Reactive Oxygen Species Are Involved in Arsenic Trioxide Inhibition of

Dehydrogenase Activity. Thangavel Samikkannu, Chien-Hung Chen, Ling-Huei Yih,. Alexander S. S. Wang, Shu-Yu Lin, Tsen-Chien Chen, and Kun-Yan Jan*...
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Chem. Res. Toxicol. 2003, 16, 409-414

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Reactive Oxygen Species Are Involved in Arsenic Trioxide Inhibition of Pyruvate Dehydrogenase Activity Thangavel Samikkannu, Chien-Hung Chen, Ling-Huei Yih, Alexander S. S. Wang, Shu-Yu Lin, Tsen-Chien Chen, and Kun-Yan Jan* Institute of Zoology, Academia Sinica, Taipei 11529, Taiwan, ROC Received September 8, 2002

Arsenite was shown to inhibit pyruvate dehydrogenase (PDH) activity through binding to vicinal dithiols in pure enzyme and tissue extract. However, no data are available on how arsenite inhibits PDH activity in human cells. The IC50 values for arsenic trioxide (As2O3) to inhibit the PDH activity in porcine heart pure enzyme preparation and in human leukemia cell line HL60 cells were estimated to be 182 and 2 µM, respectively. Thus, As2O3 inactivation of PDH activity was about 90 times more potent in HL60 cells than in purified enzyme preparation. The IC50 values for As2O3 and phenylarsine oxide to reduce the vicinal thiol content in HL60 cells were estimated to be 81.7 and 1.9 µM, respectively. Thus, As2O3 is a potent PDH inhibitor but a weak vicinal thiol reacting agent in HL60 cells. Antioxidants but not dithiol compounds suppressed As2O3 inhibition of PDH activity in HL60 cells. Conversely, dithiol compounds but not antioxidants suppressed the inhibition of PDH activity by phenylarsine oxide. As2O3 increased H2O2 level in HL60 cells, but this was not observed for phenylarsine oxide. Mitochondrial respiration inhibitors suppressed the As2O3-induced H2O2 production and As2O3 inhibition of PDH activity. Moreover, metal chelators ameliorated whereas Fenton metals aggravated As2O3 inhibition of PDH activity. Treatment with H2O2 plus Fenton metals also decreased the PDH activity in HL60 cells. Therefore, it seems that As2O3 elevates H2O2 production in mitochondria and this may produce hydroxyl through the Fenton reaction and result in oxidative damage to the protein of PDH. The present results suggest that arsenite may cause protein oxidation to inactivate an enzyme and this can occur at a much lower concentration than arsenite binding directly to the critical thiols.

Introduction Arsenic, an environmental toxicant, is associated with several human diseases, including Blackfoot disease (1), diabetes (2), hypertension (3), and cancers of the skin, lung, bladder, and liver (4, 5). Although several hypotheses have been proposed, the mechanism of arsenic toxicity has not been established. Recent studies indicated that reactive oxygen species (ROS) are involved in arsenite-induced cell signaling (6), DNA damage (6, 7), gene mutation (8), micronuclei (9), apoptosis (10, 11), and cell proliferation (12). Similarly, the production of nitric oxide is involved in arsenite-induced DNA damage (13), poly(ADP-ribosylation) (14), micronuclei (15), and inhibition of pyrimidine dimer excision (16). However, a more popular hypothesis comes from a well-established concept that arsenite has a high affinity to vicinal thiols, and arsenite may bind critical thiols to interfere normal cellular functions. Mammalian cells possess a system that regulates redox status of cellular thiols and protects SH-containing protein from excessive oxidation. It includes low molecular weight donors of SH groups and enzymes, which can catalyze the reduction of SH groups in proteins and detoxify prooxidants by conjugation with glutathione. The protective effects of glutathione (17) and dithiol (18) against the toxic effects of arsenic suggest that arsenic toxicity results from * To whom correspondence should be addressed. Tel: +886-227899513. Fax: +886-2-27858059. E-mail: [email protected].

forming reversible bonds with the thiol groups of proteins. This view is also consistent with the report that the whole blood nonprotein sulfhydryl level in arsenicexposed subjects was 60% of that of controls (19). Moreover, the active sites of many phosphatases contain adjacent sulfhydryl residues (20), and phosphatases are inhibited by phenylarsine oxide (PAO), a vicinal thiol specific reagent (21). PAO is a membrane-permeable trivalent arsenical that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures. Pyruvate dehydrogenase (EC 1.2.4.1; PDH) is often considered a primary target for the toxic actions of arsenicals. PDH catalyzes the oxidative decarboxylation of the end product of glycolysis, pyruvate, to form acetylCoA while reducing NAD to NADH. Any disruption of the action of this enzyme undermines the ability of the cell to meet its energy requirements and could therefore result in cellular damage and death. Inhibition of this enzyme’s activity by arsenite is believed to be a consequence of the binding of the arsenical to dithiols in the lipoamide component of the multienzyme complex. This concept is mainly based on in vitro studies in which bifunctional reagent, p-[(bromoacetyl)amino]phenyl arsenoxide, has been shown to selectively inactivate the lipoamide dehydrogenase purified from Escherichia coli (22). Moreover, the sodium arsenite inhibition of PDH activity in mouse kidney extract can be prevented and reversed by several dimercapto compounds (18). No data

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are available regarding the exact mechanism of how arsenic inhibits PDH activity in mammalian cells. However, PDH was reported to be susceptible to damage and inactivation by ROS (23). Mitochondrial respiration usually consumes about 90% of O2 utilized by cells and is generally considered the major source of cellular ROS (24). Mitochondrial proteins such as E2 subunits of PDH are major targets of oxidative stress (25). The purpose of this research was to investigate how arsenic trioxide (As2O3) inhibits the PDH activity in human cells. Does As2O3 inactivate PDH activity in human cells via binding to the dithiols of the enzyme complex or via ROS?

Materials and Methods Cell Culture. HL60 cells (kindly provided by Dr. C. Y. Liu of Veterans Hospital, Taipei) were cultured in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 µg/mL), and 0.03% glutamate. Cultures were incubated at 37 °C in a watersaturated atmosphere containing 5% CO2. Cells were subcultured every 3 days, and cultures were maintained within a cell density range of 2-10 × 105/mL. Chemicals. PAO was purchased from Aldrich Chem. Co. (Milwaukee, WI). DTT was purchased from Bio-Rad (Hercules, CA). Sodium pyruvate was purchased from Calbiochem (San Diego, U.S.A.). As2O3, hydrogen peroxide, and copper sulfate were purchased from Merck (Darmstadt, Germany). Monobromobimane and 10-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red) were purchased from Molecular Probes (Eugene, OR). Dimercaptosuccinic acid (DMSA), dimercaptopropanol (DMP), sodium selenite, catalase, ferrous sulfate, neocuproine, deferoxaminemesylate, oligomycin, rotenone, myxothiazol, PDH (from porcine heart), and N-ethylmaleimide were purchased from Sigma (St. Louis, MO). Arsenic trioxide predissolved in 1 M NaOH and neocuproine predissolved in chloroform were diluted in PBS. The other stock solutions were prepared by dissolving the drugs in milliQ-filtered water. The drug in stock solution was added to the complete medium, and the old culture medium was replaced with the drug-added medium. Determination of PDH Activity. Cells (1 × 106) resuspended in 50 mM Tris-HCl and 0.5 mM EDTA (pH 7.4) were sonicated for 3 min in an ice bath with a 9 s pulse and 1 s off and centrifuged at 12 000 rpm for 15 min. Supernatant was used for determination of PDH activity according to the method of Robertson et al. (26) with slight modification. Briefly, cell supernatant (100 µg) was resuspended in reaction buffer TrisHCl (pH 7.4) containing 1 mM NADP+, 0.5 mM thiamin pyrophosphate, 1 mM MgCl2, 0.5 mM CoA, 0.5 mM dithiothreitol, 5 mM pyruvate, and an appropriate volume of buffer. The reaction was measured with an ELISA reader at 340 nm. Protein content was determined by the Bio-Rad protein assay kit using bovine serum albumin as a standard. PDH activity was expressed as mU/mg protein. We used NADP+ instead of NAD+ because our pilot experiments indicated that NADP+ gave higher ∆340 than NAD+. Determination of Cellular Thiols. The cellular contents of total thiol and vicinal thiol were measured with the method of Sen et al. (27). Briefly, each sample was divided into three groups: (i) untreated group; (ii) vicinal thiol blocked group, in which cells were treated with 20 µM PAO for 10 min; and (iii) total thiol blocked group, in which cells were treated with 250 µM N-ethylmaleimide for 10 min. The cells were then washed and resuspended at 106 cell/mL PBS containing 40 µM monobromobimane for 15 min at room temperature. The fluorescence (350/488 nm) was measured with a Hitachi fluorescence spectrophotometer (model F-4010, Tokyo, Japan). The cellular total thiol content was estimated from the fluorescence intensity of i-iii, and the cellular vicinal thiol content was estimated from the fluorescence intensity of i and ii.

Samikkannu et al. Determination of H2O2. The cellular level of H2O2 was measured with the aid of the fluorogenic probe, Amplex Red, as described by Zhou et al. (28). Fluorescence of 2 mL of reaction buffer containing 10 µM Amplex Red, 10 mM Tris-HCl, pH 7.5, 1 U/mL horseradish peroxidase, and the sample solution was measured with a Hitachi fluorescence spectrophotometer model F-4010 with excitation and emission at 560 and 590 nm, respectively. A H2O2 concentration-dependent increase of fluorescence intensity was established (data not shown). Cells after treatment were washed, suspended in 200 µL of ice-cold doubledistilled water, and sonicated for 3 min with a 9 s pulse and 1 s off. The cell lysate was added to the reaction buffer and incubated at 37 °C for 5 min before the fluorescence measurement. Statistical Analysis. The experiments were performed independently 2-6 times. Results are expressed as mean ( standard deviation. The significance between means was analyzed with one way ANOVA followed by Tukey’s test. A comparison between As2O3 or PAO with vs without modulator was considered to be statistically significant and is indicated by * when p < 0.01.

Results As2O3 Was a Much More Potent PDH Inhibitor Adding to Cells Than Adding to Pure Enzyme Preparation. The IC50 values for As2O3 to inhibit the PDH activity in porcine heart pure enzyme preparation and in HL60 cells were estimated to be 80.0 and 2.1 µM, respectively (Figure 1A,B,C,G). Thus, inactivation of PDH activity by As2O3 was about 38 times more potent in cells than in pure enzyme preparation. These data suggest that while As2O3 inactivates the PDH activity in pure enzyme preparation by binding to the dithiols in the lipoamide component of the multienzyme complex, this same mechanism may not be applicable in HL60 cells. To test this notion, the inhibitory effects of As2O3 on the total thiol content and the vicinal thiol content in HL60 cells were studied. The IC50 values for As2O3 to reduce the total thiol content and the vicinal thiol content were estimated to be 82.5 and 81.7 µM, respectively (Figure 1D,E,G). The IC50 values for PAO to reduce the total thiol content and the vicinal thiol content were estimated to be 2.3 and 1.9 µM, respectively (Figure 1D,E,H). Thus, As2O3 was a weaker thiol reacting agent as compared to PAO. These data also indicate that while As2O3 was a potent PDH inhibitor, it was a weak thiol reacting agent in HL60 cells. We have also checked the possibility that the reduction of PDH activity may be due to cell death. The results indicate that a 4 h treatment with 2 µM As2O3 or 1 µM PAO did not affect the trypan blue exclusion of HL60 cells (data not shown). With a 72 h treatment, the concentrations of As2O3 and PAO required to reduce the cell viability to 50% were estimated to be 9.6 and 28.8 µM, respectively (Figure 1F-H). These data indicate that PAO was less cytotoxic to HL60 cells than As2O3, and arsenic inactivation of PDH activity was unrelated to HL60 cell growth inhibition. ROS Were Involved in As2O3 Inhibition of PDH Activity. Because the sodium arsenite inhibition of PDH activity in mouse kidney extract can be prevented and reversed by several dimercapto compounds (19), the effect of dithiol agents on As2O3 inhibition of PDH activity in HL60 cells was studied. The results indicate that DTT, DMSA, and DMP suppressed PAO inhibition of PDH activity in HL60 cells, but this was not observed for As2O3 (Figure 2A). Because ROS are also involved in As2O3 toxicity, the effect of antioxidants on arsenic inhibition

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Figure 1. As2O3 was a potent PDH inhibitor in HL60 cells but not a potent dithiol reacting agent. Pure PDH from porcine heart (30 mU in a total reaction mixture of 1 mL) was incubated with various concentrations of As2O3 (triangle) or PAO (inverse triangle) at 37 °C for 0.5 h, and then, the PDH activity was measured (A). HL60 cells were incubated with 4 µM As2O3 or 0.5 µM PAO for various lengths of time, and then, the PDH activity was measured (B). HL60 cells were incubated with various concentrations of As2O3 or PAO for 4 h, and then, the PDH activity (C), the total thiol content (D), and the vicinal thiol content (E) were measured. HL60 cells were incubated with various concentrations of As2O3 or PAO for 72 h, and then, the cell number was counted with the trypan blue exclusion method (F). The IC50 values for As2O3 (G) or PAO (H) were estimated from Figure 1A,C-F.

Figure 2. Antioxidants but not dithiol compounds ameliorated As2O3 inhibition of PDH activity. HL60 cells were treated for 4 h with dithiol compounds alone, with As2O3 or PAO plus dithiol compounds (A); with antioxidants alone, with As2O3 or PAO plus antioxidants (B). The dithiol compounds were DTT, DMSA, and DMP.

of PDH activity in HL60 cells was examined. The results indicate that pyruvate, catalase, and selenite ameliorated As2O3 inhibition of PDH activity, but this was not observed for PAO (Figure 2B). These results suggest that while dithiol binding is involved in PAO inhibition of PDH activity, ROS seem to be involved in As2O3 inhibi-

Figure 3. Treating HL60 cells with As2O3 increased cellular H2O2 level. (A) HL60 cells were treated for 20 min with As2O3 alone or with As2O3 plus catalase, SOD, NAME, or MTC. The cellular H2O2 level was measured by recording the fluorescence intensity of Amplex Red. *, with vs without modulator. Cells were treated with 2 µM As2O3 (B) or with 1 µM PAO (C) for various lengths of time, and the cellular H2O2 level (open symbols) and the PDH activity (solid symbols) were measured.

tion of PDH activity. If this is true, then treatment with As2O3 will and treatment with PAO will not increase cellular oxidant level. The level of H2O2 was then measured with the aid of a fluorogenic probe, Amplex Red. The results indicate that treatment with As2O3 increased the fluorescence intensity of Amplex Red, and the As2O3-increased fluorescence intensity was sensitive

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Figure 4. Mitochondrial respiration inhibitors decreased H2O2 induction and suppressed As2O3 inhibition of PDH activity. (A) HL60 cells were treated for 4 h with 4 µM As2O3, with mitochondrial respiration inhibitors (0.4 µM rotenone, 25 µM myxothiazol, 0.2 µM oligomycin) or with As2O3 plus modulators. (B) HL60 cells were treated for 4 h with 0.4 µM rotenone, 25 µM myxothiazol, 0.2 µM oligomycin, 4 µM As2O3, 0.5 µM PAO, As2O3, or PAO plus modulators.

to antioxidants such as catalase and superoxide dismutase (SOD) but was insensitive to nitric oxide synthase inhibitors such as Nω-nitro-L-arginine methyl ester (NAME) or S-methyl-L-thiocitrulline chloride (MTC) (Figure 3A). These results indicate that treatment with As2O3 increased the H2O2 level in HL60 cells. The Amplex Red fluorescence intensity of 2 µM As2O3-treated cells increased up to 80 min after the addition of As2O3 and then declined slightly thereafter; however, the PDH activity decreased continuously upon the addition of As2O3 (Figure 3B). On the other hand, treatment with 1 µM PAO did not increase the Amplex Red fluorescence intensity; however, the PDH activity decreased continuously upon the addition of PAO (Figure 3C). These results are consistent with the notion that ROS were involved in the inhibition of PDH activity by As2O3 but not that by PAO. Mitochondrial Respiration Inhibitors Suppressed As2O3 Inhibition of PDH Activity and H2O2 Production. Because PDH is a mitochondrial enzyme and mitochondiral respiration is involved in univalent reduction of O2 to O2•-, the effect of blocking the electron transport on arsenic induction of H2O2 and inhibition of PDH activity was examined. The results indicate that rotenone (a complex I inhibitor), myxothiazol (a complex III inhibitor), and oligomycin (an ATP-synthase inhibitor) suppressed the As2O3-increased Amplex Red fluorescence intensity (Figure 4A). Rotenone, myxothiazol, and oligomycin also ameliorated As2O3 inhibition of PDH activity, but this was not observed for PAO (Figure 4B). These results are consistent with the notion that treatment with

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Figure 5. Metal chelators ameliorated whereas Fenton metals enhanced the PDH inhibition by As2O3. (A) HL60 cells were treated for 4 h with 10 µM neocuproine, 100 µM desferioxamine, 4 µM As2O3, or As2O3 plus metal chelators. (B) Cells were treated for 4 h with 20 µM CuSO4, 20 µM FeSO4, 2 µM As2O3, or As2O3 plus metals.

Figure 6. H2O2 and Fenton metals decreased the PDH activity of HL 60 cells. (A) HL60 cells were treated for 4 h with various concentrations of H2O2 (open circle), H2O2 plus 5 µM FeSO4 (filled square), or H2O2 plus 5 µM CuSO4 (filled triangle). (B) HL60 cells were treated for 4 h with various concentrations of CuSO4 (open triangle) or 10 µM H2O2 plus CuSO4 (filled triangle). (C) HL60 cells were treated for 4 h with various concentrations of FeSO4 (open square) or 10 µM H2O2 plus FeSO4 (filled square).

As2O3 increases the mitochondrial production of H2O2 and this is responsible for the As2O3 inhibition of PDH activity. Fenton Reaction Was Involved in As2O3 Inhibition of PDH Activity. Because the O2•- generated in mitochondria is subsequently dismutated to H2O2 and this molecule in combination with Fenton metals produces the highly reactive hydroxyl radical, which is known to cause protein damage (25), the involvement of Fenton reactions in As2O3 inhibition of PDH activity was examined. The results indicate that neocuproine (a copper chelator) and desferioxamine (an iron chelator) ameliorated (Figure 5A), whereas CuSO4 and FeSO4

Arsenic Trioxide Inhibits Pyruvate Dehydrogenase

enhanced As2O3 inhibition of PDH activity (Figure 5B). These results are consistent with the notion that treatment with As2O3 increased mitochondrial H2O2 production, which was then catalyzed by Fenton metals to generate hydroxyl radical to cause PDH inactivation. This notion is further supported by the observation that treating HL60 cells with H2O2 and Fenton metals decreased the PDH activity (Figure 6A-C).

Discussion The present results indicate that while PAO inhibited the PDH activity by reacting with vicinal thiols, As2O3 inhibited the PDH activity through the generation of ROS in HL60 cells. This notion came from the following observations: (i) The inactivation of PDH activity by As2O3 was about 38 times more potent in HL60 cells than in pure enzyme preparation. These data suggest that while As2O3 inactivates the PDH activity in pure enzyme preparation by binding to the dithiols, this same mechanism may not be applicable in HL60 cells. (ii) The IC50 values for As2O3 and PAO to decrease the cellular vicinal thiol content of HL60 cells were estimated to be 80.0 and 1.9 µM, respectively. Thus, As2O3 was a weak vicinal thiol reacting agent in comparison to PAO. (iii) Dithiol compounds suppressed PAO inhibition of PDH activity but not that of As2O3. (iv) Antioxidants suppressed As2O3 inhibition of PDH activity but not that of PAO. (v) As2O3 increased cellular H2O2 production, but PAO did not. (vi) Blockage of mitochondrial respiration decreased As2O3induced H2O2 production. (vii) Blockage of mitochondrial respiration also decreased As2O3 inhibition of PDH activity, and this was not observed for PAO. (viii) Fenton metal chelators decreased and Fenton metals increased As2O3 inhibition of PDH activity. (ix) Treating HL60 cells with H2O2 and Fenton metals decreased the PDH activity. The present results suggest that As2O3 can inactivate enzyme via ROS-mediated mechanisms at much lower concentrations than by binding directly to vicinal thiols of an enzyme. Because of strong vicinal thiol reacting activity, PAO is widely used as an inhibitor for phosphatases. PAO was more potent and had a shorter lag time than As2O3 to inactivate the PDH activity in HL60 cells (Figure 1B,C); yet, PAO was less cytotoxic than As2O3 to HL60 cells (Figure 1F-H). Therefore, arsenic inactivation of PDH activity was unrelated to HL60 cell growth inhibition. Moreover, As2O3 induces apoptosis in NB4 cells through the generation of ROS (11) and sodium arsenite inhibits pyrimidine dimer excision via the generation of nitric oxide production in Chinese hamster ovary cells (16), but these are not observed for PAO. In contrast, PAO even inhibits superoxide generation in human neutrophils and human monocytes (29). PDH, a mitochondrial multienzyme complex, contains multiple sulfhydryl groups including the essential dithiol, lipoic acid, located at the thiamine pyrophosphate binding sites. PDH is susceptible to oxidative damage and can be inactivated by ROS (23, 25). Free radical attack on proteins modifies amino acids, fragments chains, and generates cross-links, thereby destabilizing protein tertiary structure and increasing susceptibility to proteolysis (30). Previous studies have demonstrated that both NADH-ubiqunone reductase (complex I) and ubiqunolcytochrome c reductase (complex III) of the mitochondrial electron transport chain are involved in univalent reduc-

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tion of O2 to O2•- (31, 32). The present results indicate that the cellular H2O2 level increased continuously up to 80 min upon the addition of 2 µM As2O3 and then declined slightly but still maintained at a level higher than untreated cells after 4 h. The present results are consistent with the notion that treating HL60 cells with As2O3 stimulates O2•- production in mitochondria. The H2O2 generated from dismutation of O2•- may have reacted with Fenton metals to produce hydroxyl radicals, which are responsible for protein oxidation and resulted in PDH inactivation. Experiments are undergoing to look for structural evidence that arsenite actually causes protein oxidation to inactivate the activity of a protein.

Acknowledgment. We thank the National Science Council (NSC 90-2320-B-001-037) and Academia Sinica, ROC, for supporting grants.

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