone (4-POBN), were purchased from Aldrich Chemical Co. (Milwaukee, WI), and were used without further purification. smce very weak or no spin adduct signal was obtained from the purchased sample when used alone. The phosphate buffer solution of pH = 7.2 was purchased from Fisher while those of pH 4.0-6.5 were obtained from Aldrich. ESR spectra were obtained at X-band (- 9.7 GHz) using a Bruker ER 200D ESR spectrometer. For accurate measurements of g-values and hyperfine splittings, the magnetic field was calibrated with a self-tracking NMR gaussmeter (Bruker. Model ER035A) and the microwave frequency was measured with a digital frequency counter (Hewlett-Packard, Model 5340A). An ASPECT 2000 computer was used for data acquisition and analysis. The concentrations given in the Figure legends are final concentrations. All experiments were carried out at room temperature.
RESULTS ESR Evidence
for Cr(V) Formation
An aqueous Cr(V1) solution containing a low concentration (0.9 mM) of NADPH did not give a detectable ESR signal (Fig. la). An aqueous solution of Cr(V1) containing CSSG-R gave a weak ESR signal (Fig. lb). but that containing both NADPH and GSSG-R at physiological pH (7.2) gave a much stronger ESR spectrum (Fig. lc). In order to better analyze the spectrum in Figure lc, a spectrum was recorded using a
REDUCTION
OF Cr(V1) BY GLUTATHIONE
REDUCTASE
(a)
)
Cr(VI) + NADPH
@)
s
Cr(VI) +GSSG-R
3
(a)+GSSG-R
/ I--1
p1.9785.
CrW
(a) +GSSG-R
20G .H
(f)
)
~~~THYLMALEI~VIIDE 5G
’
I~H
’
FIGURE 1. ESR spectrarecorded 2 min after mixing 1.9 mM K2Crz07 buffer solution (pH = 7.2) with (a) 0.9 mM NADPH; (b) 12 units/ml GSSG-R; (c) 0.9 mM NADPH and 12 units/ml GSSG-R; (d) same as (c) but the spectrum recorded at a higher gain and a wider scan width; (e) same as (c) but the spectrum recorded 10 min after mixing; (f) same as (c) but with 200 mM Nethylmaleimide added.
higher modulation amplitude and a wider scan width. The spectrum obtained (Fig. Id) was similar to those reported earlier for Cr(V) complexes of ethylene glycol [39-421, ascorbic acid [25], glucose or lactose [26], and galacturonic acid [27] : the center of the spectrum centered at g = 1.9785 with four satellite signals due to 53Cr (9.55% abundance, I = 3/2) hyperfine structure. The observed 53Cr hyperfine coupling of 17.8 G is very similar to those observed for Cr(V) complexes with oxygen ligands [25-27,41-42]. The spectra in Figure lc and Id were thus assigned to Cr(V) species. When the spectrum was measured 10 minutes after the reaction initiation, the overall
4
Xianglin Shi and Nar S. Dalal
11
_.___I--------
(a)
__-i
(b)
(c) __---____
~~~.~~~~
__-.-_ ji
Cr(VI) + NADPH + GSSG -R AT PH = 7.2
6.5
_. _-.~-.~--- 6.0
le)
_.--.--- _-.. 4.0 5G -l-i
j/ /i
(0
/1
20G -H
4+ ._-- -.--
4.0
ii
i
II FIGURE 2. Effect of reaction medium acidity on Cr(V) formation. The reaction mixture contains 0.9 mM NADPH. 12 units/ml GSSG-R, and 1.9 mM IizCrz07 in buffer solutions of (a) pH = 7.2; (b) pH = 6.5; (c) pH = 6.0; (d) pH = 5.0: (e) pH = 4.0: (f) same as (e1 but the spectrum recorded at a higher modulation amplitude and a w-ider xan width.
signal intensity was found to decrease significantly (Fig. le), showing that the Cr(V) species are long-lived but reactive intermediates. Fig. If shows the effect of Nethylmaleimide, the GSSG-R inhibitor [34, 351. By comparing the spectra in Figure lc and If, it can be seen that N-ethylmaleimide significantly inhibits the Cr(V) formation. showing that the reaction is an enzymatic process. Figure 2 shows ESR spectra recorded from an aqueous solution containing Cr(VI). NADPH and GSSG-R as a function of pH. It can be seen that as the pH decreases the relative intensities of two major Cr(V) peaks change. At pH lower than 6.0, the peak at g = 1.9772 becomes dominant (Fig. 2~). At pH = 4.0 only one peak (g = i .9772) is detectable (Fig. 2e). Again, a spectrum recorded at a higher modulation amplitude and a wider scan width (Fig. 2f) was found to be that tyfricai f and *OH radicals, providing evidence that the generation of
*OH radical
is enzymatic.
Effect of HzOz on the *OH Radical
Formation
Figure 5a shows the ESR spectrum from a mixture containing Hz02, GSSG-R and DMPO while Figure 5b from a reaction mixture containing H202, DMPO, and Cr(V1) while Figure 5c was that from a reaction mixture containing H202, DMPO, GSSG-R, and Cr(V1). Figure 5d shows the ESR spectrum from a reaction mixture containing H,O*, DMPO, Cr(VI), and NADPH. The 1:2:2: 1 quartet in Figure Sd was assigned to the DMPO-OH adduct, as discussed earlier. On adding GSSG-R, the intensity of the DMPO-OH adduct (Fig. Se) increased substantially. For the convenience of direct comparison of the effect of H202 on the -OH radical generation, the ESR spectrum observed from the reaction containing DMPO, GSSG-R, KzCrZOT, and NADPH without H202 is included in Figure 5f. From the spectra obtained with and without Hz02 (Fig. 5e and 5f), it can be noted that H201 greatly enhances the *OH radical formation (7 times), and reduces the Cr(V) formation, indicating that the formation of *OH radicals involves a reaction between the Cr(V) species and H$&. To further verify the trapping of *OH radicals (Fig. 5) in the presence of’N202, another spin trap, 4-POBN, was employed, The ESR spectrum obtained with this trap exhibits a triplet of
REDUCTION
OF Cr(V1) BY GLUTATHIONE
REDUCTASE
9
doublets with aN = 14.9 G and ai = 1.6 G (Fig. 6a). When recorded with a small modulation (0.2 G), the spectrum shows additional doublet splitting,‘arising from the coupling of a; = 0.3 G with hydroxyl hydrogen (Fi,.0 6b). These hyperfine splittings are typical those of 4-POBN-OH adduct [44, 53-551, confirming the trapping of *OH radicals in the above discussed reaction. DISCUSSION GSSG-R, a ubiquitous FAD-containing enzyme, uses NADPH to reduce oxidized glutathione (GSSG) and generate glutathione (GSH) according to: GSSG + NADPH + H + -+ 2GSH + NADP + . This enzyme’s general function is to maintain a high level of GSH in the cytosol, but it might have additional roles. Earlier investigators have reported a NADPH dependent inhibition of GSSG-R by Cr(V1) [34, 351. Experiments with mixtures of inhibited and native enzyme revealed no evidence of inhibitor formation or activator loss, suggesting that the GSSG-R inhibition by Cr(VI) results from a direct effect on the enzyme protein [34, 351. The NADPH dependent inhibition of GSSG-R by Cr(V1) has been suggested to be linked to the reduction of chromium from the hexavalent to trivalent form [34, 351. The mechanism remains unknown. The results reported here provide direct evidence for a NADPH dependent reduction of Cr(V1) by GSSG-R with Cr(V) as an intermediate, thus indicating that the initial step in the Cr(V1) reduction involves a one-electron transfer process. It may be noted that in the oxidized form of the enzyme the active site was found to have a disulfide formed by Cys-46 and Cys-41 1561. The mechanism of the enzymatic function of GSSG-R was proposed to involve the action of the thiolate ion at Cys46 which is stabilized by a nearby protonated His in the stable reduced form of the enzyme. The reduced Cys-41 was proposed to attack GSSG, release GSH and form a mixed disulflde and GSH [56]. The inhibitive effect of N-ethylmaleimide suggests that the adjacent Cys residues are involved in the reaction of GSSG-R with Cr(VI). It has been reported that certain enzymes of the cytoplasm, endoplasmic reticulum, and mitochondria of mammalian cells catalyze Cr(V1) reduction [2, 32, 28-30, 571. Jennette reported Cr(V) formation in the incubation of Cr(V1) with liver microsomes in the presence of NADPH [30]. Even though the details were not clear, Jennette suggested that a direct one-electron transfer from the microsomal electron-transport cytochrome P-450 system to Cr(V1) is a likely mechanism [30]. Since Cr(V) complexes are generally characterized as being labile and reactive, whereas Cr(III) complexes are substitutionally inert, the detection of Cr(V) formation led Jennette [30] to suggest that the Cr(V) intermediates are the likely candidates for the “ultimate” carcinogenic forms of the carcinogenic chromium species. This suggestion has led to many further investigations of the formation of Cr(V) in the reduction of Cr(V1) under biologically relevant conditions [ 15-23,25-27, 57, 581. To our knowledge, however, the present work represents the first detailed evidence for Cr(V) formation in a well characterized enzymatic Cr(V1) reduction process [58]. Recently, Kawanishi and coworkers [59] have studied the reactivity of chromium compounds with DNA by the DNA sequencing technique using 32P-5 ‘-end-labeled DNA fragments of a known sequence. They argue that it is not Cr(V) itself that is carcinogenic, but it is the reactive oxygenated species (O,- , i02, *OH) (produced by the decomposition of the CrV(02)43- ) that cause the DNA base modification.
10
Xianglin Shi and Nar S. Dalaf
However, the relevance of this mechanism to Cr(V1) toxicity does not seem to be significant for the following reasons. First, CrV(02)43 has been detected at highly basic pH, well above the physiological value [59-611. Second, in the presence of diol molecules, CrV(02)43 is known to undergo fast ligand exchange to form Cr(V)-diol complexes [39]. In none of our reactions did we ever observe a significant amount of CrV(02)43 and the dominant Cr(V) species always exhibited ligand superfine interactions or g-values characteristic of the complexes irather than the uncomplexed) CrV(02)43 ion, In our earlier work, we reported that reduction of Cr(V1) by biologically relevant diols or glutathione generates Cr(V) complexes, which subscquently react with Hz02 to generate *OH radicals 162, 631. The present work provides evidence of Cr(V) and ‘OH radical formation in an enzymatic CriVI) reduction. Moreover, addition of H202 to this reaction system yields much higher concentrations of -OH radicals, being 7 times as high as without H202, simultaneously suppressing the Cr(V) formation. These results show that Cr(V) complexes generated in the enzymatic Cr(V1) reduction also have an ability to react with Hz& to generate “OH radicals. Since Cr(V) species can also be generated in the reduction of Cr(V1) by various biological reductants [IS-23, 25-27, 5’71 and H202 is a normal biological metabolite, the reaction of Cr(V) complexes with Hz02 might be the major source of -OH radicals. We thus hypothesize that after Cr(V1) enters the cell, it is reduced by various cellular reductants (for example, GSSG-R, diol and thiol molecules) to form Cr(V) complexes which then react with Hz02 to generate “OH radicals and initiate Cr(V1) carcinogenesis. Part of this research has been supported by the Department of the Interior’s Mineral Institute Program administered by the Bureau of Mines through the Generic Mineral Technology Center of Respirable Dust under grant no. G11.35142.
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REDUCTION
OF Cr(V1) BY GLUTATHIONE
11
REDUCTASE
17. P. O’Brein, Znorg. Chim. Acfu 108, L19 (1985). 18. P. Arslan, M. Beltrame, and A. Tomasi, Biochim. Biophys. Actu 931, 10 (1987). 19. D. M. L. Goodgame and A. M. Joy, .Z. Znorg. Biochem. 26, 219 (1986). 20. X. Shi and N. S. Dalal, Biochem. Biophys. Res. Commun. 156, 137 (1988). 21. X. Shi and N. S. Dalal, in Proc. VZZZntl. Pneumoconiosis Conf., Pittsburgh, Pennsylvania, USA (in press) (1989). 22. N. S. Dalal and X. Shi, in Medical, Biochemical and Chemical Aspects of Free Radicufs, 0. Hayaishi, E. Niki, M. Kondo, and T. Yoshikawa, Eds., Elsevier, Amsterdam, 1989; pp. 547-550. 23. X. Shi., Dissertation, Department of Chemistry, West Virginia University, 1989. 24. S. Kitagawa, H. Seki, F. Kametani, and Sakurai, Chem. Biol. Interactions 40, 265
(1982). 25. 26. 27. 28. 29. 30. 31. 32.
D. M. L. Goodgame and A. M. Joy, Znorg. Chim. Actu 135, 115 (1987). D. M. L. Goodgame and A. M. Joy, Znorg. Chim. Acta 135, L5 (1987). M. Branca and G. Micera, Znorg. Chim. Acta 153, 61 (1988). J. E. Gruber and K. W. Jennette, Biochem. Biophys. Res. Common. 82, 700 (1978). J. D. Garcia and K. W. Jennette, J. Znorg. Biochem. 14, 281 (1981). K. W. Jennette, J. Am. Chem. Sot. 184, 874 (1982). A. Mikalsen, J. Alexander, and D. Ryberg, Chem. Biof. Znteructions 69, 175 (1989). S. DeFlora, A. Morelli, C. Basso, M. Romano, D. Serra, and A. DeFlora, Cancer Res. 45, 3188 (1985). 33. R. B. Banks and R. T. Cooke, Biochem. Biophys. Res. Common. 137, 8 (1986). 34. G. A. Koutras, M. Hattori, A. S. Schneider, F. G. Ebaugh, and W. N. Valentine, J.
Clin. Invest. 43, 323 (1964). 35. G. A. Koutras, A. S. Schneider,
M. Hattori, and W. N. Valentine,
Brit. J. Haemat.
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Carcinogen&s
P. Zanacchi,
A. Camoirano,
A. Morelli, and A. De Flora,
5, 504 (1984).
37. M. Sugiyama, A. Ando, and R. Ogura, Curcinogenesis 10, 737 (1989). 38. A. Meister and M. E. Anderson, Ann. Rev. Biochem. 52, 711 (1983). 39. N. S. Garifyanov, B. M. Kozyrev, and V. N. Fedotov, Dokl. Akad. Nauk.
USSR
178, 808 (1968). 40. N. S. Garifyanov and N. F. Usachera, Dokl. Akad. Nauk USSR 145, 565 (1962). 41. E. G. Derouane and T. Guhadi, Chem. Phys. Lett. 31, 70 (1975). 42. M. Mitewa, A. Malinovski, P. R. Bontchev, and K. Kabassanov, Znorg. Chim. Acta
8, 17 (1974). 43. J. R. Harbour, V. Chow, and J. R. Bolton, Can. J. Chem. 52, 3549 (1974). 44. G. R. Buettner, Free Radical Biol. Med. 3, 259 (1987). 45. K. M. Morehouse and R. P. Mason, J. Biol. Chem. 263, 1204 (1988). 46. G. Czapski, Zsr. J. Chem. 24, 29 (1984). 47. E. Finkelstein, G. M. Rosen, and E. J. Rauckman, Mol. Pharmacol. 21, 262 (1982). 48. J. R. Harbour and J. R. Bolton, Photochem. Photobiol. 28, 231 (1978). 49. P. R. Harriott, M. J. Perkins, and D. Griller, Can. J. Chem. 58, 803 (1980). 50. E. G. Janzen, C. A. Evans, and J. I. P. Liu, J. Mugn. Reson. 9, 513 (1973). 51. E. G. Janzen and J. I. P. Liu, J. Mugn. Reson. 9, 5 10 (1973). 52. N. S. Dalal and X. Shi. Biochemistry 28, 780 (1989). 53. E. G. Janzen, Y. Y. Wang, and R. V. Shetty, J. Am. Chem. Sot. 100, 2923 (1978). 54. A. Leaustic, F. Rabonneau, and J. Livage, J. Phys. Chem. 98, 4193 (1986). 55. N. Takahashi, N. Mikami, H. Yamada, and J. Miyamoto, J. Pesticide Sci. ld, 247 (1985). 56. P. G. Schulz, R. H. Schirmer,
W. Sachsenheimer,
and E. F. Pai, Nature 273, 120
(1978). 57. D. M. L. Goodgame,
P. B. Hayman, and D. E. Hathway, Ployhedron
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12 Xianglin Shi and Nar S. Dalai
58. X. Shi and N. S. Dalal, Biochem. Biophys. Res. Commun. 163. 627 (1989). 59. S. Kawanishi, S. Inoue, and S. Sano, J. Biol. Chem. 261. 5952 (1986j. 60. N. S. Dalal. J. M. Millar. M. S. Jagadeesh, and M. S. Seehra. J, Chem. Ph.ys. 74. 1916 (1981). 61. K. Singh, Dissertation, Department of Chemistry. West Virginia University. 1988 62. X. Shi and N. S. Dalal, Free Radical Res. Commun. (in press) (19891 63. X. Shi and N. S. Dalal. .4rch. Biochem. Biophys. (in press! (1990?.
Received September I, 1989; accepted October IO. 1989
West Virginia University, Morgantown,
West Virginia
ABSTRACT Electron spin resonance (ESR) measurements provide evidence for the formation of Cr(V) intermediates in the enzymatic reduction of Cr(V1) by glutathione reductase (GSSG-R) in the presence of NADPH, indicating an initial single-electron transfer step in the reduction mechanism. Depending on the pH, at least two different Cr(V) species are generated which are relatively long-lived. In addition, we have detected the hydroxyl ( *OH) radical formation during the GSSG-R catalyzed reduction of Cr(V1) by spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and a-(4-pyridyl-l-oxide)-N-tert-butylnitrone (CPOBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the *OH radical and Cr(V) formation, indicating that the Oz- is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the -OH radical formation, indicating the involvement of hydrogen peroxide (H20z) in the reaction mechanism. Addition of HZ02 suppresses Cr(V) and enhances the *OH radical formation. Measurements involving N-ethylmaleimide show that the Cr(V) species, produced enzymatically by the reduction of Cr(V1) by GSSG-R, react with H20z to generate .OH radicals, which might participate in the initiation of Cr(V1) carcinogenicity.
INTRODUCTION C(V1) compounds have been found to exert serious toxic and carcinogenic effects on humans and animals [l-4] and to cause mutations in bacteria and transformation of mammalian cells [5-81. In contrast, most Cr(II1) compounds are relatively nontoxic, noncarcinogenic, and nonmutagenic [9, lo]. Cr(V1) and Cr(II1) oxidation states are different in their metabolic pathways: Cr(V1) ions are rapidly transported across cellular membranes [2, 1 l-131, while Cr(II1) moieties do not easily penetrate cells and are not oxidized by cellular constituents [3]. Since it is known that the reduction of Cr(VI) is required for its reaction with DNA [2], the molecular mechanisms for the
Address reprint requests to: Professor Morgantown, WV 26506. Journal of Inorganic Biochemistry 0 1990 Elsevier Science Publishing
N. S. Dalal, Department
of Chemistry,
40, 1-12 (1990) Co., Inc., 655 Avenue of the Americas,
West Virginia University,
1 NY, NY 10010
0162-0134/90/$3.50
2 Xianglin Shi and Nar S. Dalal
intracellular Cr(V1) reduction have been the focus of studies during the past decade but the details are still not understood. Earlier studies on the mechanism of Cr(V1) reduction include those involving glutathione [ 1, 14-241, ascorbic acid [25], glucose [26], lactose [26], galacturonic acid [27], microsomes 128-3 11, rat liver DT-diaphorase [32], and aldehyde oxidase [33]. Glutathione reductase (GSSG-R) has been shown to be selectively inhibited by Cr(V1) in erythrocytes and also in liver in a NADPH dependent manner (34-361. Recently, Sugiyama et al. [37] reported that treatment of Chinese hamster V-79 cells with Cr(VI) results in a decrease of GSSG-R activity. They suggested that the inhibition of GSSGR may be involved in the toxic effect of Cr(VI). While the inhibition of GSSG-R by Cr(V1) was hypothesized to be linked to the reduction of chromium from Cr(V1) to Cr(III), the mechanism remains unclear 134, 35, 371. Since GSSG-II is ubiquitously present in cellular systems and has important biological functions f38], studies on the possible reduction of Cr(V1) by this enzyme and its mechanism should be of fundamental interest. Thus we carried out a detailed electron spin resonance (ESR) spectroscopic investigation of the reduction of Cr(V1) by CJSSG-R. It is shown that the reduction process involves a single electron transfer step, as well as the formation of hydroxyl (‘OH) radical, in this well defined enzymatic system.
MATERIALS
AND METHODS
Dimethyl sulfoxide (DMSO), 1,3-dimethyl-2-thiourea (DMTU), ethanol, H>Oz, and potassium dichromate were purchased from Fisher (Pittsburgh, PA). Glutathione reductase (GSSG-R) from bovine intestinal mucosa, superoxide dismutase (SOD) from bovine blood, catalase from bovine liver. sodium formate. NADPH. and Nethylmaleimide were purchased from Sigma Chemical Co. (St. Louis, MO) and used as received. Spin traps, S,5-dimethyl- 1-pyrroline-N-oxide (DMPO) a a-(4-.pyridyl- I oxide)-N-tert-butylnitrone (4-POBN), were purchased from Aldrich Chemical Co. (Milwaukee, WI), and were used without further purification. smce very weak or no spin adduct signal was obtained from the purchased sample when used alone. The phosphate buffer solution of pH = 7.2 was purchased from Fisher while those of pH 4.0-6.5 were obtained from Aldrich. ESR spectra were obtained at X-band (- 9.7 GHz) using a Bruker ER 200D ESR spectrometer. For accurate measurements of g-values and hyperfine splittings, the magnetic field was calibrated with a self-tracking NMR gaussmeter (Bruker. Model ER035A) and the microwave frequency was measured with a digital frequency counter (Hewlett-Packard, Model 5340A). An ASPECT 2000 computer was used for data acquisition and analysis. The concentrations given in the Figure legends are final concentrations. All experiments were carried out at room temperature.
RESULTS ESR Evidence
for Cr(V) Formation
An aqueous Cr(V1) solution containing a low concentration (0.9 mM) of NADPH did not give a detectable ESR signal (Fig. la). An aqueous solution of Cr(V1) containing CSSG-R gave a weak ESR signal (Fig. lb). but that containing both NADPH and GSSG-R at physiological pH (7.2) gave a much stronger ESR spectrum (Fig. lc). In order to better analyze the spectrum in Figure lc, a spectrum was recorded using a
REDUCTION
OF Cr(V1) BY GLUTATHIONE
REDUCTASE
(a)
)
Cr(VI) + NADPH
@)
s
Cr(VI) +GSSG-R
3
(a)+GSSG-R
/ I--1
p1.9785.
CrW
(a) +GSSG-R
20G .H
(f)
)
~~~THYLMALEI~VIIDE 5G
’
I~H
’
FIGURE 1. ESR spectrarecorded 2 min after mixing 1.9 mM K2Crz07 buffer solution (pH = 7.2) with (a) 0.9 mM NADPH; (b) 12 units/ml GSSG-R; (c) 0.9 mM NADPH and 12 units/ml GSSG-R; (d) same as (c) but the spectrum recorded at a higher gain and a wider scan width; (e) same as (c) but the spectrum recorded 10 min after mixing; (f) same as (c) but with 200 mM Nethylmaleimide added.
higher modulation amplitude and a wider scan width. The spectrum obtained (Fig. Id) was similar to those reported earlier for Cr(V) complexes of ethylene glycol [39-421, ascorbic acid [25], glucose or lactose [26], and galacturonic acid [27] : the center of the spectrum centered at g = 1.9785 with four satellite signals due to 53Cr (9.55% abundance, I = 3/2) hyperfine structure. The observed 53Cr hyperfine coupling of 17.8 G is very similar to those observed for Cr(V) complexes with oxygen ligands [25-27,41-42]. The spectra in Figure lc and Id were thus assigned to Cr(V) species. When the spectrum was measured 10 minutes after the reaction initiation, the overall
4
Xianglin Shi and Nar S. Dalal
11
_.___I--------
(a)
__-i
(b)
(c) __---____
~~~.~~~~
__-.-_ ji
Cr(VI) + NADPH + GSSG -R AT PH = 7.2
6.5
_. _-.~-.~--- 6.0
le)
_.--.--- _-.. 4.0 5G -l-i
j/ /i
(0
/1
20G -H
4+ ._-- -.--
4.0
ii
i
II FIGURE 2. Effect of reaction medium acidity on Cr(V) formation. The reaction mixture contains 0.9 mM NADPH. 12 units/ml GSSG-R, and 1.9 mM IizCrz07 in buffer solutions of (a) pH = 7.2; (b) pH = 6.5; (c) pH = 6.0; (d) pH = 5.0: (e) pH = 4.0: (f) same as (e1 but the spectrum recorded at a higher modulation amplitude and a w-ider xan width.
signal intensity was found to decrease significantly (Fig. le), showing that the Cr(V) species are long-lived but reactive intermediates. Fig. If shows the effect of Nethylmaleimide, the GSSG-R inhibitor [34, 351. By comparing the spectra in Figure lc and If, it can be seen that N-ethylmaleimide significantly inhibits the Cr(V) formation. showing that the reaction is an enzymatic process. Figure 2 shows ESR spectra recorded from an aqueous solution containing Cr(VI). NADPH and GSSG-R as a function of pH. It can be seen that as the pH decreases the relative intensities of two major Cr(V) peaks change. At pH lower than 6.0, the peak at g = 1.9772 becomes dominant (Fig. 2~). At pH = 4.0 only one peak (g = i .9772) is detectable (Fig. 2e). Again, a spectrum recorded at a higher modulation amplitude and a wider scan width (Fig. 2f) was found to be that tyfricai f and *OH radicals, providing evidence that the generation of
*OH radical
is enzymatic.
Effect of HzOz on the *OH Radical
Formation
Figure 5a shows the ESR spectrum from a mixture containing Hz02, GSSG-R and DMPO while Figure 5b from a reaction mixture containing H202, DMPO, and Cr(V1) while Figure 5c was that from a reaction mixture containing H202, DMPO, GSSG-R, and Cr(V1). Figure 5d shows the ESR spectrum from a reaction mixture containing H,O*, DMPO, Cr(VI), and NADPH. The 1:2:2: 1 quartet in Figure Sd was assigned to the DMPO-OH adduct, as discussed earlier. On adding GSSG-R, the intensity of the DMPO-OH adduct (Fig. Se) increased substantially. For the convenience of direct comparison of the effect of H202 on the -OH radical generation, the ESR spectrum observed from the reaction containing DMPO, GSSG-R, KzCrZOT, and NADPH without H202 is included in Figure 5f. From the spectra obtained with and without Hz02 (Fig. 5e and 5f), it can be noted that H201 greatly enhances the *OH radical formation (7 times), and reduces the Cr(V) formation, indicating that the formation of *OH radicals involves a reaction between the Cr(V) species and H$&. To further verify the trapping of *OH radicals (Fig. 5) in the presence of’N202, another spin trap, 4-POBN, was employed, The ESR spectrum obtained with this trap exhibits a triplet of
REDUCTION
OF Cr(V1) BY GLUTATHIONE
REDUCTASE
9
doublets with aN = 14.9 G and ai = 1.6 G (Fig. 6a). When recorded with a small modulation (0.2 G), the spectrum shows additional doublet splitting,‘arising from the coupling of a; = 0.3 G with hydroxyl hydrogen (Fi,.0 6b). These hyperfine splittings are typical those of 4-POBN-OH adduct [44, 53-551, confirming the trapping of *OH radicals in the above discussed reaction. DISCUSSION GSSG-R, a ubiquitous FAD-containing enzyme, uses NADPH to reduce oxidized glutathione (GSSG) and generate glutathione (GSH) according to: GSSG + NADPH + H + -+ 2GSH + NADP + . This enzyme’s general function is to maintain a high level of GSH in the cytosol, but it might have additional roles. Earlier investigators have reported a NADPH dependent inhibition of GSSG-R by Cr(V1) [34, 351. Experiments with mixtures of inhibited and native enzyme revealed no evidence of inhibitor formation or activator loss, suggesting that the GSSG-R inhibition by Cr(VI) results from a direct effect on the enzyme protein [34, 351. The NADPH dependent inhibition of GSSG-R by Cr(V1) has been suggested to be linked to the reduction of chromium from the hexavalent to trivalent form [34, 351. The mechanism remains unknown. The results reported here provide direct evidence for a NADPH dependent reduction of Cr(V1) by GSSG-R with Cr(V) as an intermediate, thus indicating that the initial step in the Cr(V1) reduction involves a one-electron transfer process. It may be noted that in the oxidized form of the enzyme the active site was found to have a disulfide formed by Cys-46 and Cys-41 1561. The mechanism of the enzymatic function of GSSG-R was proposed to involve the action of the thiolate ion at Cys46 which is stabilized by a nearby protonated His in the stable reduced form of the enzyme. The reduced Cys-41 was proposed to attack GSSG, release GSH and form a mixed disulflde and GSH [56]. The inhibitive effect of N-ethylmaleimide suggests that the adjacent Cys residues are involved in the reaction of GSSG-R with Cr(VI). It has been reported that certain enzymes of the cytoplasm, endoplasmic reticulum, and mitochondria of mammalian cells catalyze Cr(V1) reduction [2, 32, 28-30, 571. Jennette reported Cr(V) formation in the incubation of Cr(V1) with liver microsomes in the presence of NADPH [30]. Even though the details were not clear, Jennette suggested that a direct one-electron transfer from the microsomal electron-transport cytochrome P-450 system to Cr(V1) is a likely mechanism [30]. Since Cr(V) complexes are generally characterized as being labile and reactive, whereas Cr(III) complexes are substitutionally inert, the detection of Cr(V) formation led Jennette [30] to suggest that the Cr(V) intermediates are the likely candidates for the “ultimate” carcinogenic forms of the carcinogenic chromium species. This suggestion has led to many further investigations of the formation of Cr(V) in the reduction of Cr(V1) under biologically relevant conditions [ 15-23,25-27, 57, 581. To our knowledge, however, the present work represents the first detailed evidence for Cr(V) formation in a well characterized enzymatic Cr(V1) reduction process [58]. Recently, Kawanishi and coworkers [59] have studied the reactivity of chromium compounds with DNA by the DNA sequencing technique using 32P-5 ‘-end-labeled DNA fragments of a known sequence. They argue that it is not Cr(V) itself that is carcinogenic, but it is the reactive oxygenated species (O,- , i02, *OH) (produced by the decomposition of the CrV(02)43- ) that cause the DNA base modification.
10
Xianglin Shi and Nar S. Dalaf
However, the relevance of this mechanism to Cr(V1) toxicity does not seem to be significant for the following reasons. First, CrV(02)43 has been detected at highly basic pH, well above the physiological value [59-611. Second, in the presence of diol molecules, CrV(02)43 is known to undergo fast ligand exchange to form Cr(V)-diol complexes [39]. In none of our reactions did we ever observe a significant amount of CrV(02)43 and the dominant Cr(V) species always exhibited ligand superfine interactions or g-values characteristic of the complexes irather than the uncomplexed) CrV(02)43 ion, In our earlier work, we reported that reduction of Cr(V1) by biologically relevant diols or glutathione generates Cr(V) complexes, which subscquently react with Hz02 to generate *OH radicals 162, 631. The present work provides evidence of Cr(V) and ‘OH radical formation in an enzymatic CriVI) reduction. Moreover, addition of H202 to this reaction system yields much higher concentrations of -OH radicals, being 7 times as high as without H202, simultaneously suppressing the Cr(V) formation. These results show that Cr(V) complexes generated in the enzymatic Cr(V1) reduction also have an ability to react with Hz& to generate “OH radicals. Since Cr(V) species can also be generated in the reduction of Cr(V1) by various biological reductants [IS-23, 25-27, 5’71 and H202 is a normal biological metabolite, the reaction of Cr(V) complexes with Hz02 might be the major source of -OH radicals. We thus hypothesize that after Cr(V1) enters the cell, it is reduced by various cellular reductants (for example, GSSG-R, diol and thiol molecules) to form Cr(V) complexes which then react with Hz02 to generate “OH radicals and initiate Cr(V1) carcinogenesis. Part of this research has been supported by the Department of the Interior’s Mineral Institute Program administered by the Bureau of Mines through the Generic Mineral Technology Center of Respirable Dust under grant no. G11.35142.
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Received September I, 1989; accepted October IO. 1989
