Environ. Sci. Technol. 2008, 42, 2189–2194
Avoidance of Aluminum Toxicity in Freshwater Snails Involves Intracellular Silicon-Aluminum Biointeraction KEITH N. WHITE,† ABRAHAM I. EJIM,† RACHEL C. WALTON,† ANDREW P. BROWN,‡ RAVIN JUGDAOHSINGH,§ JONATHAN J. POWELL,§ AND C A T H E R I N E R . M C C R O H A N †,* Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom, and Institute for Materials Research, University of Leeds, Leeds LS2 9JT, United Kingdom, and MRC Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge CB1 9NL, United Kingdom
Received November 13, 2007. Revised manuscript received December 17, 2007. Accepted December 17, 2007.
Silicon (Si) ameliorates aluminum (Al) toxicity to a range of organisms, but in almost all cases this is due to ex vivo Si-Al interactions forming inert hydroxyaluminosilicates (HAS). We hypothesized a Si-specific intracellular mechanism for Al detoxification in aquatic snails, involving regulation of orthosilicic acid [Si(OH)4]. However, the possibility of ex vivo formation and uptake of soluble HAS could not be ruled out. Here we provide unequivocal evidence for Si-Al interaction in vivo, including their intracellular colocalization. In snails preloaded with Si(OH)4, behavioral toxicity in response to subsequent exposure to Al was abolished. Similarly, recovery from Al-induced toxicity was faster when Si(OH)4 was provided, together with rapid loss of Al from the major detoxificatory organ (digestive gland). Temporal separation of Al and Si exposure excluded the possibility of their interaction ex vivo. Elemental mapping using analytical transmission electron microscopy revealed nanometre-scale colocalization of Si and Al within excretory granules in the digestive gland, consistent with recruitment of Si(OH)4, followed by high-affinity Al binding to form particles similar to allophane, an amorphous HAS. Given the environmental abundance of both elements, we anticipate this to be a widespread phenomenon, providing a cellular defense against the profoundly toxic Al(III) ion.
Introduction The utilization of Si in biological systems is well-documented, including a structural role in microorganisms and plants (1), increased resistance to stress in plants (2) and beneficial effects on connective tissue and bone in mammals (3). However, despite its abundance in the biosphere, no intracellular pathway involving Si has been reported. It has been * Corresponding author address: Faculty of Life Sciences, University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester M13 9PT, U.K.; tel: 44 (0)161 275 5375; fax: 44 (0)161 275 3938; e-mail:
[email protected]. † University of Manchester. ‡ University of Leeds. § Elsie Widdowson Laboratory. 10.1021/es7028608 CCC: $40.75
Published on Web 02/15/2008
2008 American Chemical Society
proposed that the essentiality of Si in higher organisms is related to its influence on the bioavailability of metals, both essential and nonessential (4, 5). In particular, Si appears to limit the availability and toxicity of aluminum (Al). Aluminum is ubiquitous in the environment and, if it gains access to intracellular compartments, is highly toxic - for example, disrupting signaling pathways (e.g., calcium homeostasis) (6). Silicic acid restricts the uptake of Al from the environment in plants (7), fish (8) and humans (9), and this can be readily explained by the exogenous formation of hydroxyaluminosilicates (HAS) with low bioavailability (9, 10). However, some aquatic invertebrates accumulate substantial amounts of Al from the natural environment (11), including at circumneutral pH (12). Concentration factors in their tissues can be very high; for example, up to 104 times environmental levels in the freshwater snail Lymnaea stagnalis (12). Despite this, Al-induced toxicity in the snail is manifest only as sublethal behavioral depression that normalizes over time, at least over the short-term, even with continued Al(III) exposure (13), suggesting an effective detoxification mechanism. We have shown that Al preferentially accumulates in cells of the snail’s detoxificatory organ (digestive gland) where it is localized to excretory granules of lysosomal origin (14). This accumulated Al is accompanied by coaccumulation of Si, even when no additional exogenous Si is provided (14). Increased Si in digestive gland cells is a specific response to Al since it does not occur in response to loading with the heavy metals Cd or Zn (14). This led us to hypothesize the existence of a Si-specific mechanism for the in vivo detoxification of Al which, intracellularly, may mirror much betterdescribed ex vivo Si-Al interactions (10). However, even under experimental conditions the aqueous environment cannot be depleted of all traces of Si and thus, in previous work, we could not rule out the possibility of ex vivo Si-Al interactions, leading to uptake of preformed, nontoxic HAS. Here we sought to test our hypothesis in a different fashion, by presenting snails with Al and Si consecutively rather than concurrently; thus, differences from control (i.e., response to Al(III) with no added Si) were solely attributed to pre or postconditioning with Si(OH)4. In addition we have used nanometer-resolution analytical transmission electron microscopy to examine the nature of the Si-Al interaction in the lysosomal granules of digestive gland cells. Using TEM with energy dispersive X-ray spectrometry (EDX) and electron energy loss spectrometry (EELS) to undertake elemental analysis, we sought to localize Si and Al in situ within digestive gland lysosomal granules in snails exposed to Al.
Materials and Methods Animals and Experimental Conditions. Mature Lymnaea stagnalis (2.5–3.5 cm shell length; Blades Biologicals, UK), collected from the wild, were acclimatized to aerated simulated, defined pond water (SDPW: 222 mg L-1 CaCl2, 9.6 mg L-1 MgSO4, 4 mg L-1 KHCO3, 5.1 mg L-1 KNO3, 58 mg L-1 NaHCO3, adjusted daily to pH 7.3) at 12 °C and on a 12:12 h light:dark regime for at least two weeks prior to the start of experiments. All solutions were prepared using ultrahigh purity (UHP) water (Elga water purifier, High Wycombe U.K.) and high-purity chemicals (Merck or Aldrich). Polypropylene plasticware used throughout was acid washed and rinsed before use. During exposure experiments in 20 L tanks, SDPW was renewed every two days. Orthosilicic acid (7 mg L-1 in SDPW) was prepared from dilution of a 7 M stock solution of basic sodium silicate and neutralization to pH 7.3, and then left to equilibrate for 48 h; this ensured that silicic acid was in the ubiquitous monomeric form. Aluminum (500 µg VOL. 42, NO. 6, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Pre-exposure of Lymnaea stagnalis to orthosilicic acid inhibits behavioral toxicity induced by Al. Snails were preloaded with 7 mg L-1 orthosilicic acid for 25 days, followed by exposure to 500 µg L-1 Al for 20 days in the absence of added Si (b). Control groups of snails were exposed to Al with no Si pre-exposure ((), and to neither Si nor Al (4, baseline). (A) Behavioral state score (median and interquartile range. n ) 6 for each point; a, p < 0.001; b, p < 0.025; c, p < 0.02). (B) Si concentration in the digestive gland (DG, mean ( SEM, n ) 5; a, p ) 0.05; b, p < 0.05). (C) Si concentration in the remaining soft tissue (RT, i.e., minus digestive gland). (D) Al concentration in the digestive gland (mean ( SEM, n ) 5; a, p < 0.002; b, p < 0.01; c, p < 0.04). (a) Si + Al vs baseline control; (b) Si + Al vs Al control; (c) Al control vs baseline control. L-1) was added as the nitrate from acidified (pH
