INTRODUCTION

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The cell membrane of Saccharomyces cerevisiae is a primary site of heavy metal toxicity by Cd²⁺ and Cu²⁺, with resultant loss of mobile cellular solutes, such as K⁺ (1, 10, 13, 19). Silver, in addition to loss of K⁺, has been reported to increase efflux of accumulated phosphate, mannitol, succinate, glutamine, and proline (17, 18). Mercury and silver both inhibit yeast respiration. A specific target for mercury has not been defined, but ATP content of the cell is rapidly depleted (5). Silver is reported to bind with phosphate, resulting in collapse of the proton motive force (17). Toxic metal ions, including Cu²⁺, Co²⁺, Ni²⁺, Cd²⁺, Mn²⁺, and Hg²⁺, also inhibit plasma membrane ATPase by means of various binding interactions (15). Silver and mercury have relatively high affinities for reduced thiol groups, but which of the many thiol-containing cellular constituents, such as glutathione, cysteine, or coenzyme A, and thiol-containing proteins are affected is unclear (6). The above effects lead to increased permeability of the cell by external materials, i.e., adverse effects on membrane integrity, and a reduced ability to maintain electrochemical gradients or membrane potential (2). Therefore, it is possible to use membrane damage as an indicator of heavy metal toxicity.

Propidium iodide (PI), which fluorescently stains nucleic acids in damaged or dead cells, has been widely used to indicate cell membrane integrity (8), whereas oxonols, which are lipophilic anionic dyes, accumulate in cells with reduced membrane potential (9). As long as the free dye concentration is below the saturation point for the binding sites available in the cell, the intracellular dye accumulation is membrane potential dependent (14). The relationship between changes in oxonol fluorescence and membrane potential is linear (9). The compound bis-(1,3-dibutylbarbituric acid) trimethine oxonol (Ox) has the highest degree of voltage sensitivity among oxonols (3).

This study compared the toxic effects of mercury and silver ions on two metabolically distinct species of Candida, C. albicans, an obligate commensal and opportunistic pathogen of warm-blooded animals in nature, and C. maltosa, a hydrocarbonoclastic species of industrial significance, by a flow cytometric procedure with PI and Ox. The effects of Ag(I) and Hg(II) on both yeasts differed regarding loss of membrane integrity, membrane potential, and cell recoverability.

	MATERIALS AND METHODS

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Culture maintenance and growth. Cultures of C. albicans strain GSU-30 and C. maltosa strain R-42 were obtained from the lyophilized culture collection at Georgia State University. Stock cultures were maintained on Bacto Sabouraud dextrose agar (SAB; Difco Laboratories, Detroit, Mich.) slants and transferred every 3 to 4 weeks. Working cultures were grown on the Bacto yeast nitrogen base (Difco Laboratories) supplemented with 0.5% glucose (pH 5.5) (DYNB) with agitation at room temperature (22°C) for 18 h.

Chemicals. AgNO₃ and HgCl₂ (ACS reagent grade; Sigma Chemical Co., St. Louis, Mo.) were dissolved in deionized distilled water (ddH₂O) to make 100 mM stock solutions. Working solutions of 0.005 to 0.20 mM concentrations were comprised of serial dilutions of the stock solutions in morpholineethanesulfonic acid (MES) buffer (J. T. Baker, Phillipsburg, N.J.). MES, which exhibits negligible metal-binding properties (11), was used for metal exposures at pH 5.5. MES at pH 6.8 was used for Ox staining (MES at this pH provided the greatest peak separation between heat-killed cells and live cells [data not shown]).

Exposure of cells to heavy metals. Cells grown on DYNB for 18 h at 22°C were harvested in a centrifuge, washed twice with ddH₂O, and suspended in MES buffer (pH 5.5) to an optical density at 600 nm of 0.6 in a 1.0-cm light path (equivalent to 2.33 × 10⁶ cells of C. maltosa ml¹ or 1.20 × 10⁷ cells of C. albicans ml¹). Various concentrations of Ag(I) or Hg(II) in 20 µl were added to 0.98 ml of cell suspension in 1.7-ml microcentrifuge tubes. The tubes were centrifuged and then incubated in static culture at room temperature for 1 h. These suspensions were then centrifuged at 10,000 × g for 2 min and the pellet was resuspended immediately in the staining buffers.

Fluorescent probe staining procedure. PI and Ox (Molecular Probes Inc., Eugene, Oreg.) were used separately for examination of membrane integrity and membrane potential, respectively. The protocols used for Ox and PI staining were similar to those described by Deere et al. (7). The stock solution of Ox contained 1.0 mM Ox in dimethyl sulfoxide (J. T. Baker), whereas the stock solution of PI contained 1.0 mg of PI ml¹ in phosphate-buffered saline (PBS). For Ox staining, yeast cells were suspended in MES (pH 6.8) to give approximately 10⁶ to 10⁸ cells ml¹. Stock dye solution (5.0 µl) was added to 1.0 ml of yeast suspensions. Samples were incubated for 30 min at room temperature in the dark before flow cytometric analysis. For PI staining, 20 µl of stock solution was added to 0.98 ml of yeast suspensions in PBS. The incubation time was always from 5 to 8 min before analysis.

Flow cytometric analysis of membrane potential and membrane integrity. The stained cells were analyzed with a FACScalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) equipped with a 15-mW, 488-nm argon-ion laser. Cells (10⁴) were acquired in each sample. Lower and upper fluorescence limits that included most cells in the live control (more than 99.9%) were determined. The stained cells within those limits were considered as intact or nondepolarized cells, and stained cells with higher fluorescence than the upper limit were regarded as permeabilized or depolarized cells.

Cell recoveries. Viable cell densities were determined from recoveries on SAB agar. After exposure to the heavy metals, the cell suspensions were serially diluted in SAB broth (for neutralization), and the dilutions were plated onto SAB agar. The CFU were counted after incubation at 30°C for 48 h.

Uptake of heavy metals. After a first exposure of C. maltosa cells to heavy metals in MES buffer, aliquots of the cell suspensions were centrifuged and the supernatants were exposed to a second challenge of C. maltosa with cell densities similar to those for the first challenge. Comparison of the observed sigmoidal dose-response curves of percentages of depolarized cells versus metal concentrations obtained from the successive metal exposures provided a model system for estimation of relative heavy metal uptake or binding.

Statistical analysis. The CellQuest software was used to analyze all data acquired by flow cytometry. All tests were performed in duplicate.

	RESULTS

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Both C. albicans and C. maltosa demonstrated significant reductions in membrane potential after exposure to increasing concentrations of Ag(I) and Hg(II) (Fig. 1). The loss of membrane potential was more abrupt or "stepwise" with Ag(I) than with Hg(II). The flow cytometric histogram for Ag(I) exposure showed two distinct peaks indicating viable and depolarized cells, respectively, whereas the histogram for Hg(II) had only a single peak that shifted towards a higher fluorescence with increasing concentrations of Hg(II).

View larger version (33K): [in this window] [in a new window]   FIG. 1.   Decreases in membrane potential of C. albicans (1.17 × 107 cells ml1 [A], 1.98 × 107 cells ml1 [C]) and C. maltosa (1.15 × 107 cells ml1 [B], 7.40 × 106 cells ml1 [D]) with exposure to increasing concentrations of Ag(I) and Hg(II) in MES buffer.

The depolarization rates for cells of C. albicans and C. maltosa exposed to Ag(I) and Hg(II) for different time periods are given in Fig. 2. The cells of both species showed similar responses. At a concentration of 0.02 mM Ag(I), most cells of C. maltosa lost their membrane potential within 2 min, whereas 0.02 mM Hg(II) had a negligible effect on membrane potential even at 15 min. The percentage of depolarized cells increased gradually after 15 min. At concentrations below 0.02 mM, the percentage of depolarized cells in Ag(I) reached a plateau (84%) within 15 min, but the percentage of depolarized cells in Hg(II) continued to increase with time (Fig. 2B).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   (A) Depolarization with time of cells of C. albicans in 0.002 mM Ag(I) (, 3.65 × 106 cells ml1; , 7.38 × 106 cells ml1) and 0.016 mM Hg(II) (, 3.65 × 106 cells ml1; , 7.38 × 106 cells ml1). (B) Depolarization of C. maltosa (2.33 × 106 cells ml1) in Ag(I) (, 0.020 mM; , 0.004 mM) and Hg(II) (, 0.020 mM; , 0.010 mM). Each data point represents the average obtained from duplicate independent assays.

C. albicans and C. maltosa lost membrane integrity rapidly in Ag(I) solutions and more slowly in Hg(II) solutions (Fig. 3). In contrast to C. maltosa, cells of C. albicans were not permeabilized in Hg(II) solution, even at the highest concentration tested (0.040 mM).

View larger version (32K): [in this window] [in a new window]   FIG. 3.   Decreases in membrane integrity of C. albicans (1.17 × 107 cells ml1 [A], 1.98 × 107 cells ml1 [C]) and C. maltosa (1.15 × 107 cells ml1 [B], 7.40 × 106 cells ml1[D]) with exposure to increasing concentrations of Ag(I) and Hg(II) in MES buffer.

The percentage of intact cells (retaining membrane integrity) was reduced at concentrations of Ag(I) that were similar to those required for an equivalent reduction of the percentage of nondepolarized cells (maintaining membrane potential) (Fig. 4). Ag(I) affected membrane integrity and membrane potential of both species to a greater extent than did Hg(II). Nearly a 10-fold increase in the concentration of Hg(II) over that of Ag(I) was required for a 50% reduction in the number of nondepolarized cells for C. maltosa, whereas about a fivefold increase in concentration resulted in a similar effect for C. albicans. Markedly higher concentrations of Hg(II) versus Ag(I) were required for equivalent reductions in the percentage of intact cells for the two yeasts (Fig. 4).

View larger version (20K): [in this window] [in a new window]   FIG. 4.   The percentage of nondepolarized cells (maintaining membrane potential; Ox staining; , ) and the percentage of intact cells (retaining membrane integrity; PI staining; , ) of C. albicans (A) (1.20 × 107 cells ml1) and C. maltosa (B) (2.33 × 106 cells ml1) in increasing concentrations of Ag(I) (, ) and Hg(II) (, ). Each data point represents the average obtained from duplicate independent assays.

Cell recoveries on SAB and losses in membrane potential and membrane integrity for both C. albicans and C. maltosa had similar kinetics in the presence of Ag(I) (Fig. 5A and B). In the presence of Hg(II), however, the loss of culturability preceded the loss of membrane potential and membrane integrity for both species, particularly for C. maltosa (Fig. 5C and D).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Effects of increased concentrations of Ag(I) and Hg(II) on the percentage of recoverable (, plate counting), intact (, PI staining) and nondepolarized (, Ox staining) cells of C. albicans (1.20 × 107 cells ml1) and C. maltosa (2.33 × 106 cells ml1). Each data point represents the average obtained from duplicate independent assays.

The sigmoidal curves of the percentage of depolarized cells of C. maltosa versus concentrations of both Ag(I) and Hg(II) were used to estimate the active concentrations of heavy metals that remained in the supernatants (Fig. 6). Similar sigmoidal curves were produced from the initial metal solutions and their supernatants. Higher concentrations of Ag(I) relative to Hg(II), however, were required for equivalent depolarization on the second exposure to the supernatants, an inverse of the Ag-Hg effects in the first exposure to the initial solutions (Fig. 6).

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Percentage of depolarized cells of C. maltosa produced in solutions of Ag(I) () and Hg(II) () and in supernatants [, Ag(I); , Hg(II)] of these solutions. The solutions and supernatants received equivalent inocula (2.33 × 106 cells ml1).

When an initial concentration of 0.02 mM Ag(I) was challenged for 2 min with cells of C. maltosa, there was insufficient Ag(I) remaining in the supernatant to produce depolarization with the second challenge (Fig. 7), but in the case of Hg(II) at an initial concentration of 0.02 mM, the supernatant depolarized about 60% of the cells within 15 min. At an initial concentration of 0.01 mM Hg(II), the residual Hg(II) in the supernatant still caused some cells to be depolarized by 15 min, whereas no appreciable depolarization of cells occurred with Ag(I) with these same test parameters (Fig. 7). These data indicated that uptake and binding of Ag(I) by C. maltosa was greater and more rapid than that of Hg(II) (Fig. 2B, 6, and 7).

View larger version (19K): [in this window] [in a new window]   FIG. 7.   Depolarization of cells of C. maltosa in supernatants of Ag(I) and Hg(II) solutions that had been initially challenged. Cells were exposed to the initial solutions of Ag(I) (, 0.040 mM; , 0.020 mM) and Hg(II) (, 0.020 mM; , 0.010 mM) for increasing time periods (in minutes, as indicated); cells were exposed to the supernatants for 1 h. The initial solutions and their supernatants received equivalent inocula (2.33 × 106 cells ml1). Each data point represents the average obtained from duplicate independent assays.

	DISCUSSION

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An apparent threshold level of Ag(I) reduced membrane potential and membrane integrity rapidly for the individual cells of C. albicans and C. maltosa, suggesting that a major target of silver is located in the cell membrane. The absence of such a threshold dose for Hg(II) suggested that the target molecules and their threshold levels of mercury were different from those of silver. Moreover, in Ag(I) solutions, cells lost recoverability at a rate similar to those for cell depolarization and membrane permeabilization, whereas in Hg(II) solutions, loss of cell recoverability preceded the loss of membrane potential and membrane integrity, especially for C. maltosa. C. albicans retained membrane integrity even after exposure to Hg(II) for 1 h and in this regard differed significantly from C. maltosa. A further distinction between the activities of the two ions was the fact that the uptake and binding of Ag(I) by C. maltosa were greater and more rapid than those of Hg(II).

Brown and Smith (4) showed by a cytochemical method that the Hg(II) accumulated by Cryptococcus albidus was present in various parts of the cell other than the cell wall and membranes. Passow and Rothstein (16) demonstrated that mercury ions caused irreversible membrane damage in S. cerevisiae, whereas Brunker (5) found that this metal inactivated the enzymes that are responsible for catabolic metabolism. These reports suggested that mercury ions might interact with a variety of reactive sites in both the cell membrane and intracellular targets. An interaction of Hg(II) with C. albicans and C. maltosa at multiple sites with disruption of vital cell processes might explain the observed loss of cell recoverability before the loss of membrane potential and membrane integrity. Ag(I) and Hg(II) may act similarly for both yeasts and possibly with different targets, but the more-rapid binding of Ag(I) may overshadow any threshold differences between membrane function and cell recoverability.

We recognize that chemical forms of a metal in solution, which regulate metal binding to the membrane and penetration into the cell, are difficult to identify and vary under different experimental conditions (6, 12). Nevertheless, relative metal toxicity may be assessed from the equivalent biologically active metal concentrations. We found that the percentage of depolarized cells of both species increased with increasing concentrations of metals and generated a sigmoidal dose-response relationship. These sigmoidal curves permitted an estimation of metal concentrations that remained in the supernatants. For example (based on data shown in Fig. 7), with an initial concentration of both Ag(I) and Hg(II) in the first exposure at 0.05 mM, the equivalent concentration of Ag(I) remaining in the supernatant would be approximately 0.004 mM and the Hg(II) level would be about 0.016 mM. With adjustment of cell densities and exposure periods (which change the shape of the curve), a broader range of metal concentrations may be estimated. The data are supportive of determinations of relative binding rates. Therefore, even though mechanistic details of the cellular effects on membrane potential remain unknown, the loss of membrane potential upon exposure to heavy metals as determined by flow cytometry appears to be a relevant and accurate indicator of heavy metal toxicity.