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An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury.

The emergence of zinc as a potent neurotoxin has prompted the development of techniques suitable for the measurement of intracellular free zinc ([Zn(2+)](i)) in cultured cells. Accordingly, a new family of Zn(2+)-sensitive fluorophores has become available. Using ionophore-induced elevations of [Zn(2+)](i) in cultured neurons, we measured [Zn(2+)](i)-induced changes in the novel dyes FuraZin-1 and FluoZin-2 and compared them with the established [Zn(2+)](i)-sensitive fluorophores mag-fura-2 and Newport Green. All of these dyes effectively detected [Zn(2+)](i), and FuraZin-1, FluoZin-2, and Newport Green showed selectivity for [Zn(2+)](i) over [Ca(2+)](i) and [Mg(2+)](i). However, the dyes showed little difference in their apparent sensitivity to [Zn(2+)](i), even though their in vitro affinities for Zn(2+) varied from 20 nM to 3 microM. We show herein that this is a consequence of the relatively high concentrations of intracellular dye used in experiments of this nature. Thus, for the measurement of [Zn(2+)](i), the sensitivity of the reporting system is dominated by the intracellular dye concentration, whereas dye affinity is unimportant. We extend these findings to show that calibration of dye signal to ion concentration is critically dependent on precise measurement of intracellular dye concentration.

Iron is an essential element for cells but may also be an important cytotoxin. However, very little is known about iron transport, redox status, or toxicity specifically inside cells. In this study, we exploited the sensitivity of fura-2 to quenching by ferrous iron (Fe(2+)) to detect intracellular free iron ([Fe(2+)](i)) in neurons, astrocytes, and oligodendrocytes in primary culture. All cell types exposed to Fe(2+) in the presence of the ionophore pyrithione rapidly accumulated Fe(2+) to a similar extent. The heavy-metal chelators bipyridyl and N,N,N',N'-tetrakis(2-pyridalmethyl)ethyl-enediamine rapidly reversed the increase in [Fe(2+)](i), whereas desferrioxamine had little effect. Interestingly, the Fe(2+)-mediated quenching of fura-2 fluorescence was reversed in a concentration-dependent manner by hydrogen peroxide. This was likely caused by the oxidation of Fe(2+) to Fe(3+) inside the cell. Acute exposure of cells to Fe(2+) was only toxic when the metal was applied together with pyrithione, showing that Fe(2+) is only toxic when elevated inside cells. Interestingly, only neurons and oligodendrocytes were injured by this elevation in [Fe(2+)](i), whereas astrocytes were unaffected, although [Fe(2+)](i) was elevated to the same degree in each cell type. These studies provide a novel approach for detecting [Fe(2+)](i) in a manner sensitive to the redox state of the metal. These studies also provide a model system for the study of the toxic consequences of elevated [Fe(2+)](i) in neural cells.

Using the H(+)-sensitive fluorophore 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and microfluorimetry, we investigated how elevated intracellular free zinc ([Zn(2+)](i)) altered intracellular proton concentration (pH(i)) in dissociated cultures of rat forebrain neurons. Neurons exposed to extracellular zinc (3 microM) in the presence of the Zn(2+)-selective ionophore pyrithione (20 microM) underwent intracellular acidification that was not reversed upon washout of the stimulus. Application of a membrane-permeant Zn(2+) chelator, but not an impermeant chelator, partially restored pH(i). Removal of extracellular Ca(2+) greatly inhibited [Zn(2+)](i)-induced acidification, suggesting that acidification was a secondary consequence of Ca(2+) entry. Additional experiments suggested that Ca(2+) entered through the plasma membrane sodium/calcium exchanger (NCE), because a specific inhibitor of reverse mode NCE operation, KB-R7943 (1 microM), significantly inhibited Zn(2+)-induced acidification. In addition to the phenomenon of [Zn(2+)](i)-induced acidification, we found that elevated [Zn(2+)](i) inhibited neuronal recovery from low pH(i). Neurons exposed to a protonophore underwent robust acidification, and pH(i) recovery ensued upon protonophore washout. In contrast, neurons acidified by the protonophore in the presence of Zn(2+) (3 microM) and pyrithione (20 microM) showed no ability to recover from low pH(i). Application of a membrane-permeant Zn(2+) chelator partially restored pH(i) to pre-stimulus values. Experiments designed to elucidate mechanisms responsible for pH(i) regulation revealed that neurons relied primarily on bicarbonate exchange for proton export, suggesting that elevated [Zn(2+)](i) might impede pH(i) by inhibiting proton efflux via bicarbonate exchange. These results provide novel insights into the physiological effects of raising [Zn(2+)](i), and may help illuminate the mechanisms by which Zn(2+) injures neurons.

The membrane-permeant oxidizing agent 2,2'-dithiodipyridine (DTDP) can induce Zn(2+) release from metalloproteins in cell-free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asymmetric chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad-spectrum cysteine protease inhibitor butoxy-carbonyl-aspartate-fluoromethylketone. N,N,N', N'-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and other cell-permeant metal chelators also effectively blocked DTDP-induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK-801, by the intracellular calcium release antagonist dantrolene, or by high concentrations of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc-selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura-2- and magfura-2-loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP-mediated increase in intracellular free Zn(2+) concentrations. Our studies suggest that under conditions of oxidative stress, Zn(2+) released from intracellular stores may contribute to the initiation of neuronal apoptosis.

Increased intracellular free Zn(2+) ([Zn(2+)](i)) is toxic to neurons. Glia are more resistant to Zn(2+)-mediated toxicity; however, it is not known if this is because glia are less permeable to Zn(2+) or if glia possess intrinsic mechanisms that serve to buffer or extrude excess [Zn(2+)](i). We used the Zn(2+)-selective ionophore pyrithione to directly increase [Zn(2+)](i) in both neurons and astrocytes. In neurons, a 5-min exposure to 1 microM extracellular Zn(2+) in combination with pyrithione produced widespread toxicity, whereas extensive astrocyte injury was not observed until extracellular Zn(2+) was increased to 10 microM. Measurements with magfura-2 demonstrated that pyrithione increased [Zn(2+)](i) to similar levels in both cell types. We also measured how increased [Zn(2+)](i) affects mitochondrial membrane potential (Deltapsi(m)). In astrocytes, but not in neurons, toxic [Zn(2+)](i) resulted in an acute loss of Deltapsi(m), suggesting that mitochondrial dysregulation may be an early event in [Zn(2+)](i)-induced astrocyte but not neuronal death., (Copyright 2000 Academic Press.)