Environ. Sci. Technol. 2000, 34, 3907-3913
Permeability Changes in Model and Phytoplankton Membranes in the Presence of Aquatic Humic Substances BERNARD VIGNEAULT,† ALINE PERCOT,‡ MICHEL LAFLEUR,‡ AND P E T E R G . C . C A M P B E L L * ,† INRS-Eau, Universite´ du Que´bec, Case Postale 7500, 2800 Rue Einstein, Sainte-Foy, Que´bec, Canada G1V 4C7, and Groupe de Recherche en Transport Membranaire and De´partement de Chimie, Universite´ de Montre´al, Case Postale 6128, Succursale Centre-Ville, Montre´al, Que´bec, Canada H3C 3J7
Aquatic humic and fulvic acids can increase the permeability of biological membranes to lipophilic solutes. In in vivo experiments, passive diffusion of fluorescein diacetate (FDA) into the green alga Selenastrum capricornutum increased in the presence of Suwannee River humic and fulvic acids at pH 5 (humic > fulvic) but not at pH 7. The observation of enhanced diffusion at the lower pH is consistent with adsorption measurements, which showed that the association of humic and fulvic acids with the algal surface was greater at pH 5 than at pH 7. Permeability experiments were also performed on model membranes to investigate the interaction of these humic substances with membrane lipids. In these in vitro experiments, we followed leakage of the fluorescent probe sulforhodamine-B (SRB) that had been encapsulated within 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles; this model phospholipid is representative of those found in the plasmalemma of green algae. Release of SRB from the vesicles was markedly accelerated in the presence of Suwannee River humic and fulvic acids (humic > fulvic); for the humic acid, lowering the pH from 7.6 to 5.7 enhanced this surfactant-like effect. The demonstration that humic substances can alter the permeability of phytoplankton and model membranes at natural concentrations and pH values has potential implications for the uptake and regulation of toxic and essential solutes by the phytoplankton community.
Introduction In natural waters, fulvic and humic acids compose 50-80% of the organic matter, DOM (1). These humic substances have a surfactant-like structure, containing both hydrophilic domains, such as carboxylic and phenolic groups, and hydrophobic domains, such as aliphatic and aromatic moieties. Because of this amphiphilic character, humic substances behave as natural surfactants and can adsorb on a large number of natural surfaces, including biological membranes. * Corresponding author phone: (418)654-2538; fax: (418)654-2600; e-mail:
[email protected]. † Universite ´ du Que´bec. ‡ Universite ´ de Montre´al. 10.1021/es001087r CCC: $19.00 Published on Web 08/08/2000
2000 American Chemical Society
Adsorption of humic substances on biological surfaces has been demonstrated directly, by loss of DOM from solution, and indirectly, by following changes in the electrophoretic mobility of individual cells in the presence or absence of DOM. Biological surfaces that have been studied include phytoplankton (2-5), isolated fish gill cells (2), bacteria (6), and fungi (7). This diversity suggests that the adsorption of humic substances on biological membranes is a general process. Their association with biological membranes and their surface-active properties raise the possibility that humic substances may change the structure and fluidity of the lipid bilayer to such an extent that cell membrane permeability could be affected (8). In soil science, the ability of humic substances to increase plant membrane permeability has been recognized for some time. For example, several authors concluded, on the basis of increased uptake of nutrients by plants, that humic substances increase cell membrane permeability (9-11). Samson and Visser (12) demonstrated an increase in potato cell membrane permeability toward K+ in the presence of peat humic acid. Visser (13) also studied the physiological effects of humic substances on soil bacteria and concluded, on the basis of the similarity of these effects and those caused by synthetic surfactants, that the cell membrane was a prime target for the physiological action of humic substances. Given this evidence from the soil literature, it seems plausible that aquatic humic substances could have similar effects on phytoplankton. Experimental support for such effects is however scarce. Zientara (14) studied the transmembrane potential in internodal cells of the macroalga Nitellopsis obtusa and proposed that the primary effect of humic substances was on plasmalemma permeability, especially on the electrogenic proton pump. Effects on passive diffusion are also possible; for example, Parent et al. (5) demonstrated that fulvic acid increased the uptake rate of sorbitol by the unicellular green alga Chlorella pyrenoidosa. Surfactant-like effects of aquatic humic substances on phytoplankton membrane permeability could involve interaction with the lipid fraction and/or with the proteins responsible for facilitated uptake of metabolites. In the present study, we demonstrate increased passive diffusion through intact phytoplankton membranes and model phospholipid membranes in the presence of aquatic humic substances, and we investigate the effects of pH, the nature of the humic material (humic versus fulvic acid), and its origin. Model phospholipids were selected to be representative of green algae, and membrane composition was varied. The effect on membrane permeability was related to the extent of adsorption of the humic and fulvic acids to the phytoplankton cells.
Experimental Section Two experimental approaches, both using fluorescent probes, were employed to demonstrate the effects of aquatic humic substances on passive diffusion through biological membranes. As a first step in this investigation, we used the freshwater green alga Selenastrum capricornutum and measured the diffusion of a probe into the phytoplankton cells in vivo by following the formation of a fluorescent product by the intracellular enzymatic hydrolysis of the probe. In a second set of experiments, permeability measurements were performed on phospholipid vesicles as a model for phytoplankton cell membranes. The leakage of a fluorescent probe from these liposomes was measured. This experimental system enabled us to test the influence of membrane VOL. 34, NO. 18, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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composition and demonstrate effects on phospholipid bilayers with no interference from protein or other components. Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3phosphatidylglycerol (POPG) were purchased from Avanti Polar Lipids (Birmingham, AL). The probes sulforhodamine B (SRB) and fluorescein diacetate (FDA) were obtained from Molecular Probes Inc. (Eugene, OR). Standard Suwannee River humic and fulvic acids from the International Humic Substances Society (IHSS, St. Paul, MN) were selected as representative and well-characterized aquatic humic substances (15). Finally, soil-extracted Laurentian and Armadale fulvic acids from Ecolinc Inc. (Roxboro, PQ) were chosen to compare with the standard aquatic fulvic acid. The organic carbon contents of the humic substances were obtained from IHSS for Suwannee River humic and fulvic acids, from Wang et al. (16) for Laurentian fulvic acid, and from Ghosh and Schnitzer (17) for Armadale fulvic acid. All humic substances used were lyophilized solids easily dissolved in aqueous solution at the experimental pH. Electrophoretic Mobility (EPM). S. capricornutum, chosen as a test species because of its wide use in aquatic toxicology, was obtained from the University of Toronto Culture Collection (UTCC-37). To assess the role of the surface charge of S. capricornutum cells in the adsorption of humic substances, the electrophoretic mobility of these cells was measured at different pH values. Cells were grown axenically in Fraquil media (18), buffered at pH 7 with 0.01 M HEPES (N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid, HEPES). The ionic strength of the culture and exposure media was increased to 0.01 M with NaNO3 for the electrophoretic mobility measurements and for the subsequent experiments regarding cell membrane permeability (see below). Cells were grown at constant temperature (20 °C) and luminosity (100-115 µEinstein m-2 s-1) with gyratory shaking (50 rpm). Exposure media consisted of fresh culture medium, without added trace metals and vitamins. Cells were harvested after 3 days, in late exponential growth phase, by gentle filtration onto 0.4-µm Poretics polycarbonate membrane filters (Osmonics Inc, Livermore, CA) and rinsed 3 times with 10 mL of exposure medium. Minimal vacuum was applied (
