Environ. Sci. Technol. 2007, 41, 4264-4270
Ni Uptake by a Green Alga. 2. Validation of Equilibrium Models for Competition Effects ISABELLE A. M. WORMS† AND K E V I N J . W I L K I N S O N * ,‡ CABE (Analytical and Biophysical Environmental Chemistry), University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland, and Department of Chemistry, University of Montreal, P.O. Box 6128, Succursale Centre-ville, Montreal, Canada H3C 3J7
It is generally admitted that the presence of major cations and H+ can attenuate trace metal uptake. Recent models such as the biotic ligand model (BLM) aim to quantify and predict this effect by determining stability constants for each of the major competitors for any given interaction of a trace metal with a biological organism. In this study, short-term Ni internalization fluxes (Jint) were used to quantitatively assess the binding of H+, Mg2+, Ca2+ (KH-Rs, KMg-Rs, KCa-Rs), and trace metals to transport sites (Rs) leading to Ni biouptake by Chlamydomonas reinhardtii. H+ and Mg2+ are shown to compete directly for the entry of Ni with affinity constants that are of the same order of magnitude (KMg-Rs ) 105.1 M-1; KH-Rs ) 105.3 M-1) as that measured for Ni (KNi-Rs ) 105.1 M-1). The Ni internalization fluxes were also strongly linked to the Mg cell status. In contrast, the role of Ca2+ could not be explained by a simple competitive equilibrium with the Ni transport sites. Aluminum (KAl-Rs ) 108 M-1), Zn (KZn-Rs ) 106.5 M-1), and Cu (KCu-Rs ) 106.6 M-1) were all shown to compete strongly with Ni for uptake. In addition to the determination of uptake constants, these studies provide insight into the transport mechanisms of Ni by the green alga, C. reinhardtii.
As a consequence, water hardness is expected (and often observed) to attenuate the biological effects of trace cations (6). Calcium and magnesium are found in relatively high concentrations in freshwaters. They are essential to the maintenance of cellular integrity and for cell signaling and reactivity. In several organisms, nickel has been shown to enter biological cells via the magnesium transporters (7). For Daphnia magna, waterborne Ni was shown to interfere with Mg transport, leading to both acute and chronic toxicity, but having little impact on Ca metabolism (8). In contrast, a 10-fold increase in the concentration of Ca2+ led to a 10fold increase of the LC50 (lethal concentration for 50%) in fathead minnows, and accumulated gill Ni appeared to be a good predictor of nickel toxicity (9). While the presence of hardness ions is generally accepted to compete with cellular binding sites for the metal of interest (10, 11), the uptake process is complex and the presence of secondary cations can also have other less predictable effects on metal uptake. For example, Cu actually increased Cd uptake by Rhodospirillum and various green alga (12-14) and increased Ni, Pb, and Zn uptake by Chlorella kesslerii (15, 16). As seen for metals, H+ has been shown to attenuate the uptake of trace metals for pH ranging from 5 to 8 (17-20). On the other hand, pH effects may also result in an increased metal biouptake due to the protonation of ligands in solution (increased concentration of free ion); shifts in the chemical speciation of the metal ion in solution (e.g., hydroxo or carbonato complexes) or to modifications of the overall surface potential of the organism (15). No current consensus appears to exist on the role of the hydroxo or carbonato complexes on Ni bioavailability (21). The objective of this paper is to quantify the effects of Ca2+, Mg2+, pH, and trace metals on short-term Ni biouptake by Chlamydomonas reinhardtii. In order to relate Ni chemistry to its bioavailability, internalization fluxes (Jint, mol cm-2 s-1) are determined. Our hypothesis is that the role of competitors on Ni biouptake fluxes can be quantitatively predicted by an equilibrium-based model (e.g., BLM). Validation of the constants is performed in a complex synthetic media and the possible role of Ca and Mg homeostasis on Ni biouptake is discussed.
Introduction
Theory
Metal bioavailability is often defined as the capacity of a metal species to be taken up by an organism, generally implying that the metal must cross a plasma membrane. Toxic metals are generally thought to enter cells by ionic or molecular mimicry (1, 2), most often by employing channels or carriers involved in ionoregulation. The Biotic Ligand Model (BLM, (3)) attempts to quantitatively evaluate the effects of competition by assigning a stability constant to each of the reactions between a metal and its corresponding uptake or toxicologically sensitive sites. Because the BLM assumes that the metal species in solution and those adsorbed to the biological surface are at thermodynamic equilibrium, i.e., biological internalization is the rate-limiting step of the overall biouptake process (4, 5), competition is necessarily antagonistic, i.e., in the presence of a second ion, lower (or unchanged) uptake or biological effects should be observed.
The BLM employs equilibrium constants to predict the magnitude of the competition among similarly charged ions for sensitive sites at the surface of the organism. While constants are often determined from biological effects data (22), they can also be determined from a plot of the internalization flux (Jint, mol cm-2 s-1) against [Ni2+] via the Michaelis-Menten equation (eq 1)
* Corresponding author phone: (514) 343 6741; fax: (514) 343 7586; e-mail:
[email protected]. † University of Geneva. ‡ University of Montreal. 4264
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 12, 2007
Jint )
Jmax [Ni2+] KM + [Ni2+]
(1)
where Jmax is the Ni uptake flux at saturation, KM is the Michaelis-Menten constant, and [Ni2+] is the equilibrium concentration of Ni2+ in solution. KM indicates the concentration at which the transport sites are half saturated. Under the equilibrium assumption, the reciprocal of the MichaelisMenten constant provides a value of the affinity constant describing the interaction of the metal with the Ni uptake sites, KNi-Rs (4). The effect of competing cations can be 10.1021/es0630341 CCC: $37.00
2007 American Chemical Society Published on Web 05/16/2007
described analytically using similar equilibrium considerations (eq 2, ref (22)).
Jint )
Jmax [Ni2+]KNi-Rs [Ni2+]KNi-Rs + [Cn+]KC-Rs + 1
(2)
where KC-Rs is the affinity constant for the competition of Ni2+ for a given uptake site and [Cn+] is its concentration. It follows that KC-Rs can be determined from the value of the Michaelis-Menten constant for the competitor, K′M, from experiments where [Ni2+] is varied and the concentration of the competitor is kept constant (eq 3).
KC-Rs )
(
KM′ -1 KM [Cn+]
)
(3)
Finally, at low metal concentrations, [Ni2+] , KM (eq 1) and Jint should be directly proportional to the concentration of free ion. In such cases, the effects of competitors can also be evaluated by following Ni2+ permeability through the biological membrane (PNi, eq 4). In the absence of competitive effects, PNi should be constant.
PNi ) Jint/[Ni2+]
FIGURE 1. (a) Effect of calcium, magnesium, and pH on internalized Ni as a function of time for 2 × 10-7 M [Ni]: without competitors (b); or in the presence of 10-5 M Ca2+ (9); 10-5 M Mg2+ (2); or 10-5 M H+, i.e., pH 5.0 (1). Error bars indicate standard deviations and are given when larger than the symbol size.
(4)
Materials and Methods Culture Conditions. Wild type 2137 mt+ Chlamydomonas reinhardtii were obtained from Professor M. GoldschmidtClermont (University of Geneva). Algae were transferred from a week-old tris-acetate-phosphate (TAP) (23) agar plate into a (4×) diluted TAP solution (I ) 10-2 M) (24). Cells were grown to a density of (2-3) × 106 cells mL-1 for 4 days using a 12 h light (50 µmol photons m-2 s-1)/12 h dark regime at 20 °C and 100 rpm agitation (Multitron incubator, Infors). Cells were then diluted in fresh media to give (1-2) × 105 cells mL-1. After two more days of growth, corresponding to the late logarithmic growth phase ((1-2) × 106 cells mL-1), cells were harvested by low-speed centrifugation (1000g, 3 min) in 50 mL sterile (polypropylene) centrifuge tubes, resuspended in 10-2 M NaMES (Sigma) buffer at pH 6 and centrifuged again (2×). Finally, (2-5) × 105 cells mL-1, corresponding to 0.8-1.2 cm2 mL-1, were transferred to an experimental media. All culture media and experimental solutions were sterilized (autoclave or 0.2 µm filtration) prior to use. All manipulations of algal cultures were performed under laminar flow (Heraus, Germany) and bottle borders were flame sterilized. In some experiments designed to get better insight into the effects of Ca and Mg homeostasis on Ni biouptake, Ca and Mg concentrations (0 to 10-4 M) were manipulated in the diluted TAP growth media. Experimental Conditions. Experimental solutions contained 10-2 M NaMES (Sigma) and the appropriate Ni2+ (2.5 × 10-8 to 10-3 M) and competitor ([Ca2+] 0-10-3 M; [Mg2+] 0-10-3 M; pH 4.5-8; trace cation 1-5 × 10-7 M) concentrations. In the pH experiments, algae were exposed to 2.5 × 10-7 M Ni2+, buffered at pH 4.5 (5 × 10-3 M acetate or succinate), 5 (5 × 10-3 M succinate or 10-2 M NaMES), 5.5 (10-2 M NaMES), 6.5 (10-2 M NaMES), 7 (10-2 M HEPES), and 8 (10-2 M EPPS or NaHCO3). The pH was adjusted using concentrated solutions of HNO3 (suprapur, Baker) or NaOH (5 M, Sigma), and ionic strength was adjusted to 10-2 M by addition of NaNO3. All polymerware was acid washed (minimum 24 h in 0.1% HNO3), rinsed 3× with deionized water and 3× in Milli-Q water (R > 18 MΩ cm; TOC < 2 µg L-1). Determination of Internalization Fluxes. Metal internalization fluxes were determined in short-term experiments
designed to minimize Ni depletion (
