Mechanisms for the Cytotoxicity of Cysteamine

Thomas M. Jeitner1, and David A. Lawrence

Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, New York 12201–0509

Received January 10, 2001; accepted April 16, 2001

ABSTRACT

The major aim of this study was to quantitatively assess the contribution of H2O2 generation to the cytotoxicity induced by cysteamine. Cysteamine produces H2O2 at levels that correlate with its toxicity between 23 and 160 µM. A maximum of 6.9 µM H2O2 is generated by 625 µM cysteamine. When compared to the toxicity of exogenous H2O2, cysteamine-derived peroxide accounted for 57% of its toxicity. This corresponded to the percent toxicity due to 23 to 91 µM cysteamine. The remaining 43% toxicity appears to involve the inhibition of glutathione peroxidase, because activity of both the cellular and purified enzyme were inhibited by 200 µM cysteamine concentrations. CCRF-CEM cells have no catalase activity, so the inhibition of glutathione peroxidase may sensitize these cells to the less than toxic levels of peroxide generated by this aminothiol. Cysteamine also stimulated the production of cellular glutathione in a manner that was not related to its H2O2 generation. The production of glutathione did not influence toxicity but may reflect the accumulation of cysteamine to levels that inhibit glutathione peroxidase.

Key Words: aminothiol; cysteamine; leukemia; neoplasia; peroxide; glutathione peroxidase; catalase; buthionine sulfoximine.

Cysteamine (CySH, ß-mercaptoethylamine) is an aminothiol compound that is cytotoxic at 10–4 to 10–3 M concentrations. Jeitner et al. (1998) established that 10–4 M levels of CySH kill cells by apoptosis. Catalase completely ameliorates the toxicity of CySH (Biaglow et al., 1984Go; Held and Biaglow, 1994Go; Meier and Issels 1995aGo, bGo; Takagi et al., 1974Go), which suggests that H2O2 is the primary toxicant produced by this agent. Thiols (RSH) can generate H2O2 if transition metals (Mn, with n referring to the oxidation state) are available to catalyze the oxidation of the thiols to their corresponding disulfide (RSSR) and if the thiols are present at below 10–3 M concentrations (Reactions 1–3; Biaglow et al., 1984; Capozzi and Modena, 1974; Held and Biaglow, 1994; Meier and Issels, 1995a; Staite et al., 1985; Starkebaum and Root, 1985; Tahsildar et al., 1988).


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These reactions are restricted to less than 10–3 M thiol concentrations because at millimolar concentrations, thiols react with the generated H2O2 rapidly enough to remove it from solution. Held and Biaglow (Held and Biaglow, 1994Go) have proposed that the thiol-derived H2O2 then goes on to react with reduced transition metals to produce hydroxyl radicals via the transition metal-catalyzed Harber-Weiss reaction (Halliwell and Gutteridge, 1989Go) and that these radicals are the final arbitrator of thiol toxicity.

While it is established that thiols can generate H2O2, it is not known if the amounts generated are sufficient to kill cells. Amelioration of thiol toxicity by catalase clearly indicates that H2O2 is involved in the toxicity but does not negate the involvement of other factors. Polargraphic measurements indicate that CySH generates appreciable initial rates of peroxide production (Biaglow et al., 1984Go; Jeitner et al., 1998Go), but these rates are sustained for no longer than 15 to 30 min (Jeitner, unpublished observations). The implication of these studies is that 10–4 to 10–3 M CySH produces micromolar amounts of H2O2 and raises the question of whether this is a sufficiently cytotoxic quantity, given that some cells, such as fibroblasts, are able to survive mM H2O2. It has been difficult to address the question of the stoichiometery of H2O2 production by thiols using common peroxidase-based methods. In these methods, the reduction of H2O2 is coupled to the oxidation of a dye that can be monitored (Keston and Brandt, 1965Go). Unfortunately, thiols act as oxidizable substrates for the heme-centered peroxidase used in these assays (Svensson, 1988Go) and so compete with the dyes during the oxidation reaction, resulting in an underestimation of the amount of H2O2. Jiang et al. (1990) have published a method for the measurement of peroxide that is not affected by thiols and therefore can be used to assess the stoichiometry of H2O2 formation by thiol-bearing compounds. The following report describes the measurement of H2O2 generation by CySH in the conditions under which this thiol-bearing compound kills cells.

In addition to generating H2O2, CySH also stimulates intracellular glutathione (GSH) production (Meier and Issels, 1995aGo, bGo), which could protect the cells against the thiol-derived peroxide. GSH is a major component of the cellular defenses against oxidizing species. It acts as an oxidizable substrate of GSH peroxidase (GSH Px) and as a direct scavenging agent (Halliwell and Gutteridge, 1989Go). CySH (Meier and Issels, 1995bGo) or ß-mercaptoethanol (Messina and Lawrence, 1992Go) stimulates GSH synthesis by increasing the rate of cellular cysteine uptake through the formation of mixed disulfides with cysteine. Mixed disulfides of cysteine and CySH enter cells via transport system L and are reduced intracellularly to release both thiol compounds. The cysteine is then utilized in GSH synthesis (Meier and Issels, 1995bGo). As described above, this additional GSH could limit the effects of CySH-derived H2O2 Thus, the aim of these studies was to examine the contributions of CySH-derived H2O2 and GSH production to the cytotoxicity associated with CySH exposure. The study also addresses the need to understand the biochemistry of cell culture medium constituents and the fundamental characteristics of the cell lines utilized when assessing in vitro cytotoxicity of chemicals.

MATERIALS AND METHODS

Chemicals.
Catalase (20,000 units/mg), superoxide dismutase (25,000 units/mg), GSH peroxidase, CySH, 5,5'-dithio-bis-(2-nitrobenzoic acid), phenol red-free RPMI medium, xylene orange, tert-butyl hydrogen peroxide, and H2O2 were purchased from Sigma (St. Louis, MO). Chelex 100 resin was purchased from BioRad (Melville, NY). All of the thiol compounds with the exception of GSH were dissolved in Chelex-treated 100 mM sodium phosphate buffer and buffered to pH 7.2 with NaOH. GSH was dissolved in 30% (w/v) sulfosalicyclic acid. The thiol groups concentration was determined with 5,5'-dithio-bis-(2-nitrobenzoic acid) according to the protocol of Collier (Collier, 1973Go), and adjusted to 1.25 mM for GSH and 100 mM for the other thiols prior to their storage at –80°C. L-Buthionine-(S,R)-sulfoximine (BSO) was purchased from Schweizerhall (South Plainfield, NJ) and prepared as a sterile 100 mM solution in Chelex-treated 100 mM NaH2PO4/Na2HPO4, pH 7.2. Fetal bovine serum (FBS) was purchased from Gibco (Gaithersburg, MD). CCRF-CEM cells were purchased from the American Type Tissue Culture Collection (Manassas, VA) and Raji, Sup-T1, J45.01, Pinarowicz, and RL cells were supplied by the Wadsworth Center Cell Culture Facility (Albany, NY). Finally, the concentrations of the stock H2O2 and tert-butyl hydrogen peroxide were determined by potassium iodide titration as described by Beutler (Beutler, 1984Go).

Cell culture.
The cell lines CCRF-CEM, Raji, SUP-T1 [VB], J45.01, Pinarowicz, and RL, derived from human leukemias, were maintained in RPMI-1640 medium supplemented with 2 mM glutamine, 10 µg/mL gentamycin (RPMI), and 10% (v/v) fetal calf serum, at 37°C in 5% CO2:95% (v/v) air.

Drug treatments.
Prior to drug incubations, the cells were collected by centrifugation at 500 x g for 5 min at room temperature and resuspended at a final density of 2.5 x 105 cells/mL in RPMI 1640, 10 mM HEPES, 4.167 mM NaHCO3 (pH 7.4), 10 % (v/v) FBS, 10 µg/mL gentamycin, and 2 mM glutamine at 37°C. CySH, H2O2, or BSO was added to the cells in a solution of Chelex-treated 100 mM NaH2PO4/Na2HPO4, pH 7.2, at a ratio of 1:19 for drug solution to cell suspension. Finally, the tubes containing the treated cells were placed on a rotating wheel at 2.5 revolutions per min for the duration of the drug incubation.

Toxicity measurement.
For viability studies, cells were incubated as described above in a final volume of 2 mL per replicate. At the end of the incubation period (typically 2 h), catalase was added to a final concentration of 50 units/mL. The cells were then centrifuged at 500 x g for 5 min at room temperature and resuspended in Hank's Buffered Salt Solution warmed to 37°C. This was followed by centrifugation as before and resuspension in RPMI-1640 media, 10% fetal calf serum, 10 µg/mL gentamycin, and 2 mM glutamine at 37°C, to give a density of 2.5 x 105 cells/mL. The cells from each replicate were then aliquoted into 4 wells on a flat-bottom, 96-multiwell plate in 100 µL lots and incubated at 37°C in 5% CO2:95% air for 72 h. During the final 4 h of incubation, 50 µL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide in RPMI 1640 (without phenol red) was added to each well. Finally, 100 µL 50% (v/v) N,N-dimethylformamide, 10 mM EDTA, 10% sodium dodecyl sulphate, 200 mM acetic acid was added to each well and incubated overnight at room temperature. The final color was quantified at 570 nm. Toxicity was expressed as a percentage where the color due to the untreated cells was taken as 0% and the color of the wells containing no cells as 100%.

Total glutathione determination.
The measurement of GSH was performed on 5 mL cultures prepared and treated as described above in Drug treatments. GSH content was measured as described by Messina and Lawrence (1992).

Hydrogen peroxide assay.
The generation of hydrogen peroxide by CySH, in the presence of FBS-containing medium, was measured by a modification of the method of Jiang et al. (Jiang et al., 1990Go). In brief, 0.5 mL of sample was reacted with 0.5 mL 500 µM ammonium ferrous sulfate, 200 µM xylene orange, 200 mM sorbitol in 170 mM H2SO4. The reactants were incubated for 30 min at room temperature, centrifuged at 10,000 x g for 5 min, and the absorbance of the supernatant was read at 560 nm. A standard curve of H2O2 prepared in media was prepared for each assay and was used to determine the H2O2 concentrations in the test samples. The standard curves were linear from 39 nM to 5 µM H2O2 (final concentration). CySH up to mM concentrations does not interfere with the assay (Winterbourn and Metodeiwa, 1999Go).

Enzyme measurements.
For these experiments, each replicate required 100 mL of cells as described in Drug treatments. At the end of the 2-h drug incubation, the cells were centrifuged twice at 500 x g for 5 min at 4°C and resuspended in ice-cold phosphate-buffered saline (PBS). The cells were then centrifuged once more and resuspended in 100 µL ice-cold H2O, snap-frozen, and stored at –80°C. Catalase and GSH peroxidase (GSH Px) activity were assayed by the methods of Beutler (Beutler, 1984Go). Protein content was measured using the bicinchoninic acid procedure from Pierce (Rockford, IL) according to their instructions.

Statistics.
The differences between the means of treated and untreated groups were examined by Student's t-test. Lines of best fit were obtained by the least-squares fit method using KalidaGraph (Synergy Software, Reading, PA) data analysis.

RESULTS

Toxicity of CySH
CCRF-CEM cells were incubated with graded concentrations of CySH for 2 h at 37°C and assessed for viability 72 h later. Two-hour exposure to CySH at 39 µM to 1.25 mM caused a decrease in the number of viable cells with a plateau at about 160 µM. Moreover, cytotoxicity correlated linearly with 23 to 160 mM concentrations of CySH (r2 = 0.96). The effective concentration to produce a 50% change (EC50) and EC90 values for the toxicity of CySH equaled 88.5 ± 5.1 µM (n = 8) and 129 ± 4.7 µM (n = 6), respectively. Concentrations greater than 1.25 mM were progressively less toxic (Fig 1aGo). Since oxygen consumption measurements indicated that the production of H2O2 from CySH in the medium was essentially complete within 30 min (Jeitner, unpublished observation), it was pertinent to investigate the minimum period of CySH exposure required for cytotoxicity. Thus, cells were exposed to a toxic concentration of CySH (1.25 mM) for periods ranging from 20 min to 2 h (Fig. 1bGo). These studies demonstrated that 2 h is the minimum period of exposure required to achieve maximum toxicity. The toxicity of 1.25 mM CySH, also resulted in a toxicity of 98.9 ± 0.3% viability (n = 10), that was completely ameliorated by catalase (50 units/mL). This amelioration indicates that H2O2 is a major contributor to the toxicity of CySH. Catalase also had no effect on viability of CCRF-CEM cells in the absence of CySH.



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FIG. 1. Toxicity of cysteamine. CCRF-CEM cells were incubated with the indicated concentrations of CySH for 2 h and toxicity assessed 72 h later (a). The % toxicity due to CySH from 23 to 160 µM fits the following expression: y = –1.7(10–3)M + 7.5(101)Mx, r2= 0.96. Shown are the means and SEM of at least 7 separate determinations. In (b) CCRF-CEM cells were incubated with 1.25 mM CySH for the indicated periods and toxicity assessed 3 days later. The mean values of quadruplicates from 2 separate experiments (filled circle, open circle) are shown.

 
The toxicity of 200 µM CySH was also tested in a number of cell lines besides CCRF-CEM (Table 1Go). Not all cell lines were equally sensitive to CySH. For example, Raji, SUP-T1 [VB], and J45.01 were less vulnerable to the toxicity of CySH than RL, Pinarowicz, and CCRF-CEM cells, which were equally affected by this agent. There was no apparent correlation between the sensitivity of these cells to CySH and their cellular lineage (Table 1Go).


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TABLE 1 Toxicity of Cysteamine to Cells Derived from Human Leukemias
 
Role of Glutathione in the Toxicity of CySH
Two-hour exposure to CySH increases the GSH content of CCRF-CEM cells (Fig. 2Go). The maximal response was equivalent to a 3-fold increase in GSH, over the basal levels in CCRF-CEM cells. There was also a significant decrement at 625 µM CySH (p < 0.01), after the maximum at 160 µM CySH, for which we have no ready explanation. The GSH values in Fig 2aGo probably also represent the maximum production within the 2-h exposure period, because the evolution of GSH due to 80 µM CySH increased steadily up to 80 min, after which little additional GSH was made (Fig. 2bGo).



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FIG. 2. Promotion of cellular glutathione by cysteamine in CCRF-CEM cells. The cellular GSH concentration increased after a 2 h exposure to CySH; results represent the mean ± SEM of 7 individual experiments (a). All of the increases in GSH due to CySH were significantly different (p < 0.0001) from basal GSH level in cells, which was 1.25 ± 0.07 nmol/106 cells. The open symbols GSH production due to CySH fitted the expression: y = 1.3 + 1.5 (104)x (r2= 0.90). In (b), CCRF-CEM cells were incubated with 80 µM CySH for the indicated times and then harvested for the determination of GSH. The data for 3 individual experiments are shown. In (c), the loss of viability due to 23 to 160 µM CySH is plotted against the corresponding increase in cellular GSH concentration and fits the following linear equation: y = 1.7 + 2.0(10–2)x, r2= 0.91.

 
The promotion of GSH synthesis was also increased linearly with respect to CySH concentration over the range of 23 to 160 (r2 = 0.90, Fig. 2aGo). Since cellular GSH content was linearly correlated to CySH over concentrations that were associated with CySH's cytotoxicity (Fig. 1aGo), it was decided to test the correlation between the rise in GSH and % toxicity. Figure 2cGo shows that the CySH-induced changes in cellular GSH content and % toxicity were correlated (r2 = 0.91). This correlation suggested the possibility—albeit unlikely—that the increase in GSH might be involved with the toxicity of CySH.

The possibility that the increase in GSH played a role in the toxicity of CySH was tested by blocking GSH biosynthesis with BSO and evaluating the effect on the toxicity of CySH at 90 µM (Griffith and Meister, 1979Go). BSO attenuated the CySH-induced rise in cellular GSH, and 25 µM BSO was sufficient to produce comparable levels of GSH in both BSO-treated and BSO + CySH-treated cells (Fig. 3aGo); 25 µM BSO alone was not toxic to CCRF-CEM cells (Fig. 3bGo). Although 25 µM BSO inhibited the CySH-induced stimulation of cellular GSH synthesis, it did not attenuate the cytotoxicity (Fig. 3bGo). Thus, the stimulation of GSH synthesis by CySH did not contribute to the CySH-induced cytotoxicity.



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FIG. 3. Inhibition of cysteamine-induced glutathione synthesis. CCRF-CEM cells were incubated with the indicated concentrations of BSO for 15 min, followed by the addition of CySH (in the continued presence of BSO) for a further 2 h, after which total cellular GSH content was measured (a). The effects of 25 µM BSO alone (filled circle) and in combination with 90 µM CySH (open circle) are shown. The basal level of GSH of 1.1 ± 0.1 nmol GSH/106 cells. In (b), cells were treated with 25 µM BSO and/or 90 µM CySH as described above, with the exception that the cells were processed for the assessment of toxicity 72 h later. The results for 3 individual experiments are shown for both panels.

 
Role of Peroxide in the Toxicity of CySH
As noted earlier, the abrogation of the toxicity of CySH by catalase indicated that H2O2 is a major toxin produced by this thiol; however, it is unclear how much H2O2 is generated by CySH and whether its concentration is enough to kill cells. CySH-mediated H2O2 production was measured in the medium used for the incubations to determine whether FBS contains a catalyst for H2O2 production (Jeitner et al., 1998Go) and to emulate the toxicity studies (Fig. 1Go). Our initial studies indicated that the assay described by Jiang et al. (1990) was unsuitable for measuring H2O2 in FBS-containing medium. Apparently, the buffering capacity of the cell culture medium interfered with the assay. Consequently, the concentration of H2SO4 was increased from 25 mM to 170 mM, in order to restore the assay pH to 2.3. Using this modification, it was shown that CySH generated H2O2 in the presence of FBS, and the peroxide production was abrogated by catalase (data not shown). Additional studies showed that the likely catalyst for the production of H2O2 was a transition metal in the FBS such as copper (data not shown).

CySH generated H2O2 in a concentration-dependent manner, to reach a maximum of 6.9 µM H2O2 following a 2-h incubation with 625 µM CySH (Fig. 4aGo). Higher concentrations of CySH generated successively less H2O2. The maximum of H2O2 production of 6.9 µM at 2 h is probably an underestimate because time-course studies showed that the apex of H2O2 production occurred between 30 and 60 min and declined thereafter (Fig. 4bGo). Approximately 20% of the peroxide produced by 80 and 200 µM CySH was lost in the final 60 min of the 2-h incubation (Fig. 4bGo). As noted for the stimulation of GSH production (Fig. 2Go), the generation of H2O2 by 23 to 160 µM CySH fitted to a linear equation (r2 = 0.92, Fig. 4aGo). Similarly, the CySH-induced H2O2 production and cytotoxicity correlated well, over the range for which they were both related (23 to 160 µM; r2 = 0.92, Fig. 4cGo). To assess whether the H2O2 generated by 23 to 625 µM CySH was sufficient to account for all of the toxicity in this concentration range, the cytotoxicity of CySH was plotted as a function of the amount of H2O2 produced by this agent and compared with the cytotoxicity of H2O2 (Fig. 5Go). The toxicity plots with CySH-generated H2O2 versus H2O2 intersect at ~3.53 µM H2O2, the amount of peroxide produced by 91 µM CySH (Fig. 4aGo). Concentrations of CySH less than 91 µM clearly produce enough peroxide to account for up to 57% of the toxicity of this agent, because less reagent H2O2 was required to produce the equivalent change in cytotoxicity. This was also indicated in Figure 4cGo. By the same analysis, the H2O2 generated by CySH at concentrations greater than 91 µM was less than the amount of reagent H2O2 required to cause the same change in cell viability. The toxicity at these concentrations was due to H2O2, because it was completely abrogated by catalase, suggesting that CySH inhibits cellular peroxidases thereby sensitizing the cells to peroxide generated by reactions induced by CySH. Of the 2 cellular peroxidases tested, catalase and GSH PX, only the latter was active in CCRF-CEM cells (Table 2Go). CySH at 200 µM, but not at 80 µM, inhibited cellular GSH Px activity (Table 2Go). In addition, CySH at concentrations greater than 1.25 mM significantly (p < 0.0005) inhibited highly purified GSH Px activity with an effective concentration to produce a 50% inhibition (IC50) of 1.7 mM (data not shown).



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FIG. 4. Generation of H2O2 by cysteamine in FBS-containing medium. CySH was incubated at 37°C for 2 h after which H2O2 was measured (a). Shown are the data for 5 individual experiments. Peroxide production correlated with CySH concentration over the range of 23 to <= 160 µM (y = 1.7 + 1.8 (104)x, r2= 0.93). The production of H2O2 by 80 (filled circle) and 200 (open circle) µM CySH, as a function of time, is shown in (b) and represents 3 separate experiments. No H2O2 was produced in the absence of CySH (data not shown). In (c), the loss of viability due 23 to 160 µM CySH is plotted against the corresponding increases in H2O2 production and fits the following equation: y = 2.2 + 2.3(10–2)x, r2= 0.92.

 


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FIG. 5. Comparison of the toxicity of cysteamine and H2O2. The loss of viability due 23 to 625 µM CySH as a function of the H2O2 produced by these concentrations of CySH is shown (filled circle). Percent toxicity due to exogenous H2O2 is also shown (open circle). Five experiments were performed in each case.

 

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TABLE 2 Effect of CySH on the Activities of GSH Px and Catalase in CCRF-CEM Cells
 
DISCUSSION

The major aim of this study was to quantitatively assess the contribution of H2O2 generation by CySH to its cytotoxicity. It has been shown for the first time that the H2O2 produced by 23–91 µM CySH was sufficient to account of 57% to the toxicity of this agent, but not of 91–625 µM CySH. In the latter range, CySH also inhibits GSH Px. This inhibition, coupled with the complete deficit of catalase in CCRF-CEM cells, suggests that the cells were sensitized to the less than toxic amounts of H2O2 produced by 91–625 µM CySH and killed. GSH Px was not inhibited by 80 µM CySH. The inhibition of cellular and purified GSH PX by CySH is the first such demonstration of such an action by this aminothiol. These observations highlight a number of important observations with regards to the assessment of in vitro toxicity. First, the use of catalase to solely determine the role of H2O2 in toxicity does not take into account factors that sensitize cells to peroxide, such as the inhibition of cellular peroxidase. Second, the characteristics of the cell culture medium and the cell line are critical to the toxicity assessment and chemicals at different concentrations may generate reactions with differential effects on cell viability.

The stoichiometry of H2O2 production induced by CySH is complex. CySH in solution consumes O2, produces O2. (as measured by the reduction of nitro-blue tetrazolium, Jeitner unpublished observation), and generates H2O2 (Fig. 4Go and Takagi et al., 1974). All of these reactions require transition metals that can be reduced by thiols. These observations indicate the reaction scheme for the production of H2O2 by CySH, described in the Introduction, is essentially correct. The amount of peroxide produced by CySH is less than what would be predicted by Reactions 1–3 (Fig. 4aGo) (Jeitner et al., 1998Go; Takagi et al., 1974Go). The chemistries that could limit H2O2 production include superoxide scavenging by CySH, the reaction of H2O2 with serum-associated {alpha}-keto acids (Jocelyn, 1972Go) or CySH, peroxidase activity in the media, and the Fenton reaction (Halliwell and Gutteridge, 1989Go):


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The second-order rate constants for the reaction of CySH with OH, O2•, and H2O2 are 5.9 x 109 (Aruoma et al., 1988Go), 102—103, and 2.9 M–1.s–1 (Winterbourn and Metodeiwa, 1999Go), respectively. Since the rate constant for the reaction of CySH and •OH is several orders of magnitude greater than the constants for O2• and H2O2, the oxidation of CySH by Fenton-derived .OH is the most likely reaction to limit thiol-dependent H2O2 production. It is unlikely that components in the media or serum significantly interfered with CySH-dependent H2O2 production because 200 µM CySH and 1 µM Cu2+ generated 5 µM H2O2 in Chelex-treated 100 mM phosphate buffer (pH 7.2), an amount comparable to that shown in Fig. 4bGo (Jeitner, unpublished observation).

Takagi et al. (1974) also directly measured H2O2 production by CySH in a FBS-containing solution. They were able to measure a maximum production of 80 µM H2O2 due to 3 mM CySH within 10 min. In the present study, there was no H2O2 present in the FBS-containing medium after 2 h with 2.5 and 5 mM CySH. Thus, any H2O2 that had been produced by these concentrations of CySH had presumably been consumed by the end of 2 h when the measurements were made. This is plausible because the time-course studies with 80 and 200 µM CySH (Fig. 4bGo) showed a loss of approximately 20 % of the H2O2 towards the end of the incubation. In addition, even though the second order rate constant for the reaction of H2O2 and CySH is relatively slow at 2.9 M–1.s–1 (Winterbourn and Metodeiwa, 1999Go), the ~103-fold excess of CySH over H2O2 suggests that these species would have reacted within 2 h. This points out the importance of understanding the kinetics of reactions as well as the transient nature of reactive toxic products. The preceding discussion also offers some explanation for the attenuated toxicity of CySH at concentrations greater that 625 µM (Fig 1aGo), in that CySH probably scavenged a significant proportion of the toxic H2O2.

The inhibition of GSH Px appears to be one of the determinants of the toxicity of CySH. This inhibition requires mM amounts of CySH based on studies with the purified enzyme (IC50~ 1.69 mM). Millimolar CySH concentrations may accumulate within cells, based on the following concept. The cysteine that enters the cells as a mixed disulfide of cysteine and CySH is predominantly used for GSH synthesis (Meier and Issels, 1995bGo). Therefore, the increases in cellular GSH content reflect intracellular CySH concentrations, assuming that CySH is metabolized slowly (Huxtable, 1989Go). The latter assumption is reasonable because the activity of CySH dioxygenase, which catabolizes CySH, is low in tissues other than the kidney (Huxtable, 1989Go). Since CySH stimulates the production of mM amounts of GSH, CySH could conceivably attain mM levels within cells. For example, the addition of 2.5 mM CySH results in a net increase of GSH of 2.8 nmol/106 cells (Fig. 2aGo). This is equivalent to an intracellular concentration of 2.8 mM, assuming a volume of ~1 µL/106 cells (Lawrence, unpublished observation). Thus, if intracellular GSH and CySH concentrations are equivalent then CySH could accumulate to the mM levels required to inhibit GSH Px.

Some of the cellular selectivity reported herein as well as the complexity of CySH kinetics with plasma factors may relate to the selective antineoplastic effects of CySH. CySH appears to be selectively toxic or cytostatic to a variety of neoplastic cells, including those derived from neuroblastomas, gliomas, and leukemias (Table 1Go), as well as experimentally-induced neoplasias (Jeitner et al. 1998 and references therein). The lack of catalase activity in CCRF-CEM cells implies that these cells rely on GSH Px for defense against peroxides, and so are sensitive to inhibition of this enzyme. It is possible that the previous reports of selective antineoplastic effects of CySH may have involved neoplastic cells with little active catalase or GSH Px. Decrements in the catalase or GSH Px activities of neoplastic cells have been noted in other studies, including losses of up to 95% of catalase activity (Railloud et al., 1990Go; Sieron et al., 1988Go; Tisdale and Mahmoud, 1983Go; Turleau et al., 1984Go; Vuillaume et al., 1992Go).

The loss of catalase activity and the attenuation of GSH Px activity by a medicinal aminothiol such as CySH suggests that the inhibition of this enzyme may be an achievable aim for the eradication of neoplasia with low catalase activity. Based on previous studies (Jeitner et al. 1998 and references therein), it is unlikely that CySH would be beneficial as a general antineoplastic agent, but it could possibly be useful as an adjunct to chemotherapy and radiotherapy, particularly at sites such as the brain that are readily accessed by this drug and not others. In any case, this report indicates that an exogenous thiol compound can enhance generation of an oxidative product, which can be cytotoxic. It cannot be assumed that thiol-containing compounds are always antioxidants promoting cell survival in vitro.

ACKNOWLEDGMENTS

The authors would like to thank Drs. N. H. Hunt, J. A. Melendez, B. S. Kristal, and A. J. L. Cooper for their comments on the studies and the manuscript, Dr. S. A. Rich for his help with the cell lines, and Ms. L. D'Adolf and F. J. Jeitner for their support. In addition, the assistance of the Biochemistry Core (Dr. R. MacColl and Ms. L. E. Eisele) of the Wadsworth Center, is gratefully acknowledged. These studies were supported by NIEHS ES 038778.

NOTES

1 To whom correspondence should be addressed at Dementia Research, Burke Rehabilitation Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605. Fax: (914) 597-2757. E-mail: tjeitner{at}burke.org. Back

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