A mechanism for acute aluminium toxicity in fish

J. theor. Biol. (1991) 151, 417-428

A Mechanism for Acute Aluminium Toxicity in Fish C. EXLEYt, J. S. CHAPPELL:~ AND J. D. BIRCHALL§

t Institute of Aquaculture, University of Stifling, Stirling FK 9 4LA, Scotland, ~ Bioengineering, University of Washington, Seattle, WA 98195, U.S.A. and § ICI Chemicals and Polymers Ltd, P.O. Box 11, The Heath, Runcorn, Cheshire WA7 4QE, U.K. (Received on 29 November 1990, Accepted on 13 February 1991) Aluminium is acutely toxic to fish in acid waters. The gill is the principal target organ and death is due to a combination of ionoregulatory, osmoregulatory and respiratory dysfunction. The toxic mechanism has hitherto received little direct consideration and is unknown. In this paper the mechanism of acute aluminium toxicity is approached from a chemical perspective. Symptomatic evidence of toxicity is taken from the literature and combined with our own research to elucidate a biochemically sound model to describe a possible mechanism of acute aluminium toxicity in fish. The proposed model delineates the chemical conditions immediately adjacent to the gill surface and emphasizes their importance in aluminium's toxic mode of action. The mechanism is shown to be bipartite. Aluminium binding to functional groups both apically located at the gill surface and intracellularly located within lamellar epithelial cells disrupts the barrier properties of the gill epithelium. The concomitant iono- and osmoregulatory dysfunction results in accelerated cell necrosis, sloughing and death of the fish. The mechanism of epithelial cell death is proposed as a general mechanism of aluminium-induced accelerated cell death.

Introduction

A significant effect of the acidification of surface waters has been the decline of natural fish populations (Schofield, 1976). Acidity per se has only rarely been the pre-eminent factor in such decline and aluminium is now recognized as the principal toxicant of acid waters (Dickson, 1978). Aluminium is the third most abundant element in the earth's crust. Complexed in mineral ores and soil strata it is precluded from biology. The acidification of catchment geology, often due to acidic deposition, leaches aluminium from its edaphic origin and concentrates it in the aquatic environments of pore and surface waters (Cronan & Schofield, 1979). The solubility and speciation of aqueous aluminium together determine its bioavailability (see Exley & Phillips, 1988). In freshwater in the absence of naturally occurring ligators of aluminium including fluoride, silicic acid, humic and fulvic acids, aluminium solubility is controlled by both a m o r p h o u s and crystalline aluminium hydroxide (see Fig. 1). In acid water, ( p H < 6 . 0 ) the concentration of soluble aluminium may exceed 20 txM 1-1 and is acutely toxic to fish (see Exley & Phillips, 1988). Acute toxicity is ameliorated in the presence of fluoride (Driscoll et al., 1980) calcium (Brown, 1983) and sodium (Dietrich et al., 1989) and abolished through the complexation of aluminium by 417 0022-5193/91/150417+ 12 $03.00/0 ~ 1991 Academic Press Limited

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citrate (Driscoll et al., 1980) or silicic acid (Birchall et al., 1989). Surface water pH is critical and toxicity is most severe c. pH 5.0 (Baker & Schofield, 1982). The term "acute" aluminium toxicity is used when death occurs within a short time interval, (usually less than 96 hr) subsequent to either continuous or episodic exposure to an aluminium challenge. Moribund fish are typically characterized by macroscopic and/or microscopic damage to gill tissue. The symptoms might include; (1) ionoregulatory disturbances often measured as a net loss of plasma electrolytes (e.g. NaCI and Ca 2+) and a net gain of plasma protons and aluminium, (ii) respiratory dysfunction characterized by a plasma acidosis and concomitant hypoxia and hypercapnia, and (iii) osmoregulatory breakdown resulting in a net flux of water into the fish.

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pH FiG. 1. Negative logarithm of the total molar concentration of aluminium allowed by amorphous and crystalline aluminium hydroxide. A!3+ is the dominant soluble species below pH 5.0 and AI(OH)2 predominates above pH 6.2, the minimum solubility point for both curves (Martin, 1986).

Additional observations associated with acute aluminium toxicity include the excessive production of mucous (Muniz & Leivestad, 1980) the inhibition of the activities of the enzymes carbonic anhydrase and Na-K-ATPase (Staurnes et aL, 1984) and both the apical and intracellular accumulation of aluminium at the gill epithelium (Youson & Neville, 1987; Exley, 1989). Rapid necrosis and sloughing of epithelial cells often precludes this last observation (Exley, 1989). The relative contributions of these dysfunctions to an acute response depends on the water chemistry (see Table 1). Research to-date implicates very strongly the rapid etflux of plasma NaCI across the gill as a major contributor to the death of fish living in ion-deficient waters of catchments susceptible to acidification.

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Acute aluminium toxicity in fish is clearly the result of the breakdown of the barrier properties of the gill epithelium. How then might aluminium instigate such disequilibrium across the gill?

Present Theories of Acute Aluminium Toxicity The symptoms of acute aluminium toxicity offer some insight into aluminium's toxic mode of action. The differentiation, however, between those effects that are unique to an aluminium challenge and those that are the consequence of secondary stress response is very fine and has confused previous attempts to describe the mechanism of aluminium toxicity in fish. Two theories, not mutually exclusive, presently prevail in the literature; the physical effect (Muniz & Leivestad, 1980) and the surface binding theory (Sadler & Lynam, 1987; Neville & Campbell, 1988). The former describes the inflammation of gill epithelial cells caused by the precipitation of aluminium hydroxide at the gill surface. This condition is exacerbated by the excessive production of mucous at the gill surface. In the surface binding theory aluminium at the gill surface binds to functional groups at the gill epithelium. It was initially postulated that the products of aluminium hydrolysis, principally AI(H20) 3+, AI(H20)5(OH) 2+, AI(H20)4(OH)~, AI(H20)3(OH) 3 and AI(HzO)2(OH)~, competed for binding to groups integral to membrane structure and function (see e.g. Sadler & Lynam, 1987). Toxicity was observed to be most acute when AI(H20)5 (OH) 2+ was calculated to be the dominant species in solution and thereafter this species was the suggested toxic moeity. This chemically naive model has since been revised (Neville & Campbell, 1988) and the free ion toxicity 3+ • o model (see Pagenkopf, 1983) attributes toxicity to Al(aq) binding at the apical surface of the gill lamellar epithelium. Together, these models corroborate many of the observations of acute aluminium toxicity in fish. They do not however account for; the lack of toxic response to an extended exposure of fish to low aluminium concentrations (<5 ~M 1-~) in acid water (Exley, 1989); the irreversibility of an exposure of fish to an acutely toxic concentration of aluminium on their return to optimal water conditions (Potts et al., 1989); and the unlikelihood that apical, (extracellular) aluminium interactions alone could account for the severe disruption to gill barrier properties typical of acute aluminium toxicity. Any new model must take these additional and critical factors into account.

New Concepts in Toxicity The consensus of research to-date recognizes the gill as the principal target organ in acute aluminium toxicity. Aluminium binding to specific functional groups at the gill epithelium is a prerequisite to acute toxicity and is confirmed in studies using citrate (Driscoll et al., 1980) and silicic acid (Birchall et al., 1989) to prevent aluminium binding and hence toxicity (see Exley, 1989).

TABLE 1

(2) Aluminium, 1-10 I.t.M 1-I. Ca 2+, >50 I.I.M1-1, (<250 t~M I-I). Na +, <4mM I-l. No competing anions

plasma electrolytes

(a) pH 4-5-
(c) pH 5.6-pH6-0

(b) pH 5-0-pH 5-5

(i) Reduction in the inhibition of active uptake of Na+CI (ii) Some reduction in efflux of plasma electrolytes (iii) Moderate/low respiratory dysfunction (iv) Some plasma acidosis

(iii) Negligible respiratory dysfunction (iv) Insignificant plasma acidosis (i) Moderate/tow inhibition of active Na+C1- uptake (ii) Efflux of plasma electrolytes is potentiated (iii) Negligible respiratory dysfunction (iv) Insignificant plasma acidosis (i) No effect on active Na+CI uptake (ii) Low/insignificant efflux of plasma electrolytes

(i) Inhibition of the active uptake of Na+CI - is potentiated (ii) Moderate/low efflux of

(1) Aluminium, 1-10 ~.M I-~. Ca 2+ and/or Na +, <25 IZM 1-~. Negligible competing anions

(a) pH 4.5-
Toxic symptoms

General water chemistry

Toxicity

(a) Some overall reduction in toxicity from elevated calcium. Probably still acute at higher aluminium. Ionoregulatory dysfunction dominates

(c) Low, rarely acute

(b) High, toxicity is potentiated in this pH range. lonoregulatory dysfunction dominates

(a) Moderate, though potentially acute, lonoregulatory dysfunction dominates. (Acute toxicity infers death within 96 hr of exposure)

Summary of the major aluminium-induced symptoms of acute toxicity

m X tin ,< r~

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(i) Significant ionoregulatory and respiratory dysfunction including an acute plasma acidosis (ii) Toxic symptoms as above

(a) Ca 2÷ a n d / o r Na ÷ < 25 p.M 1-t. pH c. 5"0

(4) Aluminium, >20 p.M 1-1. No competing anions

(c) Condition invariably still acute due to respiratory dysfunction

(b) Acutely toxic. No mitigatory effect of Ca 2+ a n d / o r Na +

(a) Acutely toxic conditions Respiratory dysfunction dominates

(a) The permeability of the gill to Na+CI - is unaffected. No evidence of respiratory dysfunction in this aluminium range

(c) Toxicity, rarely acute. Respiratory dysfunction dominates

(b) Significant reduction in overall toxicity, though often still acture at high aluminium. Cmntributions from both respiratory and ionoregulatory dysfunction to acute toxicity

Information adapted from: Brown (1983), Dietrich et al. (1989), Baker & Schofield (1982), Sadler & Lynam (1987), Potts et al. (1989), Dalziel et al. (1987), Wood et al. (1988), Neville (1985), Booth et al. (1988), Walker et al. (1988), Malte (1986), Jensen & Weber (1987), Malte & Weber (1988), Witters (1986), Battram (1988).

(b) C a 2 + > 5 . 0 p . M l -I, Na + < 4 mM 1-t pH c. 5.0 (c) Ca 2+ c. 2.5 mM 1-t (iii) Significant reduction in ionoregulatory effects though a n d / o r Na + c. 150 mM I-I respiratory dysfunction and pH c. 5"0 plasma acidosis still acute

(i) No inhibition of active Na÷CI - uptake (ii) No promotion of efflux of plasma electrolytes

(a) pH 5-0-pH 5.5

(i) Low/insignificant inhibition of active Na÷CI - uptake (ii) Significant reduction in elilux of plasma electrolytes (iii) Moderate/high respiratory dysfunction (iv) Moderate plasma acidosis (i) Insignificant inhibition of Na+CI active uptake (ii) Insignificant emux of plasma electrolytes (iii) High respiratory dysfunction (iv) Severe plasma acidosis

(3) Aluminium, 1-10 I~M I-I. Ca z+ c. 2.5 mM 1-1 a n d / o r Na + c. 150 mM 1-I. No competing anions

(c) pH 5-6-pH 6-0

(b) pH 5.0-pH 5-5

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Aluminium binds via the trivalent cation, Al(H20)3~-aql, the only oxidation state available to biology (Martin, 1986). The nature of this ion, in particular the high charge to radius ratio, (c. 6e/~), dictates binding via small electronegative species such as oxygen (Pauling, 1960). Oxygen-based functional groups, for example, phosphates, carboxylates, carbonyls and hydroxyls are ubiquitous in biology and the fish gill is no exception to this. The gill epithelium constitutes a phospholipid bilayer partially and fully bisected by membrane inclusions such as transport proteins (Bolis et al., 1984). The pre-eminent phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and sphyngomielen are charged and both the choline and serine derivatives have been shown to bind aluminium in vitro (Shi & Haug, 1988). Aluminium binding to membrane phospholipids reduces membrane fluidity and alters membrane barrier properties (Viersta & Haug, 1978). The steriochemistry of membrane transport proteins is orientated by metal co-factors (Wallace, 1989) and such proteins thus offer potential binding sites for aluminium. In addition to the gill epithelium, the overlying mucous is polyanionic (Kirshcner, 1978) and may act as a sink for alumirlium. Aluminium binding close to the surface of the gill is thus dictated by a number of competing chemical equilibria. Al--(bulk water ligand x) ~ Al--(gill surface ligand y) X--aquo, fluoride, silicic acid citrate, hydroxyl

Y--carboxylates, phosphates carbonyls etc

In addition aluminium competes with and substitutes for a number of cations of which Ca 2+, Na ÷, Mg 2+, and H + may be important at the gill surface. The stability of aluminium binding is not only dependent on the relative concentrations of competing cations and anions but also on the intrinsic binding strength of aluminium to gill and mucous ligands. This binding might be weak electrostatic or strong co-ordination. The intrinsic binding strength is influenced by the orientation of the ligating groups and is potentiated when geometric orientation offers multiple co-ordination to a single aluminium ion (Birchall & Chappell, 1988a). Aluminium will thus bind to functional groups at the gill surface. Stable co-ordination may result in toxicity perhaps as a consequence of aluminiums' notoriously slow ligand exchange kinetics (Womack & Colowick, 1979) an order of magnitude slower than for Na, Ca or Mg, and/or aluminium altering the steriochemistry of accepting membrane biomolecules (Wallace, 1989). Aluminium binding to functional groups that might typically be found associated with gill, (see earlier) is predicted to be potentiated in the pH range 6-7 where competition from both hydroxyl groups and protons is reduced. Contrary to this, acute aluminium toxicity in fish is most severe in the pH range 4.5-5.5 and is rarely documented above pH 6.0 (Exley, 1989). This discrepancy is resolved in acid waters where the pH immediately adjacent the gill lamellar epithelium is less acidic than the bulk water, The solution phase closest to the gill epithelium, (the gill boundary layer, gbl) is both chemically and physically dissimilar to the bulk water, (bw), The chemistry of this phase is determined by differences in ion flux rates between transmembranal transport and simple diffusion into the bw. The gbl pH is determined by the hydration

ACUTE ALUMINIUM TOXICITY IN FISH

423

equilibria of expiration products, principally CO2 and NH3, and diffusion into and out of the boundary layer of acid and base equivalents from both the bw and intracellular sources. The passive efflux of physically dissolved CO2, the predominant expiration product from the blood, contributes acidity to the gbl: CO2 + HzO ~ caH2CO3 ~ H + + HCO~ pka at 15°C c. 6.08 (Boutilier et al., 1985), ca--carbonic anhydrase. Hydration of CO2 at the gill surface is slow, tl/2 c. 60 sec, and only 5-15% of the total expired CO2 is initially hydrated (Perry, 1986). The reaction continues more rapidly in the mucous where an excess of the enzyme carbonic anhydrase catalyses the hydration towards completion (Wright et al., 1986). CO2-derived acidity is neutralized in the gbl by NH3 freely diffusing across the gill epithelium from the blood at concentrations equivalent to 10-20% of the mole fraction of total expired CO2 (Wright & Randall, 1987). NH3 + H + ~ NH~- pka at 15°C c. 9.58 (Thurston et al., 1979). The result, at circumneutral bw pH, of approximately equimolar concentrations and NH3 in the gbl is a gbl pH in the range 7.0-8.0. A buffering potential

of H2CO3

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i

4

5

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5

__1.__t 6 7 pH of bulk woler

~.L...8

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FIG. 2. Predicted relationship between the pH of the bulk water (bw) and the corresponding pH immediately adjacent the gill lamellar epithelium (gbl). X--Gill boundary layer pH regime in which aluminium is predicted to bind successfully to gill-associated ligands in competition with both protons and hydroxide groups. Y--Bulk water pH regime in which aluminium is documented to be acutely toxic.

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for the gbl is also predicted (see Fig. 2). The stability of the microenvironment of the gbl is enhanced by the overlying mucous which acts as a hydrodynamic barrier (Shephard, 1982) and supports ion gradients between the gbl and the bw (Takeuchi & Silen, 1985). Our observation that silicic acid protects against acute aluminium toxicity at a bw pH of 5.0 (Birchall et al., 1989) corroborates the existence of a circumneutral gbl pH. Aluminosilicate chemistry predicts that protection by silicic acid would be incomplete at a pH below 6.5 (Chappel & Birchall, 1988; Exley & Birchall, in press). The facility to maintain a circumneutral gbl pH protects fish in acid waters from the deleterious effects of acidity p e r se. In salmonids acid toxicity is rarely documented at bw pH above 4.5 (McDonald, 1983). Thus, in dilute acid waters, pH range 4.5-5.5, the corresponding gbl pH favours aluminium binding to oxygen-based functional groups (see Fig. 2). A New Mechanism of Toxicity

In acute aluminium toxicity aluminium is bound to apically located groups in the gill lamellar epithelium. Specific binding sites are implicated (see Fig. 3). For example, aluminium might complex the phospholipid phosphatidylserine (Shi & Haug, 1988) neutralizing the charge on one or more carboxylate and/or phosphate ligand and subsequently reducing membrane fluidity (Viersta & Haug, 1978). Similarly aluminium substitution for the metal co-factors of transport proteins would ,be expected to alter membrane permeability. Aluminium is known to inhibit the active uptake of NaC1, (Dalziel et al., 1987) and the inhibitive mechanism might involve aluminium substitution for either a metal co-factor in the functional domain of an active transport protein or the transport species per se. Aluminium substitution for Ca 2+ in intercellular cement would also be expected to alter paracellular membrane permeability. Thus, aluminium binding to extracellular ligands at the gill epithelium will alter membrane barrier properties and is a prerequisite to acute toxicity (Exley, 1989). Chemistry predicts such apical aluminium binding to be reversible, however, the effects of an acute challenge are rarely documented to be so and death is often the result even after the return of fish to optimal water quality. We suggest that apically bound aluminium alters membrane permeability to allow the intracellular accumulation of aluminium (see Fig. 3). Such an effect is documented in phospholipid vesicles in vitro (Shi & Haug, 1988). The initial fate of intracellular aluminium is unknown, however citrate is the pre-eminent intracellular ligand, c. 0.1 mM l -j, and at physiological pH, competes successfully for aluminium binding against alternative ligands such as inorganic phosphate and ATP (McDonald & Mamrack, 1988). The citrate complex itself is unlikely to be directly toxic but citrate will act as an intermediate chelator, passing aluminium to groups with higher affinity (Birchall & Chappell, 1989). The rapid and continued intracellular accumulation of aluminium favours competition for aluminium with citrate from minority high affinity cytosolic ligands. Such ligands will include highly phosphorylated proteins and the phosphate esters of inositol (Birchall & Chappell, 1988a). Myoinositol hexaphosphate, for example, binds aluminium ten times more strongly than

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mbl

Woter flo~

l! Blood flow

FIG. 3. Schematic representation of the potential sites of aluminium interaction at the lamellar epithelium of the gill. mbl, Mucous boundary layer; ml, mucous layer; gbl, gill boundary layer; ij, intercellular junction; ap, ATPase pump; cc, chloride cell; ac, accessory cell; ec. epithelial cell. Numbers denote interaction sites. (1) Apical surface of chloride cell. (2) Basally-located active transport system. (3) Narrow apical junction connecting chloride and accessory cells. (4) Wide apical junction connecting accessory and epithelial cells. (5) Polyanionic mucous layer including the enzyme carbonic anhydrase. (6) Apical surface of epithelial cell. (7) Intracellular effects on intercellular junctions. (8) lntracellular accumulation of aluminium. (9) Apically-located active transport system. (10) Apical membrane channels. (11) Extracellular effect on intercellular junctions.

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an e q u i m o l a r c o n c e n t r a t i o n o f citrate at physiological p H (Birchall & Chappell, 1988b). The biochemistry o f these biomolecules is such that a l u m i n i u m binding w o u l d have severe biochemical repercussions (Berridge & Irvine, 1989). For example, the s e c o n d messenger system is integral to the control o f intracellular calcium homeostasis, (Carafoli, 1987). Recently a l u m i n i u m has been s h o w n to interfere with p h o s p h a t i d y l inositol hydrolysis in vitro ( M c D o n a l d & M a m r a c k , 1988) and with calcium homeostasis in b o t h isolated rat hepatocytes (Schrfl et al., 1990) and m o u s e pancreatic acinar cells (Wakui et al., 1990). Cytosolic calcium, often in t a n d e m with protein kinase C is a f u n d a m e n t a l trigger o f cellular activity and is integral to the control o f both intra and intercellular ion transport (Nishizuka, 1986). A l u m i n i u m interference in the s e c o n d messenger system o f gill epithelial cells w o u l d have catastrophic effects on the barrier properties, (trans- and paracellular transport) o f the gill lamellar epithelium. Such effects are consistently d o c u m e n t e d in acute a l u m i n i u m toxicity, (see Table 1) and represent the m a j o r contribution to an acute response. The c o n c l u s i o n is that a l u m i n i u m is not merely an irritant to fish in acid waters but exerts two principal effects. It induces an increase in gill epithelium permeability with c o n s e q u e n t alterations in t r a n s m e m b r a n a l ion fluxes. A l u m i n i u m is allowed to a c c u m u l a t e intracellularly in epithelial cells a n d o n a p p r o a c h i n g a toxic threshold illicits deleterious disruptions in cytosolic calcium homeostasis. S u b s e q u e n t accelerated cell death exacerbates the b r e a k d o w n in the barrier properties o f the gill and the death o f fish results. A l u m i n i u m effects on gill m e m b r a n e permeability resulting in accelerated cell death m a y be general features o f a l u m i n i u m toxicity in cells. C. Exley is an ICI Research Fellow at the University of Stirling.

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